key: cord- -e im go authors: taschuk, ryan; scruten, erin; woodbury, murray; cashman, neil; potter, andrew; griebel, philip; tikoo, suresh k.; napper, scott title: induction of prp(sc)-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease date: - - journal: prion doi: . / . . sha: doc_id: cord_uid: e im go the ongoing epidemic of chronic wasting disease (cwd) within cervid populations indicates the need for novel approaches for disease management. a vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. the development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. to address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (dses), whose exposure for antibody binding depends on disease-associated misfolding of prp(c) into prp(sc). here, a dse corresponding to the rigid loop (rl) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. this vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein g recombinant fusion with the rl epitope (had :tgg-rl). oral immunization of white-tailed deer with had :tgg-rl induced prp(sc)-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. by building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular prp(sc)-specific oral vaccine for cwd as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based cwd vaccines. chronic wasting disease (cwd) is a prion disease that is currently spreading through north american cervid populations. incidence of cwd has largely been limited to north america, but reported cases in south korea and norway indicate the potential for global consequences. , cwd has had a devastating impact on the farmed elk industry and its ongoing spread through wild populations threatens a natural resource that is of considerable economic, ecological and social importance. the circumstantial evidence that human prion diseases can result from exposure to cwd, as well as a recent demonstration of oral transmission of cwd to cynomolgus macaques, add concern of the zoonotic potential of cwd. , at the current time, culling is the primary tool available for management of cwd. while there has been some success in limiting the spread of cwd in wild populations through strategic culling, this is an incomplete and controversial strategy for long-term disease management. , management of cwd, in particular within wild populations, will likely depend on the coordinated application of several disease management strategies. one important component of this strategy may be the use of vaccines to either reduce prp sc shedding or prevent infection. in particular, the unique ability of prions to cause long-term environmental contamination, lasting from years to decades, supports vaccination as a strategy to both achieve prophylactic prevention and reduce shedding towards mitigating exposure and infection of animals in contaminated environments. , emerging evidence of the uptake and presentation of environmentally-shed prions by plants further support the importance of limiting shedding by infected animals. development of a prion vaccine is challenged by the unique nature of the infectious agent; the misfolding of a self-protein (prp c ) into an infectious and pathological conformation (prp sc ). while an effective vaccine for cwd remains elusive, there is proof of principle evidence that this is an achievable goal as antibodies to prp c neutralize prion propagation in vitro and in vivo. [ ] [ ] [ ] [ ] [ ] [ ] [ ] numerous studies have shown the benefits of immunization to abrogate infectivity, reduce prp sc loads within relevant tissue, increase survival time, and provide partial protection following infection. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] further, the paradigms of vaccine success are shifting such that reduced shedding, rather than outright prevention of infection, may be a relevant and achievable measure of vaccine efficacy. while these results provide optimism for the development of a prion vaccine, there are concerns of potentially deleterious consequences associated with the induction of antibodies with reactivity towards a widely-expressed self-protein. while many passive and active immunization trials involving antibodies against prp c do not cause pathological reactions, other early experiments suggested that antibodies against prp c trigger neuronal apoptosis or activation of inappropriate signalling cascades. [ ] [ ] [ ] further investigation suggested that antibodies to a variety epitopes of the globular domain of prp c have varying degrees of neurotoxicity. mechanistically, this neurotoxicity seems to mimic the cellular events associated with prion infection and may involve antibody-induced conformational changes in prp c . , by targeting epitopes that are specifically exposed upon pathological misfolding of prp c to prp sc , it is possible to induce antibody responses specific for the pathological conformation, thereby sparing the healthy form of the protein and prioritizing the immune response to the problematic conformation. to date, three dses have been translated into injected vaccines that induce prp sc antibody responses. , of these, a rigid loop (rl) dse is considered particularly attractive for a cwd vaccine based on unique structural features of this loop in cervid prp including its predicted ability to convert from a compact loop into an unstructured region that resembles a free peptide immunogen. , the majority of efforts to develop prion vaccines have been in the context of injected delivery which could have application for farmed animals. for wild animals, an effective vaccine would need to be deployed without direct contact with animals, making oral vaccines the best delivery option. the potential benefits of oral vaccination are perhaps best exemplified by the highly-successful example of rabies that demonstrated it is possible to achieve protective immunity through oral, self-administered vaccines. , different molecular and biological vectors have been investigated as oral cwd vaccines and these studies have demonstrated that oral vaccine formulations can induce both mucosal and systemic antibody responses in deer and mice. [ ] [ ] [ ] [ ] perhaps most relevant, goni et al, using a salmonella-based oral delivery platform in deer, were able to induce partially protective immune responses to cwd. this, however, was achieved with an intensive immunization protocol ( immunizations, including specific manual application of the vaccine to the tonsils) that would limit the potential for application of this approach in a wildlife setting. optimistically this study provides proof-of-principle support for the development of an oral cwd vaccine while highlighting the need for delivery platforms that are compatible with a real-world wildlife vaccine from economic, regulatory and practical considerations. in this work, we describe the construction and immunogenic characterization of an oral prion vaccine based on a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein g recombinant fusion with the rl epitope (had :tgg-rl). oral delivery of had : tgg-rl to white-tailed deer induced prp sc -specific systemic and mucosal immune responses after two immunizations. the vaccine showed a satisfactory safety profile as characterized by limited vector shedding and non-reactivity of the induced antibodies to prp c . collectively, through strategies of formulation that have proven effective for other wildlife vaccines, these efforts provide a potential prp sc -specific oral cwd vaccine as well as providing a platform for the generation of other peptide-based oral cwd vaccines. recombinant virus was assayed for both incorporation of the tgg-rl open reading frame as well as expression of the tgg-rl fusion antigen. total dna was isolated from either mock-infected hek cells or those infected with either had , had :tgg-rl and subjected to restriction digest analysis. a restriction fragment of the appropriate size ( bp) corresponding to the tgg-rl expression cassette and flanking homologous recombination sequence was recovered only from cells infected with had :tgg-rl [ fig. a ]. an approximately kb fragment is observed within the viral infected cultures representing the adjacent genomic sequence coding for various structural adenovirus proteins, and is absent from mock-infected isolates [ fig. a ]. to confirm expression of the heterologous fusion protein, conditioned media from hek cells infected with either had or had :tgg-rl were analyzed by western blot. membranes were probed with polyclonal anti-tgg or anti-rl sera to confirm the composition of expressed antigen. from the media of the had :tgg-rl infected cells both the anti-tgg and anti-rl sera recognized a kda protein corresponding to the mature, processed form of tgg-rl [ fig. b ]. to assess the immunogenicity of had :tgg-rl white-tail deer (n d /group) were orally immunized and epitope-specific antibody titers in serum and feces were quantified with a peptide elisa. serum titers from of animals receiving the had :tgg-rl oral vaccine displayed seroconversion following primary immunization [ fig. a ]. peptide-specific antibody titers in serum continued to rise following a second immunization weeks later, plateauing at week and the gradually decreased during the remainder of the trial. one of the vaccinated animals failed to seroconvert at any time during the trial. the ability of oral had : tgg-rl vaccine to induce mucosal responses was also evaluated following oral immunization. dse-specific antibody responses were detected in of the animals receiving had : tgg-rl [ fig. b ]. the overall kinetics of the fecal antibody responses mirrored the serum antibody response. notably, the same animal that failed to develop detectable serum also failed to develop fecal antibody responses. these results confirm that oral delivery of a recombinant viral vector, expressing an appropriate dse and carrier molecule, is capable of inducing both systemic and mucosal antibody responses in white-tailed deer. splenocyte proliferative responses were analyzed to corroborate the induction of serum antibody responses. splenocytes were isolated weeks after the initial oral immunization, and were co-cultured with purified tgg protein to assess their responsiveness to the tgg carrier protein. splenocytes from all animals exhibited proliferative responses (si > . ) to concanavalin a, a polyclonal t cell mitogen (data not shown). splenocytes from of animals displayed moderate to strong proliferative responses to tgg that were significantly greater (p < . ) than that observed for animals receiving had [ fig. ]. splenocytes from the animal failing to seroconvert following oral immunization with had :tgg-rl also failed to respond to in vitro tgg stimulation. proliferative response data corroborate that the figure . vaccine production. (a) dna was isolated from hek cells infected with had and had :tgg-rl and subjected to paci digestion to identify the presence of tgg-rl coding sequence. (b) western blot of conditioned media from either mock infected or had :tgg-rl infected hek cells. proteins were resolved by sds-page, blotted to nitrocellulose and probed with either anti-tgg or anti-rl sera at : or : , respectively. protein-antibody complexes were probed with alkaline phosphatase labeled secondary antibody, and visualized following development with bcip/nbt. peptide-specific antibody responses in animals receiving had :tgg-rl immunizations could be supported by t-helper cells recognizing the tgg carrier protein. to determine if the antibodies induced by oral immunization retained the same prp sc specificity as previously characterized for the parenteral vaccine, serum from had :tgg-rl vaccinated animals was assayed for prp c reactivity by elisa, using recombinant cervid prp - for antibody capture. serum samples corresponding to peak antibody titers at week post-vaccination was selected based on the assumption that this provided sufficient time for affinity maturation of antibody responses. prp c reactive monoclonal antibodies, d and m , were used as positive controls, while a polyclonal antibody, affinity-purified from serum samples collected following parenteral vaccination with the rl construct, was included as a negative control. antibody titers for serum samples collected from animals receiving had were designated and were considered to reflect background activity when assaying serum samples in elisa [ fig. a ]. serum samples from animals receiving had : tgg-rl did not display detectable reactivity with cervid prp c as the group titers were calculated to be less than , similar to the control serum samples [fig. a ]. the prp sc specificity and reactivity of the serum antibodies induced by had :tgg-rl were also analyzed with immunoprecipitation assays, using both healthy and prion-infected brain tissue. a prp c/sc reactive monoclonal figure . systemic and mucosal epitope-specific antibody responses. white-tailed deer received an oral administration of . £ viral particles of either had (n d ) or had : tgg-rl (n d ). the animal who failed to mount an antibody response to the had :tgg-rl vaccine was excluded from this consideration. animals were orally immunized twice with a twoweek interval. serum antibody titers (a) and fecal antibody titers (b) were quantified with a capture elisa using rl peptide to coat the wells. data presented are as the mean § sd. antibody, ah b, as well as polyclonal antibodies affinity-purified from serum samples collected following parenteral vaccination with a yml construct were included as controls. the yml antibody has demonstrated reactivity with prp sc in immunoprecipitation assays pooled serum samples collected from animals six weeks after had :tgg-rl vaccination demonstrated reactivity with prp sc brain tissue but failed to react with prp c in healthy brain material [ fig. b ]. further, pre-immune serum from the had :tgg-rl vaccinated animals failed to react with prp sc . to assess the duration of had vector shedding, fecal samples were assayed for viral dna using an adenovirus hexon-specific pcr. fecal samples collected prior to immunization were negative for adenovirus [ fig. ]. at three days post immunization, fecal samples from all animals vaccinated with either had :tgg-rl or had were positive for the adenovirus hexon sequence. this vector shedding reflects passive transmission of the oral vector through the animal since the had vector was not replication competent. at days after primary (a) antibody titers were quantified by capture elisa using cervid prp c - as coating antigen, and are reported as mean values § sd. monoclonal antibodies d and m served as positive controls, while polyov.rl sera served as a negative control. (b) immunoprocipitation of prp from healthy and prioninfected brains. pooled serum from deer orally immunized with had :tgg-rl was assessed for reactivity with prp sc and prp c . serum antibodies were cross-linked to magnetic beads and incubated with non-infected and infected % brain homogenate. a prp c/sc reactive monoclonal antibody, ah b, as well as polyclonal antibodies affinity-purified from serum samples collected following parenteral vaccination with a yml construct were included as controls. the yml antibody has demonstrated reactivity with prp sc in immunoprecipitation assays. figure . vector shedding. ten white-tailed deer (n d /group) orally received . £ viral particles of either had or had :tgg-rl. animals were orally immunized at d and d . total dna was isolated from fecal samples on individual animals, and served as template for diagnostic pcr to identify viral sequence coding for the ad hexon protein. dna concentration was standardized for each ul pcr, and entire reactions were resolved in % agarose gels. immunization all animals were pcr negative for ad . similarly, three days after the secondary vaccination, fecal samples from all animals were again positive for adenoviral dna [fig. ]. finally, all animals were confirmed to not be shedding detectable levels of virus when the trial concluded days post-vaccination. sequencing of the pcr product generated from day fecal samples confirmed the amplified sequence matched human group c adenovirus hexon sequence (ac_ . ). prion vaccine development by our group has prioritized epitopes that induce antibody responses specific to the misfolded prp sc species. to date, peptide-based vaccines, corresponding to three different dses, have been delivered parenterally and their immunogenicity and specificity has been characterized. , , the priority of the current investigation was to translate the dse corresponding to the prp rigid loop region to an oral vaccine vector. in terms of philosophy and molecular construction, these efforts build upon the highly successful example of oral vaccines for rabies. the haptenic nature of peptides requires the use of an immunogenic carrier protein to generate the t-cell help required for the induction of an antibody response. our previous cwd vaccines utilized the leuktoxin (lkt) protein of mannheimia haemolytica to support the induction of t-cell dependent antibody responses. however, this large prokaryotic protein is poorly suited for expression in eukaryotic systems and viral vectors. in contrast, rabies glycoprotein g (gg) is an extremely potent immunogen capable inducing robust and prolonged immune responses. as a carrier, gg is capable of inducing immune responses to heterologous antigens and peptide epitopes. in a direct comparison with lkt, a truncated version of gg (tgg) induced significantly higher titre, and longer duration, dse-specific antibody responses. for these reasons, the tgg-rl fusion antigen was selected for translation into an oral delivery vector. human adenovirus serotype (had ) was selected as a mucosal vaccine vector for its broad species and tissue tropism, as well as propensity to induce both systemic and mucosal humoral immunity to heterologous antigens. , many mammals share habitats with cervids, making a replication-incompetent had a priority to ensure limited environmental contamination by the vaccine. shedding of the had vector was limited to three days immediately following vaccination. in contrast, shedding could be detected for weeks following oral delivery of a replication-competent vector. that the replication-defective vector was able to infect cells in the gastro-intestinal tract of white-tailed deer and express the transgene was evident in the induction of rl-specific systemic and mucosal antibody responses. although larger antigen expression, and therefore greater immune responses would likely be possible with the use of a replication-competent virus, safety issues surrounding environmental contamination or secondary immunization of non-target species influenced our choice of a replication-defective virus. within these efforts, only four of the five animals receiving the had-tgg-rl vaccine demonstrated detectable epitope-specific antibody responses. based on virus shedding, we can be confident that the non-responding animal (an ) received the vaccine but (based on the splenocyte proliferation assays and antibody titres) did not mount an immunological response to the vaccine. there are a number of potential explanations for this vaccine non-responder. firstly, it is important to appreciate that the purpose of this investigation was to provide proof-of-principle for the development of an oral vaccine for cervids utilizing vectors that are consistent with a real-world vaccine from financial and regulatory perspectives. this did not include efforts to optimize either the dose or timing of the vaccinations which could certainly impact the magnitude and consistency of the induced responses. as such, it may be possible to achieve a more prominent and uniform response through optimizing the dose and/or timing of vaccination. secondly, within outbred populations, even for commercialized vaccines, it is not unusual to observe variability in vaccine responsiveness, including non-responders. r. taschuk et al. further to that, the antibody titres induced following oral vaccination with the had :tgg-rl vector were approximately two orders of magnitude lower than those achieved when using an adjuvanted parenteral vaccine with the same dse. , this may be due to the delivery of an unadjuvanted oral vaccine which was not dose optimized as well as the use of a replicationincompetent vector that generated a limited amount of antigen. further, with the oral vaccine, there wasn't a significant anamnestic response following the secondary immunization. this may be due to the induction of had -specifc immunity, which prevented virus infection or uptake by mucosa-associated lymphoid tissue. the parenteral dse-specific vaccine induced antibody responses that were also detectable in nasal secretions and cerebrospinal fluid. the failure of the oral vaccine to induce detectable humoral responses within either nasal secretions or csf [data not shown] may be attributable to the induction of a much lower systemic antibody response. there are several options to increase the immunogenicity, and potentially protective capacity, of the had vector. these include optimizing the dose and timing of vaccination as well as incorporating accessory immunostimulatory proteins in the transgene, chemical modification or encapsulation of the recombinant virus to decrease degradation in gastrointestinal tract, and possibly reducing interference by vector immunity by increasing the interval between multiple immunizations. [ ] [ ] [ ] while developing a cwd vaccine for use in wild cervids was the primary objective of this study, these results may also be applicable in captive cervids and domestic livestock. a properly formulated and sufficiently immunogenic vaccine could be delivered in the feed of animals, thereby providing a method to eliminate the handling of individual animals during vaccination. while oral immunization is the only option for implementing a cervid-specific wildlife vaccination program, a commensurate benefit to mucosal vaccination is the induction of a broad anti-prp humoral response. recent studies indicate the importance of inducing both systemic and mucosal immunity, where high levels of igg and iga correlated to increased survival. [ ] [ ] [ ] while the current efforts prioritized a particular dse, the platform could easily be adapted for other peptide epitopes, including those associated with antibody responses against prp c . total rna was isolated from rabies-positive fox brain tissue using rneasy mini kit (qiagen) and a cdna library was synthesized using superscript iii cdna library construction kit (life technologies). a truncated version of the rabies glycoprotein g (gg) was amplified using primers that truncated the region to eliminate the transmembrane and cytosolic domains, as well as to facilitate the introduction the codon-optimized sequence corresponding to the rigid-loop epitope. the resulting gene product encodes a product in which the rl epitope is presented as a c-terminal fusion of the truncated tgg protein. dna sequence was synthesized (genscript) and further amplified to contain a stop codon and flanking restriction sites to facilitate further cloning. both tgg and rl were restricted and co-cloned into ph l vector containing signal transcription and regulatory elements to facilitate antigen expression. paci-digested ph l:tgg-rl and ph r were co-transfected into hek cells using calcium-phosphate hek transfection kit (promega) to facilitate homologous recombination and the production of recombinant had : tgg-rl virus. an e /partial e -deleted had lacking any heterologous protein expression was included as a negative control. both had and had :tgg-rl viruses was amplified following several passages in hek cells, purified by cscl gradient centrifugation and concentration was determined from the formula: viral particles/ml d (optical density at nm) £ (dilution factor) £ ). isolation of recombinant adenoviral dna from infected tissue culture hek cells in t format were infected ( £ vp) with had :tgg-rl, had control virus, or mock infected. cells exhibiting full cpe h post infection were harvested, pelleted and incubated in extraction buffer ( mm nacl, . % triton x- , mm tris-hcl ph . ) for min at c. following centrifugation, supernatant was incubated with % sds and . mg/ml proteinase k at c for h. dna isolated by phenol:chloroform: isoamyl alcohol extraction and ethanol precipitation. isolated dna was restricted with paci to identify the tgg-rl open reading frame. restriction digest products were subjected to electrophoresis in % agarose gels. hek cells were infected with either £ vp had :tgg-rl, had control or mock infected. conditioned media containing ug total protein was isolated from all cultures h post infection and subject to electrophoresis in % sds-polyacrylamide gels, and transferred to a nitrocellulose membrane (bio-rad). membranes were probed with either anti-tgg or anti-rl polyclonal serum at : or : , respectively. immune complexes were further probed with alkaline phosphatase-conjugated goat anti-mouse igg (hcl) (kpl) at : . specific proteins were visualized following the addition of sigmafast bcip/nbt substrate (sigma-aldrich). ten six-week old female white-tailed deer (n d /group) orally received £ viral particles of ad :tgg-rl or ad virus in ml unsupplemented eagles minimum essential medium at days and . animals were housed under biosafety level containment for the duration of the trial. individual serum, nasal swabs, feces, and cerebrospinal fluid were isolated, and stored at ¡ c. an independent research team performed all animal work and experiments were done according to the guide to the care and use of experimental animals, provided by the canadian council on animal care. the saskatchewan animal care committee approved all experimental protocols. fecal samples were thawed and suspended to . g/ml in chilled extraction buffer (pbs, . % tween , x complete protease inhibitor cocktail (roche)). suspensions were vortexed, nutated for min at c, cleared of cellular debris by centrifugation at , £ g for min at c, and stored at ¡ c. dna was isolated from approximately mg of fecal material of individual animals using qiaamp dna stool mini kit (qiagen) to manufacturers specifications. eluted dna was supplemented with . ug/ul bsa and served as template for pcr to identify viral dna. template dna was standardized to ng/ ul reaction, and pcrs were conducted using phusion high-fidelity polymerase, to manufacturers specifications (neb). had primers were constructed based on the published sequence of the human adenovirus serotype hexon (ac_ . ): had -hexf : ggaca tggcttccacgtact had -hexr : gcct gttgggcaatagattgt. genomic human adenovirus serotype dna purified from spiked white-tailed deer fecal samples served as positive control template, and dna purifications from non-spiked samples served as negative control. serum and mucosal epitope-specific antibody responses were quantified by elisa through previously described protocols. serum prp c -specific antibody responses were quantified by elisa using samples from peak titer at week , performed similarly as previously described, using affinity purified truncated cervid prp - as coating antigen. control antibodies d , m , and ovine polyclonal anti-rl serum were used at : initial dilution. elisa titres are expressed as the reciprocal of the highest serum dilution resulting in an od reading exceeding two standard r. taschuk et al. deviations above the value for the pre-immune serum. serum from immunized deer were evaluated for interaction with prp sc and prp c . immunoglobulin isolated using protein-a column-affinity purification was conjugated to magnetic beads for brain homogenate immunoprecipitation assays as described. a prp c/sc reactive monoclonal antibody, ah b, as well as polyclonal antibodies affinity-purified from serum samples collected following parenteral vaccination with a yml construct were included as controls. the yml antibody has demonstrated reactivity with prp sc in immunoprecipitation assays. spleens were harvested from immunized animals at day post oral immunization. spenocyte isolation and culture were described previously. splenocytes were cultured for h in triplicate with affinity-purified tgg ( . and . mg/ml), concanavalin a ( . mg/ml). cells were cultured in a final volume ul and pulsed with . uci / ml [ h] thymidine. following h incubation, incorporation of [ h]-thymidine was determined using standard scintillation counting methods. proliferative responses were calculated as a stimulation index (counts per minute with stimulating antigen/counts per minute with media alone) and expressed as the mean of triplicate cultures. the data represent repeated measures of elisa antibody titres in animals over time and did not adhere to a normal distribution. to account for the repeated measures study design, data for each animal were first summed over time. summed data were then ranked to account for their non-normal distribution and a one-way anova analysis performed on the ranked sums. where appropriate, tukey's test was used to examine differences among treatment groups. p values less than . were considered significant. none. occurrence, transmission, ad zoonotic potential of chronic wasting disease strain characterization of the korean cwd cases in and first case of chronic wasting disease in europe in a norwegian free-ranging reindeer creutzfeldt-jakob disease in unusually young patients who consumed venison first evidence of intracranial and peroral transmission of chronic wasting disease (cwd) into cynomolgus macaques: a work in progress evaluation of a wild white-tailed deer population management program for controlling chronic wasting disease in illinois systematic review of management strategies to control chronic wasting disease in wild deer populations in north america modeling routes of chronic wasting disease transmission: environmental prion persistence promotes deer population decline and extinction scrapie agent (strain ) can transmit disease via the oral route after persistence in soil for years grass plants bind, retain, uptake, and transport infectious prions 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alters the magnitude, duration and type of antibody response to a peptide epitope from a selfprotein an adenovirus-simian immunodeficiency virus env vaccine elicits humoral, cellular, and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge an adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine recombinant adenovirus shedding after intratumoral gene transfer in lung cancer patients vectors encoding carcinoembryonic antigen fused to the b subunit of heat-labile entertoxin elicit antigenspecific immune responses and antitumor effects enhanced mucosal immunogenicity of prion protein following fusion with b subunit of escherichia coli heat-labile enterotoxin pegylation of a vesicular stomatitis virus g pseudotyped lentivirus vector prevents inactivation in serum severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice development of novel formulations that enhance adenoviral-mediated gene expression in the lung in vitro and in vivo ileal and jejunal peyer's patches play distinct roles in mucosal immunity of sheep key: cord- -r s gks authors: cutts, julia c.; agius, paul a.; zaw lin; powell, rosanna; moore, kerryn; draper, bridget; simpson, julie a.; fowkes, freya j. i. title: pregnancy-specific malarial immunity and risk of malaria in pregnancy and adverse birth outcomes: a systematic review date: - - journal: bmc med doi: . /s - - - sha: doc_id: cord_uid: r s gks background: in endemic areas, pregnant women are highly susceptible to plasmodium falciparum malaria characterized by the accumulation of parasitized red blood cells (prbc) in the placenta. in subsequent pregnancies, women develop protective immunity to pregnancy-associated malaria and this has been hypothesized to be due to the acquisition of antibodies to the parasite variant surface antigen var csa. in this systematic review we provide the first synthesis of the association between antibodies to pregnancy-specific p. falciparum antigens and pregnancy and birth outcomes. methods: we conducted a systematic review and meta-analysis of population-based studies (published up to june ) of pregnant women living in p. falciparum endemic areas that examined antibody responses to pregnancy-specific p. falciparum antigens and outcomes including placental malaria, low birthweight, preterm birth, peripheral parasitaemia, maternal anaemia, and severe malaria. results: we searched databases and identified studies ( from africa) that met predetermined inclusion and quality criteria: studies contributed estimates in a format enabling inclusion in meta-analysis and were included in narrative form only. estimates were mostly from cross-sectional data ( studies) and were heterogeneous in terms of magnitude and direction of effect. included studies varied in terms of antigens tested, methodology used to measure antibody responses, and epidemiological setting. antibody responses to pregnancy-specific prbc and var csa antigens, measured at delivery, were associated with placental malaria ( studies) and may therefore represent markers of infection, rather than correlates of protection. antibody responses to pregnancy-specific prbc, but not recombinant var csa antigens, were associated with trends towards protection from low birthweight ( studies). conclusions: whilst antibody responses to several antigens were positively associated with the presence of placental and peripheral infections, this review did not identify evidence that any specific antibody response is associated with protection from pregnancy-associated malaria across multiple populations. further prospective cohort studies using standardized laboratory methods to examine responses to a broad range of antigens in different epidemiological settings and throughout the gestational period, will be necessary to identify and prioritize pregnancy-specific p. falciparum antigens to advance the development of vaccines and serosurveillance tools targeting pregnant women. in malaria-endemic areas, individuals can acquire clinical immunity to plasmodium falciparum malaria after repeated exposure and symptomatic episodes in adults are relatively rare [ ] . despite this acquired immunity, pregnant women are highly susceptible to p. falciparum malaria. pregnancy-associated malaria (pam) represents a major public health problem, leading to poor outcomes for both mother and infant, including maternal mortality, maternal anaemia, miscarriage, stillbirth, low birthweight, and preterm birth [ ] [ ] [ ] [ ] [ ] [ ] . in endemic regions, primigravidae are at greatest risk of pam, and the frequency and density of both placental and peripheral p. falciparum infection decreases with successive pregnancies [ , [ ] [ ] [ ] [ ] [ ] . malaria in pregnancy is characterized by the accumulation of p. falciparum parasitized red blood cells (prbc) in the placental intervillous space, often observed with macrophage infiltration, fibrinoid, and parasite pigment deposits [ , ] . parasites taken from infected placentas display preferential binding to the glycosaminoglycan chondroitin sulfate a (csa) [ ] , present on the surface of placental syncytiotrophoblasts and intervillous spaces [ ] [ ] [ ] . this binding phenotype is rarely observed in parasites taken from non-pregnant individuals [ , [ ] [ ] [ ] [ ] , which are more likely to bind to receptors cd and icam- in the vascular endothelium. thus, the parasites that infect pregnant women are understood to constitute a distinct population to those that infect non-pregnant individuals. parasite binding to csa on syndecan- [ ] is mediated by the p. falciparum erythrocyte membrane protein (pfemp ) family member var csa [ ] [ ] [ ] [ ] [ ] , expressed on the surface of prbc. var csa is a large ( kda) protein with six duffy-binding-like (dbl) domains (dbl - ) and three interdomain (id) regions [ ] [ ] [ ] . the development of protective immunity to pam over successive pregnancies has largely been assumed to be due to the acquisition of antibodies to var csa, specifically those that block adhesion to csa. two vaccine candidates based on the n-terminal csa-binding region of var csa [ ] have entered early-stage clinical trials: pamvac is comprised of domains id -dbl x-id a from the p. falciparum strain fcr [ , ] and primvac is comprised of domains dbl x-dbl x from p.f. d [ ] . several studies have demonstrated parity-dependent increases in antibody responses to pregnancy-specific variant surface antigens on prbc [ , , [ ] [ ] [ ] , antiadhesion antibodies to csa-binding parasites [ , ] , and some, but not all, var csa domains [ ] [ ] [ ] [ ] , and a stronger correlation between parity and antibody responses has been observed in areas of more intense transmission [ ] . despite strong evidence for paritydependent acquisition of antibodies to var csa, and evidence for a role for var csa in mediating adhesion to csa in the placenta, direct epidemiological evidence for a protective effect of var csa antibodies in preventing pam and associated adverse pregnancy and birth outcomes has been inconsistent. furthermore, the specific antigenic targets, and functional responses necessary for protection against malaria in pregnancy and poor birth outcomes has not been established across multiple populations. we conducted a systematic review and meta-analysis of population-based studies examining associations between antibodies to pregnancy-specific p. falciparum antigens, and pregnancy and birth outcomes including placental malaria, low birthweight, preterm birth, maternal anaemia, and severe malaria. a more comprehensive understanding of the acquired immune response to pam will inform vaccine development and may help to identify serological correlates of immunity that could be employed in serosurveillance tools. the meta-analysis of observational studies in epidemiology (moose) working group [ ] guidelines and the preferred reporting items for systematic reviews and meta-analyses (prisma) specifications were adhered to in the conducting and reporting of this systematic review and meta-analysis [ ] . a completed prisma checklist is included in additional file . pubmed, web of science, scopus, african index medicus, lilacs (latin american and caribbean health sciences literature), and the malaria in pregnancy consortium databases were searched for studies published in all years up to and including june that examined the association of antibody responses to pregnancy-specific p. falciparum antigens and pregnancy and birth outcomes. key words included var csa, falciparum, pregnancy, parasitaemia, igg, dbl, placental infection, antibody, immunity, protection, vsa, variant surface antigen, pfemp , birth outcome, birthweight, gestational age, preterm birth, and intrauterine growth restriction. google scholar was used to identify additional studies by senior authors of some studies identified through other database searches but was not used in a systematic manner due to the unwieldy number of records returned using key words listed above. the reference lists of obtained papers were searched for further studies. studies reported in languages other than english were included and translated into english using online translation applications. we did not formally attempt to identify unpublished population studies because it would require us to provide substantial descriptions of the study design, sample testing and analysis used in the studies, and a review of ethical and other issues. the full search strategy for one database (pubmed) is provided (additional file ). criteria for considering studies for this review study designs and study participants population-based cross-sectional, case-control, cohort studies, including treatment to reinfection studies, and randomized controlled trials (rcts), excluding vaccine efficacy trials, were included. the primary criterion for study inclusion was pregnant women living in areas endemic for p. falciparum infection. all geographical locations were included. studies which included multiple population subsets were assessed on a sub-population basis to determine eligibility for inclusion. we included studies that measured immunoglobulin g (igg) responses to placental isolates, pregnancy-specific parasite strains including cs , and other strains that had been selected for binding to chondroitin sulfate/chondroitin sulfate proteoglycans, and recombinant or synthetic defined pregnancy-specific variant surface antigens. studies that employed the following types of assays to measure total antibody responses were considered: enzyme-linked immunosorbent assay (elisa), multiplex assay, and flow cytometry. we also included studies that measured functional antibody responses to pregnancy-specific prbc, including csa binding inhibition (anti-adhesion assay), prbc cell-agglutination, and phagocytosis. studies in which antibodies were measured in peripheral blood taken during pregnancy and/or the immediate postpartum period were considered. for cohort studies and rcts, if antibody responses were measured at more than two time points, results from enrolment and the latest (e.g. delivery) sampling time were extracted. estimates from cohort studies and rcts in which antibody responses were determined after the outcome measures of interest were excluded. outcome data were measured during pregnancy, at birth, or during the immediate postpartum period (within h of delivery). if not presented in the requisite format in the original papers, authors were asked to provide data for maternal p. falciparum placental malaria, maternal peripheral p. falciparum infection, low birth weight (< g), premature birth (delivery before weeks of gestation), and anaemia or severe anaemia (as defined in each study), and severe malaria, where relevant. we included estimates where women with active or active-chronic placental infection were compared to women with no placental infection, but we did not include past placental infection (often characterized by the presence of haemozoin in fibrin) as an outcome because in such cases the temporal relationship between antibody responses and infection would be difficult to ascertain, nor did we include estimates where active placental infection was compared with past infection. the minimum quality criteria for inclusion of studies were as follows: for placental malaria, confirmation of p. falciparum placental infection by slide microscopy of placental blood, polymerase chain reaction (pcr), or placental histology for the examination of p. falciparum parasites; for peripheral parasitaemia, detection by slide microscopy or pcr; for low birth weight, defined as less than g and birth weight was measured within h of birth; and for preterm birth, defined as delivery at less than weeks gestation, where gestational age must have been confirmed using crown rump length (crl) from ultrasound and robinson's chart or date of last menstrual period (lmp). studies that used rapid diagnostic tests as the sole method of diagnosis for p. falciparum infection were excluded. antibody levels must have been determined in maternal peripheral blood samples preceding or at the same time as outcome measurement. studies in which antibodies were measured in cord blood, placental blood, or infant peripheral blood were excluded. cut-offs for positive antibody responses by elisa or other means must have been defined using unexposed (malaria-naïve) controls or men/children from the malaria-endemic area. three authors (jcc, rp, and zl) identified possible studies and assessed the methodological quality of included studies independently, with discrepancies resolved by discussion with a fourth review author (fjif). for studies that analysed antibody levels as the outcome variable rather than the exposure variable, where possible, data were extracted and re-analysed with the specified maternal/birth outcome as the outcome variable. if the raw data were not presented, authors of the study were invited to re-analyse or provide data for the inclusion of their study in the systematic review. in addition, we contacted several authors whose studies did not meet the inclusion criteria but contained data that were eligible for the systematic review. contact was established through an initial email explaining the nature of the systematic review and the information required, together with a data extraction form for authors to complete (additional file ). if the corresponding author did not respond within three email attempts, then no further action was taken. for cross-sectional, rcts, and cohort studies, selection bias was assessed by reviewing inclusion and exclusion criteria of each study. for case-control studies, the comparability of cases and controls was assessed. an additional selection bias can occur in case-control studies when cases and/or controls are selected based on criteria relating to their exposure (i.e. antibody) status or there are differences in the reporting of exposure between cases and controls. however, this is unlikely because immunoassays were after enrolment into the study. information bias (resulting from flaws in measuring antibody and p. falciparum outcome data) is unlikely because antibodies are measured using immunoassays that are standardized within each study and across outcome groups. furthermore, the quality criterion of this review ensures accurate measurement of maternal and infant outcomes and it is unlikely that measurement of outcomes would differ according to antibody groups. initially, two authors (jcc and rp) independently assessed bias, with discrepancies resolved by discussion with a third review author (fjif). risk of bias assessment was collated by jc using the risk of bias in non-randomized studies-of interventions (robins-i) tool (additional file ) [ ] . the risk of bias assessment pertains to the association between antibody responses and pregnancy and birth outcomes derived from the study, rather than the study itself. measures of association between antibody responses and maternal and birth outcomes (odds ratios [ors], risk ratios [rrs], incidence rate ratios [irrs], or hazard ratios [hrs] ) and corresponding % confidence intervals (cis) were extracted or derived using reported data or unpublished data provided by authors. or, rr, hr, and irr are hereinafter denoted as rr. data extraction was performed independently by two review authors (jcc and rp) using the data extraction form (additional file ). for cross-sectional and case-control studies, odds ratios (ors) were extracted or calculated where possible. for cohort studies and rcts, risk ratios (rr), hazard ratios (hr), and incidence rate ratios (irr) were extracted or calculated where possible or unadjusted ors were converted to rr [ ] . if provided, cross-sectional data from rcts and cohort studies were extracted for inclusion in cross-sectional analyses. basic information about each study, including enrolment years, age of women, and iptp use, was extracted from individual publications where available. p. falciparum endemicity was categorized as low, intermediate, or high using information in the published papers. if insufficient information was provided in the publication, we used the malaria atlas project website (https://map.ox.ac.uk) to obtain estimates of the plasmodium falciparum parasite rate in - year olds (globally, - ) for each study site (longitude and latitude). we then categorized the endemicity of the study sites as follows: low [< %], intermediate [≥ % to < %], or high [≥ %]. measurement of antibody levels by established assays (elisa or flow cytometry) does not produce a common metric among studies. antibody data classified as "responders" or "non-responders" relative to a negative control (unexposed sera) were pooled, whereas categories based upon arbitrary cut-offs (including categories of responders based on statistical rankings) were simply reported in tables, but not included in the forest plots or meta-analyses. for studies where the antibody measures were analysed as continuous exposure variables, authors were asked to re-analyse their data by collapsing the antibody data into categories. if antibody or outcome data could not be provided in categorical form, the study's key findings on the association between antibody responses and outcomes of interest were described in table and in the text, that is, the study was included in narrative terms rather than quantitative terms. for studies in which responses to multiple allelic forms of an antigen or multiple parasite isolates or strains were analysed, estimates for the most seroprevalent antibody response were presented for that population. data on total igg responses or the most seroprevalent subclass response were also extracted. if antibody responses to the same antigen, in the same population-based study, were reported in several publications, results from the largest sample size were presented. separate estimates were obtained for the rr associated with prbc vsa, var csa: dbl , dbl + , id -id , id -id a, dbl , dbl x, dbl - , dbl , dbl , and full-length var csa (fv ). separate estimates were calculated for functional antibody responses. sub-group analyses were performed on women stratified by gravidity (primigravidae and multigravidae), where this was possible. a meta-analysis was performed, stratified by outcome, and where relevant var csa antigen and trimester of antibody and outcome determination. cross-sectional estimates from all study designs, except for case-control studies, were combined where possible; prospective estimates from rcts and cohort studies were combined where possible. where there were sufficient data, a pooled estimate for each malaria outcome was calculated using a random effects model. the standard error of the natural logarithm (ln) of the rr was calculated using the formula se(ln rr) = (ln(upper limit of % ci)ln(rr)) / . . the random effects meta-analyses were weighted using the inverse of the sum of the individual study sampling variances and a between-study variance component [ ] . the application of weights to individual study estimates in pooled effect estimation ensure (typically smaller) studies exhibiting higher standard error do not bias point estimates and contribute to under estimation of pooled effect confidence intervals. heterogeneity between studies was tested with the i statistic [ ] . if the i statistic was ≤ %, a metaanalysis based on a random effects model was conducted; when the i statistic was > % and/or the lower % confidence limit was between and %, the studies were not combined [ , ] . all analyses were performed using stata version . . database searches identified records, from which potentially relevant studies were selected based upon title and abstract. the full texts of these studies were examined to determine whether they complied with eligibility criteria: did not meet the inclusion criteria; fulfilled the inclusion and quality criteria; studies potentially met inclusion and quality criteria and authors were contacted with responses received from authors of studies (fig. ) . of the responders, authors provided data or estimates to fulfill inclusion and quality criteria, and for the remaining studies, the data were not available or did not meet inclusion/quality criteria. details of excluded studies are provided in additional file . a total of studies were included in the systematic review: studies contributed estimates in a format enabling inclusion in meta-analysis [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) and studies are included in narrative terms only because data were not available in the required format [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table ) . of those studies that contributed estimates, were crosssectional, were cohort (two of which contributed only cross-sectional data), were case-control studies, and were randomized controlled trials. the included table . c characteristics of studies included in narrative terms are presented in table cutts et al. bmc medicine ( ) : pregnancies, were ≥ years of age, and had term or premature deliveries. authors reported that all pm − women had been exposed to malaria since they had ab to dbl i infected women were individually matched by gravidity, age (± years), and date of delivery (± months) to women without evidence of current or previous placental infection according to the results of placental histological analysis and placental, peripheral, and cord blood smears, and/or to women with evidence of past placental infection (malaria pigment present by placental histological analysis but no parasites seen, together with negative placental, peripheral, and cord blood smears) j pregnant women who were blood film positive and diagnosed with one or more of the following clinical manifestations were categorized as having sm (n = ): severe anaemia, haemoglobin levels < g/dl; cerebral malaria, unrousable coma; hypoglycaemia, blood glucose levels < mg/dl; hypotension, systolic blood pressure < mmhg; jaundice, physical diagnosis or bilirubin levels > mg/dl and hyperparasitaemia, > , parasites/μl. pregnant women who were parasitaemic without these features were defined as uncomplicated malaria (um; women who were - weeks pregnant and were positive for peripheral parasitaemia by blood film examination were eligible to participate in trial and were randomized to one of three antimalarial treatment groups j same matched case-control study as beeson et al. [ ] . infected women were individually matched by gravidity, age (± years), and date of delivery (± months) to women without evidence of current or previous placental infection according to the results of placental histological analysis and placental, peripheral, and cord blood smears, and/or to women with evidence of past placental infection (malaria pigment present by placental histological analysis but no parasites seen, together with negative placental, peripheral, and cord blood smears we included estimates from nine studies that investigated the association between antibodies (abs) to pregnancy-specific prbc or var csa domains measured at delivery, and placental infection (fig. a) [ , , , , , [ ] [ ] [ ] ] . in all studies, placental infection was confirmed by slide microscopy of placental blood or placental histology. most studies demonstrated either no difference in the odds of placental infection in antibody responders compared to non-responders, or increased odds of placental infection in antibody responders at delivery (fig. a) . total igg responders to variant surface antigens (vsa) on csa-adherent parasite lines had increased odds of placental infection in pooled estimates from cross-sectional studies [ ] [ ] (reor = . , % ci . - . , i = . %) (fig. a) and in a case-control study (or . , % ci . - . ) [ ] . functional antibodies to cs (agglutinating, csa adhesion inhibitory) were associated with increased odds of placental infection [ ] in a malawian case-control study (or of and . fold, respectively). in contrast, a kenyan cross-sectional study showed that women who had antibodies that could inhibit adhesion of a placental isolate to csa had a % reduction in odds of placental infection (or . , % ci . - . ) [ ] (fig. a) . antibody responders to fv , and var csa domains, with the exception of dbl , had increased odds of placental infection compared to nonresponders [ , , , , ] (fig. a) . estimates for women sub-grouped by parity showed similar patterns of association between antibody responses at delivery and placental infection (additional file : figures s a and s a ). twelve studies included in narrative terms measured antibodies at delivery or in the third trimester and examined placental infection as an outcome [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] ] . nine of these studies found that abs to pregnancy-specific prbc and var csa antigens were positively associated with placental infection or placental parasite density [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] , but in some studies, this relationship was restricted to gravidity group, most commonly in primigravidae [ , ] ( table ) . one study reported no significant difference in total levels of var csa antibodies at delivery, but higher levels of high avidity abs to fv in women who were negative for placental infection compared to those who were positive for placental infection ( table ) [ ] . a cameroonian cross-sectional study reported that among malaria-positive women, those with high antiadhesion abs had reduced placental parasitaemia, but levels of anti-adhesion abs were similar between women positive and negative for malaria [ ] . to summarize, evidence from studies included in narrative terms suggests that whilst high avidity abs and anti-adhesion abs measured at delivery may be associated with protection from placental infection [ ] and reduced placental parasitaemia [ ] , respectively, total igg responses to var csa antigens and pregnancy-specific prbc are positively associated with the presence of placental malaria [ , , , [ ] [ ] [ ] [ ] [ ] ] . three prospective studies, including one rct [ ] and two cohort studies [ , ] , provided estimates for the association between antibody responses measured during pregnancy and risk of placental infection, but no clear pattern of association was found ( fig. b and additional file : figures s b and s b ). of the three cohort studies included in narrative terms, two provided evidence for an association between ab responses to some, but not all var csa domains, and prbc strains, measured earlier in pregnancy, and reduced risk of placental infection [ , ] , and one indicated no difference in ab levels to var csa domains measured in first or second trimester (t /t ) among women who were negative or positive for placental infection (table ) [ ] . overall, the majority of estimates included in this review, and studies included in narrative terms, indicate that when measured at delivery, antibody responses to pregnancy-specific prbc and var csa antigens are associated with the presence of placental infection and may therefore represent markers of infection, rather than correlates of protection. of the five studies that measured antibodies earlier in pregnancy, and followed women until delivery, two provided evidence for a protective effect of anti-var csa antibodies [ ] [ ] [ ] and three found no significant association with placental infection [ , , ] . peripheral p. falciparum infection seven studies provided cross-sectional estimates for the association between antibody responses to pregnancyspecific antigens, measured at various time points, and peripheral p. falciparum infection, yielding heterogeneous results ( fig. a and additional file : figures s a and s a ) [ , , , , , , ] . for many of the antigens examined, antibody responders had increased odds of peripheral infection compared to nonresponders, but these associations were rarely significant. early in pregnancy (t /t ), antibody responders to cs -adherent fcr had increased odds of peripheral infection in pooled estimates from cross-sectional studies (reor . , % ci . - . , i = . %) (fig. a) [ , ] . similarly, antibody responders to a placental isolate at delivery had twofold increased odds of peripheral infection (or . , % ci . - . ) [ ] . three studies included in narrative terms examined peripheral p. falciparum infection as an outcome [ , , ] . in an iptp trial in mozambique, women who were parasitaemic at delivery had higher abs to cs than non-parasitaemic women [ ] , but antibody levels to a suite of placental isolates and var csa domains were not associated with peripheral infection after adjusting for placental malaria [ ] . in women from benin, high fv and dbl abs early in pregnancy (t / t ) were associated with reduced risk of peripheral infection during pregnancy, but this association was not observed for the four other var csa domains analysed nor to cspg-binding inhibitory abs to fcr- [ ] . in addition to studies that provided cross-sectional estimates, four prospective studies and one nested case-control study provided estimates for the association between antibody responses earlier in pregnancy, and risk of peripheral parasitaemia later in pregnancy or at delivery (fig. b and additional file : figures s b and s b) [ , , , , ] . one study found that thai women positive for antibodies to dbl in the first trimester had increased odds of having a peripheral infection during pregnancy (or . , % ci . - . ) [ ] , but the remaining studies showed no clear association between positive ab responses to pregnancy-specific antigens and prospective risk of peripheral infection [ , , , ] . in summary, whilst some studies found an association between ab responses to pregnancy-specific prbc and var csa antigens and presence of parasitaemia, either concurrently or at a later time point in pregnancy, we found no evidence for a protective association between these antibodies and peripheral parasitaemia. thus, antibodies to var csa either early in pregnancy or at delivery do not appear to reduce the incidence or level of peripheral parasitaemia throughout pregnancy. the association of antibodies to pregnancy-specific prbc, measured at delivery, and low birthweight was examined in three studies ( fig. a and additional file : figures s a and s a ): two studies measured total igg antibodies [ , ] and one study measured csa adhesion inhibitory antibodies [ ] . pooled analyses indicated that compared to non-responders, igg responders to csa-binding prbc, measured at delivery, had a clinically significant % reduction in the odds of low birthweight delivery (reor = . , % ci . - . , i = . %), but the confidence interval was wide and captured a scenario of a small increase in odds of low birthweight (fig. a) [ , ] . kenyan women with anti-csa adhesion activity had an % reduction in odds of low birthweight birth [ ] compared to women who did not have anti-csa adhesion activity. this association was also observed in sub-group analysis of secundigravidae/multigravidae (additional file : figure s a ) but not among primigravidae (additional file : figure s a ), most likely because an insufficient number of women had acquired anti-adhesion activity in their first pregnancy in this study population. in contrast, there was no significant pattern of association between antibody responses to var csa antigens measured at delivery and low birthweight (fig. a) [ , , ] . estimates stratified by gravidity gave similarly heterogeneous results for association between antibodies to var csa at delivery and low birthweight (additional file : figures s a and s a) . when antibodies were measured earlier in pregnancy, estimates from one study of malian women indicated (see figure on previous page.) fig. forest plot of the association between antibodies to pregnancy-associated p. falciparum antigens and placental malaria. a estimates represent the odds of placental malaria in ab responders compared with ab non-responders, where antibodies were measured at delivery (cross-sectional studies). b estimates represent the relative risk of placental malaria in ab responders compared to ab non-responders, where antibodies were measured at various times during pregnancy, as indicated (prospective studies). estimates are for all gravidities included in original studies. staalsoe et al. (yaounde site) included secundigravidae and multigravidae only; babakhanyan et al. [ ] included multigravidae only; and cox et al. [ ] included primigravidae only. estimate for mclean et al. [ ] represents igg responders only as total igg was not available. meta-analysis was only performed on estimates where var csa antigen or functional assay (where applicable) and timing of antibody determination were the same. meta-analysis of ab responses to fv and odds of pm showed a high degree of heterogeneity (i = . %, p < . ) so results were not pooled. a estimate calculated by current authors from data in original publication; b data supplied by original authors and estimate calculated by current authors. cc, case-control; cs, cross-sectional; csa adhes. inhib., csa adhesion inhibition assay; flow cyto., flow cytometry; n, number of participants included in estimate; or, odds ratio; plac. isolate, placental isolate; prbc, parasitized red blood cells; rct, randomized controlled trial; rr, risk ratio that ab responders to id -id a but not dbl , dbl , dbl - , dbl , and dbl had increased risk of low birthweight births (rr . , % ci . - . ) compared to non-responders (fig. b) . in a sub-group analysis, this association remained significant in primigravidae (additional file : figure s b ) but not multigravidae (additional file : figure s b ) [ ] . of the studies included in narrative terms, high antibody responses to some var csa antigens including dbl ε [ ] , dbl -dbl , and dbl [ ] and functional antibodies to prbc [ , ] , were associated with reduced risk of low birthweight (or ab levels were correlated with birthweight), but these associations were dependent on the timing of antibody determination [ ] and the gravidity of subjects [ ] . in a senegalese cohort, no significant association was found for antibodies to dbl -x, dbl ε, and dbl ε, measured at t /t and delivery, and low birthweight [ ] (table ). in summary, several studies provided evidence for an association between antibody responses to pregnancyspecific p. falciparum antigens and decreased risk of low birthweight [ , , , , , ] . in this systematic review and meta-analysis, we aimed to synthesize the existing epidemiological evidence for the association between antibodies to pregnancy-specific antigens and the risk of malaria in pregnancy and its associated adverse maternal and birth outcomes. overall, we found that estimates for the association between pregnancy-specific p. falciparum antibody responses and the pregnancy and birth outcomes examined were heterogeneous in terms of both the direction and magnitude of effect. whilst antibody responses to several antigens were positively associated with the presence of placental and peripheral infections, this review did not identify evidence that any specific antibody response is associated with protection from pam and its clinical consequences across multiple populations. cross-sectional estimates for the association between antibody responses and p. falciparum infections during pregnancy suggest that antibody responses may serve as markers of current infections. indeed, previous studies have reported a concurrent increase in pregnancyspecific antibodies in women with placental infection [ , , , ] . we found that positive antibody responses to pregnancy-specific antigens were associated with increased odds of peripheral infection in some studies, with varying degrees of significance [ , , , ] . at delivery, antibody responders (igg) to the prbc surface [ , , ] , to full-length var csa (fv ) [ , ] , and to the vaccine candidate id -id a, which includes interdomain region , dbl x, and interdomain region a, had increased odds of placental infection compared to non-responders [ , ] (fig. a) . in contrast, antibodies to single domains (dbl , dbl , dbl , dbl ), measured at delivery, were not associated with placental infection (fig. a) . this finding suggests that reactivity to var csa epitopes in their native formation may be more relevant correlates of disease than responses to individual recombinant domains. indeed, previous in vitro studies using the full-length extracellular var csa have suggested that the overall folding of the protein may be important for ability to bind csa [ , ] . furthermore, it has been suggested that functional antibodies to var csa may have affinity to conformational epitopes not displayed by individual recombinant constructs from single alleles [ ] . further studies designed to measure antibody responses to multiple var csa antigens concurrently, will be essential to confirm which antibody responses are the best markers of infection and could therefore be utilized as serosurveillance tools [ ] . an effective vaccine against placental malaria should induce broadly active and strain-transcending antibodies to block the adhesion of var csa-expressing parasites to csa [ , ] . a single cross-sectional study found that women positive for csa-binding inhibitory antibodies had significantly decreased odds of placental infection, as well as low birthweight and preterm birth [ ] . although two vaccines based on the n-terminal csa-binding region of var csa are currently in early clinical trials, this review highlights a scarcity of evidence for a protective association between antibodies to (see figure on previous page.) fig. forest plot of the association between antibodies to pregnancy-associated p. falciparum antigens and peripheral parasitaemia. a estimates represent the odds of peripheral p. falciparum parasitaemia in ab responders compared to ab non-responders, where antibodies were measured at the same time point as parasitaemia (cross-sectional studies). b estimates of peripheral p. falciparum parasitaemia in ab responders compared to ab non-responders, where antibodies were measured at time points prior to parasitaemia determination. estimates are risk ratios for rct and cohort studies and odds ratio for the nested case-control study. estimates are for women of all gravidities included in original studies: cox et al. [ ] included primigravidae only. the timing of antibody and parasitaemia determination is indicated. estimate for mclean et al. [ ] represents igg responders only as total igg was not available. dbl estimates were only combined when study design was the same. estimates for fcr responses at t /t were not combined because i = . %. a data supplied by original authors and estimate calculated by current authors; b estimate calculated by current authors from data in original publication. cc, case control; cs, cross-sectional; es, estimate; n, number of participants included in estimate; flow cyto., flow cytometry; ncc, nested case-control; or, odds ratio; pi time, timing of determination of parasitaemia; prbc, parasitized red blood cells; rct, randomized controlled trial; t , first trimester; t , second trimester; t , third trimester recombinant var csa domains, including the vaccine candidates, and placental malaria. only two prospective cohort studies, included in narrative terms, found that antibody responses to selected var csa antigens and prbc were associated with protection from placental infection [ ] [ ] [ ] and responses to individual recombinant var csa antigens were associated with protection against low birthweight and preterm birth in individual studies only [ , ] . none of these studies reported an association between antibodies to the vaccine candidates (id -dbl x-id a and dbl x-dbl x) and protection from placental malaria, low birthweight, or preterm birth. interestingly, malian primigravidae who were positive for antibodies to id -id a at enrolment (during t / t ) were more likely to deliver preterm babies, suggesting that this antigen may be a marker of parasite exposure [ ] . it is likely that protection from placental malaria and its associated adverse pregnancy outcomes develops with the acquisition of an increasingly broad antibody response to different var csa domains and allelic variants, rather than to a single domain [ , ] . furthermore, high avidity antibodies and functional antibody responses are probably more important measures than simple quantification of igg responses to recombinant proteins [ , ] . in the absence of a robust, cross-population estimate of the effect of pregnancy-specific p. falciparum antibody responses, we are unable to quantify the fraction of clinical disease or adverse birth outcomes that may be averted by these immune responses. this has implications for understanding any impact of potential vaccines based on pregnancy-specific antigens on the burden of pregnancy-associated malaria in a given population. only nine of the studies examined antibody responses to multiple recombinant antigens concurrently in the same populations [ , , , , , , [ ] [ ] [ ] , and only three of these contributed estimates in the format required for meta-analysis [ , , ] . this precluded assessment of the relative clinical importance of antibody responses to individual var csa domains. a key strength of this systematic review was that we contacted authors directly, and for studies, we were able to obtain estimates that were not originally published. a limitation was the heterogeneity observed between estimates, which is likely due to methodological and epidemiological differences between the included studies. we included studies that collectively examined a broad range of antibody responses, including to the surface of prbc isolates from infected placentas, to csabinding prbc strains, and to recombinant var csa antigens. with respect to the antigens examined, parasite isolates differed in their geographical origin and recombinant antigens varied by allele, domain boundary, and expression system (additional file ), potentially impacting variation in antibody reactivity between studies [ , ] . importantly, the influence of the global genetic diversity of the var csa gene on antibody reactivity, and consequently, vaccine development, has not been adequately determined [ ] . furthermore, inconsistencies in estimates across studies may reflect a dual role for antibodies to var csa, whereby antibodies contribute to protective immunity via blocking of prbc adhesion to csa, but also contribute to local pathology by activating inflammatory monocytes and macrophages in the placenta [ , ] . in addition, differences in the methodology employed to measure antibody responses may have contributed to heterogeneity in estimates. furthermore, due to the limited availability of stratified data, we only presented estimates for women sub-grouped into primigravidae and secundigravidae/multigravidae groups. in some earlier studies, antibodies to pregnancyspecific p. falciparum antigens were associated with improved pregnancy outcomes in secundigravidae only [ ] , or in women with chronic pregnancy-associated malaria infections [ ] . therefore, further studies examining antibody responses in specific parity and clinical groups may be warranted. due to the breadth of study designs and antigens examined, in most cases estimates for the association between a specific antibody response and clinical outcome across different populations could not be pooled. importantly, estimates for the association between antibody responses and pam outcomes derived from a single population may not be generalizable to other malaria-endemic areas. we identified a lack of cohort studies examining associations between antibody responses and prospective risk of malaria in pregnancy. the majority of the included estimates (see figure on previous page.) fig. forest plot of the association between antibodies to pregnancy-associated p. falciparum antigens and low birthweight. a estimates represent the odds of low birthweight birth in ab responders compared with ab non-responders, where antibodies were measured at delivery (cross-sectional studies). b estimates represent the risk of low birthweight in ab responders compared with ab non-responders, where antibodies were measured at time points prior to delivery, as indicated (prospective studies). estimates are for women of all gravidities included in the original studies. teo et al. included secundigravidae and multigravidae only. a data supplied by original authors and estimate calculated by current authors; b estimate provided by original author; c estimate calculated by current authors from data in original publication. cc, case-control; cs, cross-sectional; n, number of participants included in estimate; or, odds ratio; prbc, parasitized red blood cells; rct, randomized controlled trial; rr, risk ratio; t , first trimester; t , second trimester; t , third trimester were derived from cross-sectional analyses, usually at delivery, thus limiting the ability to establish a causal relationship between antibody responses and pam outcomes. moreover, measurements of immunity inferred by antibody levels at a single time point can be misleading as antibody production is highly dynamic [ , , , ] . importantly, many of the studies were not originally designed to detect differences in the risk of placental infection and associated outcomes between antibody responders and nonresponders and were therefore not statistically powered to detect such associations. study populations varied with respect to the proportion of primigravidae versus multigravidae, the percentage of women receiving intermittent preventive treatment in pregnancy (iptp), the prevalence of hiv and other infectious diseases, the transmission intensity of the study site, and the malaria exposure history of the women. both parity and transmission intensity can influence the kinetics of anti-var csa antibody responses during pregnancy [ , , , ] . moreover, the transmission intensity may affect the relationship between antibody responses and pam [ , ] as women living in areas of stable/intense malaria transmission may develop a broader repertoire of functional antibody responses to pregnancy-associated p. falciparum earlier in pregnancy, than women facing low/seasonal transmission [ ] . because antibody responses to the same antigen were rarely examined across multiple populations using comparable study designs, it was difficult to assess the effect of transmission intensity. understanding the impact of transmission intensity on acquired immunity has implications for both serosurveillance and vaccine development. overall, this systematic review found that pregnancyspecific p. falciparum antibody responses likely serve as markers of exposure to malaria in pregnancy, rather than correlates of protection. in order to objectively identify and prioritize antigens for vaccine development, it is important to demonstrate that a candidate antigen is a specific target of immune responses associated with protection from symptomatic disease in naturally exposed populations [ , ] . rational vaccine design and the development of immuno-serosurveillance tools would benefit from additional prospective cohort studies examining antibody responses early in pregnancy and at multiple time points throughout to a broad range of pregnancy-specific p. falciparum antigens across different 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targets of opsonizing iggs against pregnancy-associated malaria massive chronic intervillositis of the placenta associated with malaria infection malaria during pregnancy. cold spring harb perspect med the relationship between anti-merozoite antibodies and incidence of plasmodium falciparum malaria: a systematic review and meta-analysis identification and prioritization of merozoite antigens as targets of protective human immunity to plasmodium falciparum malaria for vaccine and biomarker development publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we wish to thank elizabeth aitken, ricardo ataide, anna babakhanyan, james beeson supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare that they have no competing interests. key: cord- -ebl yc authors: hoppe, sebastian; bier, frank f.; von nickisch-rosenegk, markus title: identification of antigenic proteins of the nosocomial pathogen klebsiella pneumoniae date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ebl yc the continuous expansion of nosocomial infections around the globe has become a precarious situation. key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. thus, new ways to rapidly detect these infections are vital. consequently, researchers around the globe pursue innovative approaches for point-of-care devices. in many cases the specific interaction of an antigen and a corresponding antibody is pivotal. however, the knowledge about suitable antigens is lacking. the aim of this study was to identify novel antigens as specific diagnostic markers. additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. hence, a cdna-based expression library was constructed and screened via microarrays to detect novel antigens of klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (esbl). after screening clones, previously unknown immunogenic proteins were identified. subsequently, each protein was expressed in full-length and its immunodominant character examined by elisa and microarray analyses. consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. after specificity analysis, homology survey and d structural modelling, one epitope sequence gavvalsttfa of kpn_ , an ion channel protein, was identified harboring specificity for k. pneumoniae. the remaining epitopes showed ambiguous results regarding the specificity for k. pneumoniae. the approach adopted herein has been successfully utilized to discover novel antigens of campylobacter jejuni and salmonella enterica antigens before. now, we have transferred this knowledge to the key nosocomial agent, k. pneumoniae. by identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen. klebsiella pneumoniae is a gram-negative, facultative anaerobic rod-shaped bacterium belonging to the family of enterobacteriaceae. it is a non-motile, lactose fermenting organism, which has been known to cause severe lung damage if aspirated. other clinical symptoms common with klebsiella pneumoniae infections encompass urinary-tract-infections (uti) and wound infection potentially causing bacteremia and septicemia [ ] . in recent years it has become one of the most persistent nosocomial agents, especially due to the increasing distribution of multiple resistances to antibiotics. the most prominent group of k. pneumoniae harboring a broad resistance spectrum incorporates the extendedspectrum beta-lactamase (esbl) expressing strains. due to their outstanding clinical relevance and occurrence as agents of nosocomial infections, it is highly desirable to rapidly detect the presence of these organisms and to find suitable measures to effectively counter any infection in the early stages [ ] . while numerous dna-based typing methods exist [ ] , these are often laborious and time-consuming. in contrast, user-friendly point-of-care devices applying antigen-antibody interactions would allow for a quick and reliable detection [ ] . nevertheless, the knowledge about suitable antigens to be incorporated into such a device is scarce. thus, we have utilized a method to quickly assess novel immunogenic proteins of k. pneumoniae, which might serve as potential targets for a diagnostic tool. recently, we have successfully employed this approach to unveil immunogenic proteins for both campylobacter jejuni [ ] and salmonella enterica [ ] . concisely, prokaryotic cdna libraries are created, fusion proteins expressed and these constructs covalently attached to microarray surfaces via the use of a halotag (promega). subsequently, the microarrays are screened using polyclonal antibodies reactive to the donor species of the cdna. therefore, this approach enables a broad and reliable screening, while reducing cross-reactivity and background to a minimum [ ] due to the highly selective and covalent binding of halotag to its specific ligand [ ] . moreover, the high specificity of said interaction renders excessive protein purification steps normally encountered in microarray-based screening applications [ ] obsolete. thus, it is a faster and more direct approach as spotting combines both the deposition of samples and enables the immediate purification by a simple washing step. in connection with the above screening approach, cdna derived expression libraries were generated to express a vast number of proteins from k. pneumoniae. these libraries offer the advantage of a smaller sample size as compared to genomic libraries. this is mainly due to genomic libraries encompassing highly truncated dna fragments as well as dna representing regions that do not encode proteins in the original organism. contrastingly, cdna libraries generated via the in-fusion smarter directional cdna library construction kit (clontech) have been known to lead to longer fragments and possess a high abundance of full-length clones [ ] . thus, the overall number of clones required for screening is substantially reduced. still, prokaryotic cdna libraries display one main disadvantage. as bacterial mrna rarely contains a poly(a)-tail [ , ] , isolation of the mrna from other rna species is tedious. while some methods exist to isolate the mrna prior to reverse transcription [ , ] , we rather chose to normalize the cdna afterwards. consequently, the entire rna of k. pneumoniae was used for reverse transcription. next, normalization was performed using a duplex-specific nuclease [ ] . this treatment has been shown to effectively reduce the highly abundant rrna derived cdna portions without implementing a bias, thus altering the overall composition of the cdna in favour of the mrna derived molecules [ ] . in addition to this, ligation-independent cloning and electroporation were employed to enhance cloning efficiency [ ] . in this work, we have screened clones to detect the presence of previously unknown immunogenic proteins. in summary, we identified proteins that have not been described as immunogenic before. after further analyses and epitope mapping of several promising candidates, three proteins -a channel receptor, a putative transport protein and a hypothetical protein -revealed linear antigenic sites with varying specificity. our results offer the potential to be used for a wide array of applications including the generation of monoclonal antibodies that might be used in diagnostic examination. furthermore, several of the identified antigens or parts thereof might be suitable for vaccine development, either used in passive or active immunization. additionally, many virulence-associated factors harbor some immunogenic potential. thus, identification of novel immunogenic proteins might elucidate proteins involved in the pathogenicity and virulence of k. pneumoniae. consequently, this advances the understanding of this pathogen and illuminates new approaches to counter infections. the mean rin value of rna used for cdna generation and library construction was calculated to . . (n = ). after successful normalization, the cdna was cloned to create an expression library. of this library, different clones were screened using halolink slides. the known immunogenic proteins [ ] , outer membrane protein a ompf (uniprot/swiss-prot: a t ) and outer membrane protein a ompa (a t ), were used as positive references, whereas both dihyroorotase pyrc (a t d ) and glyceraldehye- -phosphate-dehydrogenase gapa (a t l ) served as negative references. after screening, the signal intensities of each sample were compared to the references and grouped accordingly. generally, three distinct groups were established. group i represents samples exceeding the intensities of both positive reference proteins, group ii encompasses samples ranging in between the different intensities of ompa and ompf, whereas group iii entails those samples that albeit showing higher intensities than the negative controls, are below both ompa and ompf. here, approximately % of samples belong to group i, while ii and iii contain % and % respectively. the remaining samples, %, fall into the same range as the negative protein references. consequently, clones or . % of the entire screening approach were selected for sequencing. these clones were all taken from group i. after sequencing, artificial fragments and known antigens were discarded to reveal potentially novel immunogenic proteins, see table s for a list of the initial identification via sequencing. some of the inserts were too heavily truncated to reliably identify the corresponding gene. moreover, in some cases subcloning the initially identified genes in full-length failed even after numerous attempts. therefore, those genes were removed from further characterization as the translated peptide fragments were too short to be significant. despite these limitations, potentially novel immunogenic proteins were expressed in full length and used for further characterization. the antigen candidates are summarized in table including their locus tag, protein name, length and size in kda. the difference in immunodominant behaviour was assessed by ten independent microarray and elisa analyses employing two different antibodies. in summary, table reveals the resulting mean q values and corresponding errors (n = ). a q value above one represents higher intensity signals than ompa, the used positive reference. the highest mean q value was obtained for kpn_ , a coa-linked acetaldehyde dehydrogenase and irondependent alcohol dehydrogenase; pyruvate-formate-lyase deactivase with . . . however, as this protein is highly conserved within all bacteria, it was not considered for further analyses regarding specific antigenic sites. the same holds true for kpn_ , the s ribosomal protein l methyltransferase. although a q value of . . was attained, the highly conserved nature of this protein renders it unsuitable within a diagnostic approach. contrastingly, kpn_ and kpn_ , two hypothetical proteins, were selected for future investigations as little is known about the function of these proteins. their mean q values were . . and . . respectively. moreover, kpn_ shows some homology to a known superantigen of yersinia pseudotuberculosis according to the ncbi protein cluster database [ ] . thus, the potential utilization of these proteins within a diagnostic tool seems plausible. in addition, kpn_ , a histidine triad protein, kpn_ , glucose- -phosphate uridylyltransferase, kpn_ , a nucleoside channel and receptor of phage t and colicin k and kpn_ , a putative transport protein, were selected for further investigations via epitope mapping. while kpn_ and kpn_ revealed mean q values above one with . . and . . , respectively, kpn_ and kpn_ failed to reach this level. rather, the q values attained were . . and . . . while the q values of the latter two proteins are lower compared to some of the other proteins identified, their functional descriptions and membraneassociation render them highly attractive within a diagnostic question. hence, they might be more easily accessible in a whole cell detection approach than cytoplasmic proteins. epitope mapping revealed the potential presence of linear epitopes within three of the six proteins investigated, namely kpn_ , kpn_ and kpn_ . for kpn_ seven distinct regions were identified with intensities above a.u., see figure . these comprised the peptides and , - , - , - , - , - and - . the highest mean value for these peptides was obtained for peptide with more than a.u. the positive reference, i.e. rabbit igg, reached a mean value of more than a.u., whereas the negative control mbp showed intensities of less than a.u. as the adjacent peptides are identical in all but amino acids in sequence, a consensus can easily be derived from two or more neighboring peptides. as figure reveals, only the first two peptides, llaagavvalsttfa and gavvalsttfaagaa showed some specificity during specificity control assays. here, the arrays were incubated with additional antibodies reactive to different bacterial species, namely campylobacter jejuni, staphylococcus aureus, e. coli and s. enterica. all remaining peptides display similar intensities when these antibodies were used as compared to the original k. pneumoniae antibodies. however, for the first two peptides a significant difference is observed. the mean value for peptide was approximately a.u. with k. pneumoniae antibodies and dropped to less than a.u. with the other antibodies. a similar trend is discernible for peptide , where a drop from a.u. to less than zero is visible. thus, the consensus sequence gavvalsttfa is likely a suitable linear epitope featuring specificity for k. pneumoniae. for the putative transport protein, kpn_ , three regions scored intensities above a.u. including peptides and , and as well as - , see figure . the positive reference again reached a mean value above a.u., while the negative control levelled out at less than a.u. after specificity assays, the peptides and revealed predominantly specific binding by the k. pneumoniae antibody as the mean values dropped from approximately and a.u. respectively, to less than zero for all other antibodies tested, see figure . thus, a consensus for this epitope can be derived to giafgavelfd. contrastingly, the remaining peptides revealed equal intensities independent of the antibody used. finally, for the third protein, kpn_ , one pair of adjacent peptides, namely and , achieved mean values of approximately a.u. within close proximity to the positive reference, as depicted in figure . additionally, peptide displayed the highest overall mean value with more than a.u., however neither of the two neighboring peptides ( and ) attained values of any significance. thus, the presence of a linear epitope in that region is rather unlikely. besides, as specificity assays illustrated, none of the three peptides showed specific interaction to the k. pneumoniae antibodies alone, see figure . rather, signal intensities with antibodies reactive to c. jejuni fell into the same scope. therefore, nonspecific binding to these peptides is probable. the remaining three proteins under investigation failed to disclose any linear peptide region with significant signal intensities to assume linear epitopes to exist. for a better understanding of the suitability of the identified linear epitopes, structural modelling was employed using the swiss model automated mode. for kpn_ a model was constructed based on the crystal structure of the bacterial nucleoside transporter tsx of e. coli. however, the derived model only spans residues to and as such does not contain the derived consensus sequence of the linear epitope gav-valsttfa. nevertheless, the model displays a beta barrel structure typical of outer membrane-spanning transport proteins, see figure for details. the first residues of the derived model, starting at position , are marked in orange and present a coiled region outside of the beta barrel. therefore, the likelihood of the gavvalsttfa region to reside within the barrel is slim. rather, an extension of the truncated coil seems plausible. consequently, the potential accessibility of the identified linear epitope appears high. furthermore, the predicted d structure of the first part of kpn_ is displayed in figure . contrary to kpn_ , two models were devised for kpn_ . however, only one encompasses the identified linear epitope giafgavelfd, which is highlighted in orange and is situated as part of an alpha helix and an adjacent loop. as the sequence is not enclosed by other residues or structures, good accessibility ought to be provided. in order to predict the potential specificity of the epitopes, homology analyses were performed. whereas giafgavelfd of kpn_ exhibits a broad homology throughout with all residues identical in closely related species, see figure s : homology of kpn_ , gavvalsttfa of kpn_ features four residues likely specific for k. pneumoniae within this particular sequence. the variance of the latter epitope's sequence considering closely related species to k. pneumoniae is summarized in figure . specifically, the following residues have been replaced: valine by leucine (position four), both threonine residues by serine residues (position and ) as well as alanine by threonine (position ). the influence of sequence variations on the binding capacity of the identified epitope gavvalsttfa was subsequently examined by performing an alanine scan. its results are summarized in figure . the original consensus epitope sequence shows a mean intensity of approximately a.u. alterations of the first, second, fifth, eighth and ninth residue lead to a significant drop of signal intensities. consequently, signals of less than a.u., in close proximity to the negative control mbp with a.u. are obtained. contrastingly, by altering residues three or six of the consensus sequence gavvalsttfa, i.e. replacing the first valine or leucine by alanine, the resulting mean signal intensities are significantly increased to more than a.u. for replacing valine and surpassing a.u. after changing leucine to alanine. additionally, specificity assays showed no significant signal intensity for antibodies reactive to closely related species, i.e. e. coli and s. enterica. incubation with antibodies reactive to either of those two bacterial species resulted in signal intensities in the neighbourhood of the negative control independent of sequence alterations, see figure . this was also true for gavlalsssft and saalaltssft. the screening of a cdna based expression library of k. pneumoniae has successfully identified a number of novel, previously unknown immunogenic proteins. this is well in accordance to previous achievements of this method, which we have employed for the identification of both c. jejuni [ ] and s. enterica [ ] antigens. consequently, in this current study we aimed to detect suitable proteins for the specific identification of k. pneumoniae. furthermore, identification of specific antigens might improve treatment opportunities including the development of suitable vaccines [ ] . finally, detecting proteins with yet unknown functional and structural information to be antigenic might be an indication of their potential involvement in the pathogenic nature of an organism. thus, these proteins might be suitable access points for future investigations to improve the understanding of the underlying pathogenicity and virulence of k. pneumoniae. within the proteins only a subset was selected for further analysis using epitope mapping. the rationale for these selections was based on three distinct features: the resulting q value, homology as well as functional and structural properties. while the q value mirrors a normalized intensity compared to a positive reference, it does not fully account for differences in expression levels, misfolding and other factors which might have had an influence on the binding reaction and thus on the overall intensity. still, it facilitates to rank the proteins and increases the likelihood of a given protein to be immunogenic if its q value is significantly high. however, proteins with outstanding q values were not chosen for epitope mapping by default. rather, the known homology and corresponding distribution of each protein was carefully evaluated. thus, proteins like kpn_ and kpn_ albeit scoring high q values were exempt from epitope mapping as they display a broad spectrum of homologous proteins across bacterial species. finally, the intrinsic properties of each protein, if available, were closely scrutinized. therefore, proteins with hypothetical character were predominantly chosen, as information on them is confined. in addition, all types of membrane-associated proteins deserved a better look, as these type of proteins offer a more direct route and accessibility, which might be of utmost importance in a future rapid point-ofcare device detecting whole organisms. after epitope mapping, three of the six proteins under investigation revealed sites with potential linear epitopes. still, the remaining proteins displayed no such sequences. this is, however, in accordance to the general prevalence of structural epitopes in comparison with linear epitopes in nature. in fact, approximately % of all epitopes are conformational rather than sequential [ , ] . despite these potential shortcomings of the used linear epitope mapping method, a number of intriguing linear epitopes have been identified. notably, three proteins, kpn_ , kpn_ and kpn_ , harbored sites with potential linear epitopes. further careful examination, however, excluded kpn_ from future applications as the identified epitope sequence were nonspecific for k. pneumoniae, which might be due to the conserved character of the protein within the family enterobacteriaceae. still, kpn_ is a membrane protein and upcoming investigations might help to elucidate applications using this protein either for prevention of k. pneumoniae infections or detection thereof. in contrast, the other two proteins displaying linear epitopes, kpn_ and kpn_ , indicated some specificity with the antibodies tested and two linear consensus sequences could be derived, gavvalsttfa and giafgavelfd, respectively. while the former sequence is present in kpn_ , an ion channel protein tsx, which is conserved among enterobacteriaceae, the latter sequence is part of kpn_ , a hypothetical protein with high similarity to a cation:proton antiport protein and conserved among proteobacteria. consequently, the homology of giafgavelfd (kpn_ ) is high throughout different bacterial species, which renders the sequence inapt for specific k. pneumoniae detection and other applications. in contrast, gavvalsttfa (kpn_ ) displays alterations within this sequence for bacterial species other than k. pneumoniae. as small as these alterations appear, they appear to have a crucial effect on the binding of antibodies to the target sequence and thus benefit specificity. this is well in line with the experimental results that indicate no binding by antibodies reactive to other bacteria to this sequence or any of its alterations. moreover, alanine scanning revealed a number of residues to be paramount for antibody binding. consequently, replacing the glycine (position ), alanine (position ), alanine (position ) or either threonine (positions and ) leads to a significant loss of antibody binding observable by a dramatic reduction in signal intensity. on the contrary, removing either the first valine or leucine and inserting an alanine residue as a replacement results in a significant increase in signal intensity, potentially hinting at an improved antibody binding. the reason for these effects remains nebulous; however, it is not caused by a simple change in secondary structure. this is apparent as both aavvalsttfa and gavvaasttfa, two sequences at opposite sides regarding signal intensity, are predicted to consist solely of an alpha-helix as compared to the original sequence, which is predicted to contain a beta-strand (position to ) and a helical part (position to ) by emboss garnier. furthermore, as the original sequence is fully constructed of uncharged amino acids, alterations via alanine or glycine do not change the overall charge of the peptide. consequently, the differences in signal intensity cannot be deduced to implementation or removal of charged residues. finally, changes in hydrophobicity albeit present are minimal at best and may not suffice to explain the observed characteristics. still, one has to bear in mind that the accuracy of prediction based tools is often lacking, especially for emboss garnier with an accuracy in the range of %. consequently, some of the predicted secondary structures might differ. despite some uncertainty as to the mechanistic cause of the altered peptides to behave as they did, one fact remains obvious. none of the peptides showed any significant binding to antibodies not raised against k. pneumoniae. therefore, after alanine scanning, gavvalsttfa remains a suitable candidate for specific k. pneumoniae detection or treatment. still, accessibility of the epitope is paramount to a quick diagnostic tool. thus, modelling of the d structure of the protein was performed. unfortunately, the d modelling only succeeded for the part of the protein, which does not contain said linear epitope sequence. nevertheless, the pronounced beta barrel structure of the channel protein is visible and the linear epitope sequence, albeit absent, likely to be an extension of the freely accessible n-terminal region outside the barrel structure. thus, accessibility of the epitope by an antibody might be pronounced without the need to enter the cell or channel. membrane proteins harboring a beta barrel like structure have been shown in bacteria to be exclusively found in the outer membrane [ ] . moreover, the mobile coils on the extracellular sides of these membrane proteins are pivotal for their function or interaction with other molecules. this renders the identified sequence an intriguing target for antibody-based detection. additionally, channel proteins harboring beta barrel like structures have been shown to be immunodominant in other bacterial species such as salmonella, haemophilus influenzae, e. coli, neisseria meningitides, shigella dysenteriae and chlamydia trachomatis [ ] [ ] [ ] [ ] [ ] [ ] . on the contrary, the model for kpn_ encompasses the identified consensus sequence of the linear epitope giafga-velfd, which is part of a loop between two alpha helices. the abundance of alpha helices suggests the protein to span the inner membrane [ ] . in this topological design, helices are mostly located within the membrane, notably as transmembrane domains, whereas loops are located either on the cytoplasmic or periplasmic side of the cell. when considering prediction based methods, such as s_tmhmm for topological domains [ , ] or emboss antigenic [ , ] , part of giafgavelfd is assumed to be extracellular. combined with the high flexibility and degrees of freedom of random coil structures, the likelihood for good accessibility is high rendering the sequence a potentially attractive target for whole cell detection despite its lack of specificity. furthermore, these findings support the d model and underline the accuracy of the specified structure. another key aspect in determining the accuracy of the d model prediction is a so-called z-score [ ] . the z-score for the model of kpn_ is . and . for kpn_ respectively. although the values are significantly below zero that does not inevitably indicate models of poor accuracy. in fact, low z-scores are often obtained if the protein under investigation is membraneassociated. this is mainly due to the inverse physicochemical properties of membrane proteins in comparison to soluble ones. hence, the low z-scores are more likely induced by this effect than caused by an insufficient accuracy of the models. in conclusion, we have successfully identified several novel antigens of k. pneumoniae and identified three proteins potentially harboring linear epitopes. subsequently, we achieved to identify two sequences displaying specificity during experimental investigations; however, one of these is doubtful as homology analysis has revealed it to be highly conserved among a broad spectrum of bacterial species. still, gavvalsttfa of kpn_ was identified to be specific both experimentally and has shown four residues within the eleven amino acid sequence to occur predominantly in k. pneumoniae only. thus, the likelihood for this linear epitope to be specific is high. this assumption was confirmed by alanine scanning revealing a number of pivotal residues for antibody binding. moreover, it was unearthed that neither e. coli nor s. enterica antibodies were able to bind to any of the sequences, original and modified. subsequent investigations might help to further nurture the insight into the suitability of this peptide for diagnostic and therapeutic applications. thus, monoclonal antibodies ought to be devised to be used for affinity investigations via biacore and to determine kinetics. furthermore, monoclonal antibodies could be used within a potential diagnostic tool and after validation ought to be tested with whole bacteria. if these antibodies are able to specifically detect intact k. pneumoniae cells, the resulting antibody might well be suited for integration into a point-of-care device. in a different approach, the identified epitope sequence could easily be produced in large quantity. this peptide might serve some role in serological screenings, especially if it proves to be immunodominant. consequently, antibodies against this epitope might be present in a plethora of patient sera. finally, all proteins identified here might be suitable candidates for vaccine development independent of the existence of a linear epitope, as structural epitopes might well be present and antigenicity ensured. nevertheless, additional in-depth analysis is required to determine a number of the key aspects of vaccine development prior to use. as a donor of rna the fully-sequenced strain k. pneumoniae mgh was grown on solid trypticase-soy-agar (tsa) for h at uc under aerobic conditions. for rna isolation a liquid culture was prepared by inoculating ml of brain-heart-infusion broth (bhi) with a single colony and incubated overnight at uc, rpm. this overnight culture was used to inoculate a flask containing ml fresh bhi medium. the cells were harvested h after inoculation. for initial screening a rabbit polyclonal igg antibody to k. pneumoniae (acris ap pu-n) was used. for further micro-array analyses of a subset of candidate proteins, elisa and epitope mapping this antibody was used as well as rabbit polyclonal igg antibody to k. pneumoniae (abcam ab ). the antibodies were generated with k. pneumoniae atcc serving as an immunogen. specificity assays were performed employing rabbit polyclonal igg antibody to c. jejuni (acris ap pu-n), s. aureus (fitzgerald c-cr rp), e. coli (abcam ab ) and s. enterica (abcam ab ). detection was achieved by usage of secondary antibodies. goat polyclonal antibody to rabbit igg conjugated with chromeo- (abcam ab ) for fluorescent and antibody conjugated with horseradish peroxidase (abcam ab ) for a colorimetric readout were applied where appropriate. the cells were harvested by centrifugation ( g, min) and the resulting supernatant discarded. the pellets were resuspended in fresh bhi medium. for stabilisation of the rna, ml of rnaprotect bacteria reagent (qiagen) was added to . ml of bacterial suspension and processed according to the manufacturer's instructions. lysis was performed with ml of lysis buffer ( mm tris-cl, mm edta, mg/ml lysozyme, . mau proteinase k) by pipetting and vortexing for s. after incubation, ml buffer rlt and ml % ethanol were added and the lysate applied to rneasy bacteria mini kit spin columns (qiagen) for rna isolation following the manufacturer's instructions. during this procedure an on-column dnase digest was performed using rnase-free dnase i solution (qiagen) according to the manufacturer's instructions. the isolated total rna was eluted in ml of rnase-free water and its concentration and purity analyzed by nanodrop (peqlab) measurements. the quality of isolated rna was assessed using the rna pico kit and bioanalyzer (agilent). the total rna was diluted to a working concentration of - pg/ml. the analysis was performed following manufacturer's instructions and the rna integrity number (rin) calculated by the expert software (agilent). the rin is defined to fall into a range of to , with a higher score indicating a more intact rna, whereas lower numbers are associated with degraded rna molecules [ ] . in order to use bacterial mrna as a substrate in cdna synthesis, polyadenylation was mandatory. the tailing was achieved using the poly(a) polymerase tailing kit (epicentre) following the alternate protocol offered by the manufacturer. briefly, up to mg of total rna were combined with ml poly(a) polymerase reaction buffer, ml mm atp, . ml riboguard rnase inhibitor and ml poly(a) polymerase ( u) in a total reaction volume of ml. the reaction was incubated for min at uc, terminated by the addition of ml . m edta and purified by rneasy mini kit (qiagen) following manufacturer's instructions. yield and purity were determined by nanodrop measurements. for cdna synthesis the in-fusion smarter directional cdna library construction kit (clontech) was used according to manufacturer's instructions with slight modifications. . ml total, polyadenylated rna were mixed with ml of in-fusion smarter cds primer, heated first for min at uc and then incubated for additional min at uc. after addition of . ml mastermix ( ml x first strand buffer, . ml mm dtt, ml mm dntps, ml mm smarter v oligonucleotide, . ml rnase inhibitor and ml smartscribe reverse transcriptase) the tubes were incubated for min at uc. the reaction was terminated at uc for min. for second strand cdna synthesis two ml aliquots of first strand reaction were used in long distance pcr using phusion polymerase (finnzymes). each pcr reaction was comprised as follows: ml first-strand reaction, ml rnase-free water, ml x phusion hf buffer and ml each of dntp mix ( mm), figure . specificity binding analysis of epitope peptides of kpn_ . bar chart representing the mean relative fluorescence intensities (n = ) of each peptide potentially harboring a linear epitope site after incubation with polyclonal antibodies reactive to k. pneumoniae (green) and c. jejuni (orange). none of the peptides shows a peculiar specific interaction; rather signal intensities are in the same vicinity for each peptide independent of the antibody used. this indicates mainly non-specific binding to occur. doi: . /journal.pone. .g pcr primer ii a ( mm), in-fusion smarter pcr primer ( mm) and phusion polymerase with a total reaction volume of ml. the pcr reactions were subjected to the cycling program with uc for min as initial denaturation followed by cycles of s denaturation at uc, s of primer annealing at uc and min extension at uc. for improved pcr results optimization was performed as follows; ml of the cycle experimental tube were transferred to a separate pcr tube, cycling commenced and aliquots of ml each were collected after , , , and cycles total. the different cycles were compared by gel electrophoresis and the experimental tubes subjected to additional cycles if necessary. finally, pcr reactions were purified using the qiaquick pcr purification kit (qiagen). the purity and yield of each reaction were analyzed by nanodrop measurements. normalization of double-stranded cdna was achieved with the trimmer- cdna normalization kit (evrogen) to reduce the number of cdna molecules derived from rrnas. briefly, ml of cdna (approx. ng/ml) were mixed with ml of x hybridization buffer. for the trimming reaction ml of this mixture were distributed to four different pcr tubes and overlaid with a drop of pcr-grade mineral oil. after centrifugation ( g, min), the tubes were incubated for min at uc followed by h at uc. next, pre-heated ( uc) duplex-specific nuclease (dsn) master buffer was added to each tube and incubation prolonged for min. dsn was added to the first three tubes in decreasing concentrations - u/ml, . u/ml and . u/ml -with the fourth tube receiving dsn storage buffer and no enzyme as a control reaction. the incubation prolonged for min at uc. after addition of ml dsn stop solution and subsequent incubation for min at uc, the tubes were placed on ice. the chilled reaction was diluted by addition of ml sterile, rnase-free water. for amplification of normalized cdna, ml of each reaction was used as template in pcr. each pcr reaction contained ml of template from the normalization reaction, ml nuclease-free water, ml x phusion hf buffer, ml mm dntp mix (neb), ml of each primer pcr primer ii a ( mm), in-fusion smarter pcr primer ( mm) and ml phusion polymerase. the pcr was performed with initial denaturation at uc for min and seven cycles of denaturation at uc for s, primer annealing at uc for s and extension at uc for min, respectively. for optimization, the control tube was subjected to , , , and cycles with ml aliquots taken every two cycles. the optimization samples were analyzed by gel electrophoresis ( % agarose, tae, v) and the optimal cycle number determined. the remaining three tubes were subjected to + x cycles with x being the differential of the optimized cycles to the originally performed seven cycles. after the second pcr, the experimental reactions were compared to the optimal control reaction using gel electrophoresis as above. reactions showing a successful normalization were combined and used in a third pcr reaction. after the final pcr, the reactions were purified by qiaquick pcr purification kit. a major feature of the given model is the prominent beta barrel structure that originates from the abundance of beta strands. this is a typical feature of transport and channel proteins spanning the outer bacterial membrane. contrastingly, the identified linear epitope gavvalsttfa is located at the very beginning of the protein and thus not included in the given model. however, it is likely an extension of the truncated n-terminal region marked in orange. doi: . /journal.pone. .g figure . d model of predicted structure of kpn_ . the model was predicted using the automated mode of the swiss model application by expasy (university basel). as a template the crystal structure of a na(+)/h(+) antiporter nhaa of e. coli was used. the resulting model spans residues to of the full-length protein and was subsequently dyed using the chimera . software. coils are depicted in light green, beta strands in purple and alpha helices in blue. the potential linear epitope giafgavelfd is highlighted in orange. it comprises part of an alpha helix, a connective coil and the start of the next alpha helix. doi: . /journal.pone. .g in-fusion cloning and cloning vector for cloning pfn a (promega) was used as a vector, as it features a n-terminal encoded halotag fusion protein, which allows for specific and covalent binding to a unique ligand, thus reducing background and minimizing cross-reactivity in immunoassays with halolink microarrays harboring the ligand on its surface. first, the vector needed to be linearized to be used with the in-fusion cloning technology. this was achieved by reverse pcr using ifs a for ( ttgataccactgcttttc-catggcgatcgcgttatc ) and ifs a rev ( tctcatcgtaccccgtgtttaaacgaattcgggctcg ). each reaction contained ml each of : diluted pfn a ( ng/ml) and the two primers, ml x phusion hf buffer, ml mm dntps, . ml phusion polymerase and . ml nuclease-free water to reach a total reaction volume of ml. the pcr was run using a cycle two-step program with uc denaturation for s and min extension at uc. after completion, ml of dpni ( u/ml) were added to the reaction and incubated at uc for h. the presence of a single band was checked by gel electrophoresis and the remaining reaction purified by qiaquick pcr purification kit. cloning of normalized cdna and linearized pfn a vector was performed following the manufacturer's instructions within the in-fusion smarter directional cdna library construction kit (clontech). electroporation ml of the cloning reaction were mixed with ml of electrocompetent acella e.coli cells (mobitec), a bl derivative, figure . homology of linear epitope sequence gavvalstffa of kpn_ . the sequence derived from k. pneumoniae mgh was used as a reference. identical residues are marked by dots, gaps by a horizontal dash and differences by the single letter amino acid code. seven of nine k. pneumoniae strains show identical epitope sequences, while two strains display changes in two residues. threonine replaces alanine at position , a change observed not only in these two strains but in almost all other bacteria within the list. additionally, threonine at position is substituted by either serine or phenylalanine. bacteria other than k. pneumoniae show an additional number of amino acid substitutions, most prominently leucine for valine at position and serine for threonine at position . in s.enterica the changes become more pronounced. glycine at position is replaced by serine, valine at position replaced by alanine and threonine inserted for serine at position . in some rare cases, other residues have also been substituted, e.g. valine replaces alanine at position in shigella dysenteriae. doi: . /journal.pone. .g figure . alanine scan of gavvalsttfa of kpn_ . box-whisker plot (n = ) of gavvalsttfa after alanine/glycine scanning. the box comprises % of the data, while the whiskers enclose %. the median is represented by a small horizontal line and the mean by a small rectangle. rabbit igg served as a positive reference, whereas mbp was used as a negative control. if alanine was present in the original present it was replaced by glycine, otherwise each amino acid was stepwise replaced by alanine. additionally, gavlalsssft and saalaltssft were included as they resemble sequences present in e. coli and s. enterica. switching glycine (position ), alanine (positions or ), or threonine (positions and ) to alanine or glycine, results in a significant drop in signal intensities to levels below or at the negative control. in contrast, substituting valine (position ) or leucine (position ) by alanine, leads to an increase in signal intensities to a.u. and more than a.u., respectively. note the different axis scales prior and after axis break at a.u. doi: . /journal.pone. .g and electroporated in mm cuvettes using the easyject plus electroporator (peqlab). conditions for electroporation were as follows: voltage = v, capacity = mf, resistance = v and a pulse duration of ms. the electroporated cells were added to ml of super optimal broth with catabolite expression (soc) and incubated at uc for h with shaking at rpm. afterwards, ml of the transformation reaction were plated on lysogeny broth (lb) agar containing ampicillin. for each reaction at least two plates were prepared and incubated at uc for h. a total number of clones were selected and transferred to . ml u deepwell plates (nunc) containing . ml lb-amp. the plates were incubated overnight at uc, rpm. on the next day, the deepwell plates were centrifuged, the supernatant discarded and the pellets resuspended in ml of lb-amp. a new set of u deepwell plates was prepared with ml of fresh lbamp and inoculated with ml each from the resuspended overnight cultures. the remaining ml of resuspended overnight culture were mixed with ml of sterile-filtered dmso and stored at uc. the newly inoculated plates were incubated for h at uc, rpm. afterwards, the temperature was reduced to uc, incubation continued for h and protein expression induced by addition of ml of . m b-d- -thiogalactopyranoside (iptg). incubation persisted overnight at uc, rpm. the cells were harvested by centrifugation ( g, min), the supernatant discarded and the pellets frozen at uc. after min the pellets were resuspended in ml of easylyse bacterial protein extraction solution (epicentre) and incubated for min at room temperature. dnase i was mixed with dnase reaction buffer ( mm tris-hcl, . mm mgcl , mm cacl ), added to the reaction and incubation was carried on for min at uc. the plates were centrifuged to collect cell debris for min at g. for each sample ml of lysate were transferred to microtiterplates (genetixx), which were used as reservoirs for the spotting procedure. the samples were spotted onto halolink slides (promega) using the qarray microarray spotter (molecular devices). different samples were spotted per slide with three replicate slides per screening. in total samples were screened on slides (n = ). each sample was spotted as quadruplicates with controls in two identical sets of eighteen subarrays each (total number of spots per slide ). the controls used included ht-ompa and ht-ompf as positive reference proteins as these have been described as immunodominant before. as specificity controls ht-argc and ht-pyrc were used, representing proteins without known immunodominant behaviour, thus binding of the polyclonal antibodies is not expected. in addition two different e.coli strains -acella electrocompetent cells and krx single-step figure . specificity assay of gavvalsttfa and derivatives. the bar chart represents the mean signal intensities (n = ) of gavvalsttfa and several modified peptides with single amino acid replacements incubated with antibodies reactive to k. pneumoniae (green), e. coli (orange) or s. enterica (purple). the sequences on the left represent the original epitope and modified versions displaying an increase in signal intensity for k. pneumoniae antibodies. in contrast, sequences on the right harbor modifications causing a significant drop in intensity for k. pneumoniae. rabbit igg is used as a positive reference and mbp serves as a negative control. none of the sequences tested displayed any significant signal intensity above the negative control when incubated with either e. coli or s. enterica antibodies. doi: . /journal.pone. .g competent cells (promega) -were spotted as further controls. as those two lack proteins expressed with a halotag, they are used as negative controls. after spotting of the samples, the slides were incubated for h at room temperature in a humidity chamber. next, slides were washed with pbs+ . % igepal ca- (pbsi, sigma aldrich) and dried by nitrogen flow. the well proplate module (grace biolabs) was attached to each slide. the top chamber was filled with . ml of rabbit-polyclonal antibody to k. pneumoniae (acris, mg/ml) in pbs. the bottom chamber was incubated with pbs only. after h of incubation at room temperature with gentle rocking, both chambers of each slide were washed three times with ml of pbsi. secondary antibody (goat-polyclonal to rabbit igg conjugated with chromeo- , abcam, mg/ml) was subjected to each chamber in pbs and the slides were incubated at room temperature for h in the dark under gentle rocking. finally, slides were washed three times with pbsi, the proplate modules removed and the slides dried by nitrogen flow. the slides were scanned on an axon genepix a laser scanner (molecular devices) with the following settings: nm laser, pmt gain , % laser power, lines to average , mm resolution and standard green emission filter at nm. the raw data sets of all the microarray analyses in this publication have been deposited in ncbi's gene expression omnibus [ ] and are accessible through geo series accession numbers gse , gse , gse , and gse . the median fluorescence intensity of each spot corrected by the local background (median f -b ) was used. further, relative fluorescence intensity (rfi) was calculated by subtracting the signals of the bottom chamber from the raw data signals of the top chamber to account for non-specific binding of secondary antibodies. for screening of expression libraries we used the contrast method with either argc or pyrc as specificity control to determine the contrast via the formula: with rf f i control the median of all rfis of the control used. clones harboring strong signals in microarray screening were selected to be sequenced. sequencing was performed externally by lgc genomics using ht f ( acatcggcccgggtct-gaatc ) and flxr ( cttcctttcgggctttgttag ) primers. after sequencing and identification of potentially immunodominant proteins, primers were designed to generate full-length clones for each identified gene, see table s for a list of the primers used. cloning was performed as mentioned above with slight modifications. the pfn a vector was linearized using the following primer set; a if linear for ( gtttaaac-gaattcgggctc ) and a if linear rev ( ggcgatcgcgttatcgctctg ) with pcr conditions as mentioned before. protein expression, lysis, and spotting of fulllength proteins were performed as described above. the slides were incubated for h at room temperature in a humidity chamber. for incubation with antibodies well or well proplate modules (grace biolabs) were attached to the halolink slides. processing of the slides was done similar to the original screening, however several different antibodies were used, see section antibodies. for testing of immunodominant characteristics with elisa, the crude lysate was first purified using halolink magnetic beads (promega) following the manufacturer's instructions. the proteins of interest were subsequently cleaved off by digestion with protev protease (promega) and concentration was determined by nanodrop measurements. the samples were diluted to a total protein content of mg/ml in pbs and ml of each sample was added to maxisorb plates (nunc). each sample was analyzed at least in triplicate. the elisa plate was covered with a lid and incubated overnight at uc in a humidity chamber. after five washing steps each with pbs+ . % tween- (pbst), the plates were blocked using ml % non fat dried milk in pbs per well for h. afterwards, plates were washed three times with pbst. ml of primary antibody solution (c = mg/ml) in pbs containing % non fat dried milk were applied to each well using the respective desired antibody or pbs for controls. the plates were incubated for h at room temperature and washed four times with pbst. next, ml of conjugated secondary antibody (goat polyclonal to rabbit igg conjugated with horseradish peroxidase, abcam ab , c = ng/ml) were added to each well and incubation carried on for h. finally, plates were washed once again four times with pbst and ml , , , -tetramethylbenzidine (tmb, sigma-aldrich) was added to each well for detection. after min of incubation at room temperature in the dark, the reaction was stopped by applying ml of m h so to each well. the optical density of each well was measured using the omega fluostar (bmg labtech) at a wavelength of nm. primers were designed using primer [ ] within geneious pro . . [ ] . the sequenced inserts were identified by blast [ ] . peptide sequence secondary structures were predicted using the emboss garnier [ ] algorithm and the transmembrane regions predicted by tmhmm . [ , ] . antigenic sites were predicted by emboss antigenic [ , ] . data evaluation was performed by originpro g (originlab) and microsoft excel. -dimensional structure predictions were performed using the swiss model automated mode [ ] [ ] [ ] [ ] [ ] and pdb files were visualized and analyzed by the ucsf chimera package [ ] .chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p -gm ). analysis of full-length proteins was achieved by combining the results from elisa and microarray data. hence, the rfi of each sample was calculated. next, a normalized rfi was generated by dividing the rfi of each sample by the median rfi of all the samples within an area of interest, i.e. incubation compartment. from these normalized rfis a median and standard deviation was calculated. if the median normalized rfi of the positive control was below the median normalized rfi of any of the negative references whilst taking the standard deviations into account, the test was rendered invalid. if the test passed the above criterion, the q values were calculated as follows: with rf f i sample the median of normalized rfis of the sample and rf f i pos:control the median of the normalized rfis of the positive control ompa respectively ompf. the resulting error was calculated by error propagation according to gauss. finally, incorporating all valid tests, the mean q value was determined along with its resulting error following error propagation by gauss, see table . several proteins were chosen for epitope mapping. these were the proteins encoded by kpn_ , kpn_ , kpn_ , kpn_ , kpn_ , and kpn_ . the proteins were divided into -mer oligopeptides with an overlap of amino acids in silico. the synthesis and coupling to microarray slides was performed externally by jpt peptide technologies gmbh. each peptide sequence was applied times to one slide. the slides were used with proplate -well chamber system (grace) allowing for incubation with different antibodies. first, the slides were blocked with superblock blocking buffer (thermo fischer) for h, washed five times with pbs+ . % tween- , primary antibodies applied, incubated overnight at uc with mild rocking, washed again, secondary antibodies applied for h in the dark and after a final washing procedure, dried and scanned as above. two different primary antibodies to k.pneumoniae were tested. the bottom chamber was always used as a control chamber, incubated only with secondary antibody. the peptide gavvalsttfa and modifications thereof created by substituting one amino acid by alanine/glycine were synthesized by jpt peptide technologies gmbh. these peptides in combination with two peptides showing closely related sequences, gavlalsssft and saalaltssfte, were applied times to slides. incubation procedure was performed as described above for epitope mapping the expression of the desired halotag fusion proteins was checked by sds-page. after lysis of cells, ml of each protein extract was mixed with ml of mm halotag alexa ligand. after addition of ml x tbs ( mm tris, mm nacl, ph . ) the reaction was incubated at room temperature for minutes. ml of each reaction were removed, mixed with ml of x loading buffer (fermentas) and ml dtt and heated for min at uc. the separation was performed on a mini-protean tgx gel (biorad, any kd, wells) in a protean ii xi cell chamber (biorad) for min at v. as a size reference benchmark fluorescent protein standard (life technologies) was used. fluorescence was measured in a fla- (fujifilm) with excitation at nm. figure s homology of giafgavelfd of kpn_ . the sequence derived from k. pneumoniae mgh was used as a reference. the best matches after blast analysis are shown in the figure with dots indicating identical residues. for differentiation of the sequences the ncbi accession number of the parent protein is given followed by the strain designation, if available. only three e. coli strains in lines , and feature a valine residue at the second position instead of the consensus isoleucine. consequently, this sequence is highly conserved within the enterobacteriaceae including e. coli, klebsiella, salmonella, and enterobacter among others. (pdf) table s list of sequenced clones after screening the clones were sequenced by lgc genomics using ht f and flx primers. clones that were not successfully sequenced are indicated by ''-'', clones carrying inserts too short to be reliably mapped to a gene are marked as truncated. additionally, a few inserts were detected deriving from primer concatamers. these are displayed as ''artificial''. the remaining clones are indicated by the corresponding locus tag and protein name. several clones apparently carry identical inserts, especially obvious for kpn_ or kpn_ . these were discarded from further analysis as the mapped inserts are very short and might have an artificial origin. inserts that were highly unlikely to garner new immunogenic proteins, antigens described previously, e.g. ompa, other molecules like trna and rrna were abolished from further analysis. table s primers used in this study. each primer is given with a name, its sequence in to direction and the target gene or vector. for each target f represents forward and r the reverse primer. the primers were used for cloning in the in-fusion smarter directional cdna library construction kit. 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peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites toward the estimation of the absolute quality of individual protein structure models the rin: an rna integrity number for assigning integrity values to rna measurements gene expression omnibus: ncbi gene expression and hybridization array data repository primer on the www for general users and for biologist programmers basic local alignment search tool analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins the swiss-model workspace: a web-based environment for protein structure homology modelling the swiss-model repository and associated resources swiss-model: an automated protein homology-modeling server swiss-model and the swiss-pdb viewer: an environment for comparative protein modeling protein modeling by email ucsf chimera-a visualization system for exploratory research and analysis the k. pneumoniae strain mgh was a kind gift of the group of s. bereswill (department of microbiology and hygiene, charité -university medicine berlin, berlin, germany). sh is greatly indebted to martina obry for her assistance during expression library construction and clone isolation. the authors would like to thank simone aubele for technical assistance. we also gratefully acknowledge michaela schellhase for microarray printing. conceived and designed the experiments: sh mvnr. performed the experiments: sh. analyzed the data: sh ffb mvnr. contributed reagents/materials/analysis tools: sh. wrote the paper: sh ffb mvnr. key: cord- - g rwsm authors: arruebo, manuel; vilaboa, nuria; sáez-gutierrez, berta; lambea, julio; tres, alejandro; valladares, mónica; gonzález-fernández, África title: assessment of the evolution of cancer treatment therapies date: - - journal: cancers (basel) doi: . /cancers sha: doc_id: cord_uid: g rwsm cancer therapy has been characterized throughout history by ups and downs, not only due to the ineffectiveness of treatments and side effects, but also by hope and the reality of complete remission and cure in many cases. within the therapeutic arsenal, alongside surgery in the case of solid tumors, are the antitumor drugs and radiation that have been the treatment of choice in some instances. in recent years, immunotherapy has become an important therapeutic alternative, and is now the first choice in many cases. nanotechnology has recently arrived on the scene, offering nanostructures as new therapeutic alternatives for controlled drug delivery, for combining imaging and treatment, applying hyperthermia, and providing directed target therapy, among others. these therapies can be applied either alone or in combination with other components (antibodies, peptides, folic acid, etc.). in addition, gene therapy is also offering promising new methods for treatment. here, we present a review of the evolution of cancer treatments, starting with chemotherapy, surgery, radiation and immunotherapy, and moving on to the most promising cutting-edge therapies (gene therapy and nanomedicine). we offer an historical point of view that covers the arrival of these therapies to clinical practice and the market, and the promises and challenges they present. chemotherapy, surgery and radiotherapy are the most common types of cancer treatments available nowadays. the history of chemotherapy began in the early th century, but its use in treating cancer began in the s. the term "chemotherapy" was coined by the german scientist paul ehrlich, who had a particular interest in alkylating agents and who came up with the term to describe the chemical treatment of disease. during the first and second world wars, it was noticed that soldiers exposed to mustard gas experienced decreased levels of leukocytes. this led to the use of nitrogen mustard as the first chemotherapy agent to treat lymphomas, a treatment used by gilman in . in the following years, alkylating drugs such as cyclophosphamide and chlorambucil were synthesized to fight cancer [ , ] . kilte and farber designed folate antagonists such as aminopterin and amethopterin, leading to the development of methotrexate, which in achieved leukemia remission in children [ ] . elion and hitchings developed -thioquanine and -mercaptopurine in for treating leukemia [ , ] . heidelberger developed a drug for solid tumors, -fluorouracil ( -fu), which is up to now an important chemotherapy agent against colorectal, head and neck cancer [ ] . the s saw the design of corticosteroids, along with the establishment of the cancer chemotherapy national service center in , whose purpose was to test cancer drugs. at that time, monotherapy drugs only achieved brief responses in some types of cancers [ ] . by , the first cancer to be cured with chemotherapy, choriocarcinoma, was reported [ ] . during the s, the main targets were hematologic cancers. better treatments were developed, with alkaloids from vinca and ibenzmethyzin (procarbazine) applied to leukemia and hodgkin's disease [ ] [ ] [ ] . in the s, advanced hodgkin's disease was made curable with chemotherapy using the momp protocol [ , ] , which combined nitrogen mustard with vincristine, methotrexate and prednisone, and the mopp protocol [ , ] , containing procarbazine but no methotrexate. patients with diffuse large b-cell lymphoma were treated with the same therapy and, in , a cure for advanced diffuse large b-cell lymphoma was reported using protocol c-mopp, which substituted cyclophosphamide for nitrogen mustard [ ] . surgery and radiotherapy were the basis for solid tumor treatment into the s. this led to a plateau in curability rates due to uncontrolled micrometastases. there were some promising publications about the use of adjuvant chemotherapy after radiotherapy or surgery in curing patients with advanced cancer. breast cancer was the first type of disease in which positive results with adjuvant therapy were obtained, and also the first example of multimodality treatment, a strategy this manuscript reviews the evolution of oncological treatments available today, together with several immunotherapeutic approaches and nanoscale-based therapeutics including successes, drawbacks and recent progress. the concept of immunotherapy in medicine incorporates the use of components of the immune system, including antibodies (abs), cytokines, and dendritic cells, to treat various illnesses, such as cancer, allergies, and autoimmune and infectious diseases. immunotherapy also includes the use of vaccines for the prevention of allergies and tumors. immunotherapy adds new dimensions to clinical practice, offering much more specificity, higher efficacy, directed therapy, less toxicity, lower secondary effects and better tolerance. although immunotherapy can be used for several illnesses (macular degeneration, autoimmune diseases, etc.), in the case of cancer, the aim of immunotherapy is to kill tumor cells (either directly or indirectly) or to help patients' immune systems destroy tumors. of all the types of anti-tumoral immunotherapy, this review will focus on the use of antibodies, their history, problems and current applications. antibodies (abs) are one of the most important defense mechanisms for vertebrate animals. they are produced by b cells, which, after antigen-mediated activation, undergo differentiation to secretory (plasma) cells thus producing soluble antibodies. antibodies are highly specific, and they recognize and eliminate pathogens and disease antigens, but can be deliberately generated to recognize different target molecules (tumor markers, bacteria, receptors, cytokines, hormones, etc.). thus, abs can be used in many applications, including diagnostic techniques, research and therapy (against infections, tumors, transplants and autoimmune diseases). antibodies were described in ( figure ) by von behring and kitasato as "anti-toxins" that appeared in the serum of animals after immunization with inactivated toxins (toxoids) [ ] . the researchers noted that protection could be transferred to other animals through the use of these antisera, thus beginning what it is known as "serum therapy" for treating infectious diseases (diphtheria and tetanus) in humans. soon after, these sera elements were described as "anti-bodies" because they could be directed not only against toxins, but also against a large variety of organisms and compounds (bacteria, proteins, chemicals, etc.). immunotherapy initially began with the use of antisera obtained from animals such as horses and sheep containing, among other things, a mixture of antibodies from the activation of different b cell clones, so-called "polyclonal antibodies" (pabs). in , felton and bailey obtained pure antibodies, but it was not until the s, thanks to the work of porter and edelman ( nobel prize winners), that the ab structure became known. after the introduction of abs to therapy, researchers observed that the transferred defense was only temporary (as opposed to vaccination, which induces long-term memory). in addition, it often incurred anaphylactic responses that were occasionally fatal and which greatly reduced their use in human therapy. however, these problems did not prevent pabs from being used successfully in diagnostic cancers , techniques and even in preventive therapies. anti-snake venom, ant-tetanus and anti-rh+ gamma globulins are still being used in clinical practice. figure . history of antibodies. in von behring and kitasato showed that it was possible to generate anti-toxins (against tetanous, diphtheria), and soon after, therapy with antiserum containing antitoxins were used in patients. it took several years to purify the antibodies ( ) and even more to know their structure. on , milstein and köhler developed the first monoclonal antibody, and the generation and application of monoclonal antibodies started (on diagnosis, research and therapy), initiating the modern immunology. in the s, the first anti-tumoral monoclonal antibody was tested and molecular biology techniques started to designed chimeric and humanized antibodies. later on, transgenic mice carrying human ig genes and other animal models were used to produce fully human antibodies. in , cesar milstein and george köhler ( nobel prize winners) succeeded in generating monoclonal antibodies (mabs) by fusing mouse b cells with b cell tumors (myeloma) to create hybrid cells, which were immortal and had the capacity to produce large quantities of a single (monoclonal) antibody [ ] . in , genetic studies by susumu tonegawa revealed the basis for the vast diversity of antibodies, identifying the process of somatic recombination in immunoglobulin genes [ ] . since the publication of the monoclonal antibody technique, mouse and rat mabs have been used in many laboratories with thousands of applications in various scientific fields, in diagnostic techniques (clinical, food, environmental), research and in therapy (antitumor, autoimmune diseases). monoclonal antibodies have helped in the discovery of new molecules (such as the identification of more than membrane proteins, grouped under the cd concept or cluster of differentiation), transcription factors, viral, plant and bacterial proteins, phosphorylated compounds involved in death by apoptosis, factors involved in enzymatic cascades and many more. as an example of their usefulness, the current classification of leukemia by the world health organization is based on the presence or absence of membrane molecules recognized by monoclonal antibodies that define leucocyte populations in various stages of differentiation. but one of the greatest achievements with monoclonal antibodies is their use in human therapy. surgery, chemotherapy and radiotherapy are not specifically directed to tumor cells and may also affect healthy tissue. antibodies can provide specificity and lower toxicity, opening new therapeutic possibilities. the first evidence of this potential came in when a patient suffering from lymphoma responded to treatment using a mouse mab directed specifically against his tumor b lymphocytes [ ] . this response rapidly encouraged research into the production of potentially therapeutic abs. however, clinical trials results revealed that many patients receiving this therapy developed an immune response directed against the therapeutic abs, a response known as hama (human anti-mouse antibodies) or hara (human anti-rat antibodies). some even developed anaphylactic reactions, especially after repeated administration. the high immunogenicity of antibodies due to their large size compared to conventional pharmaceutical drugs, and differences in the pattern of glycosylation between murine and human abs, once again led to the cessation of antibody use in therapy. completely human mabs needed to be developed to avoid immune rejection, but their production was much more complex than initially thought. in contrast to mouse or rat myeloma cells, human myeloma cells proved difficult to adapt to continuous growth in vitro. researchers tried to resolve this problem by immortalizing b cells using the epstein-barr virus (ebv) [ ] and by fusing human b cells with well-established murine myeloma (obtaining heterohybridomas) [ ] . however, the low production of antibodies in these cells, the instability of heteromyeloma cells and numerous technical problems lead to the search for alternative methods for generating human-like mabs in the mid- s. one of these methods was the modification of murine mabs through genetic engineering ( figure ). several antibody molecules and some antibody fragments are shown. chimeric (mouse-human) antibodies carry mouse heavy and light variable domains (in yellow) being the rest of the molecule of human origin (in red). in the case of humanized antibodies, only the hypervariable regions are mouse derived (in yellow). it is possible to generate bi-specific antibody molecules, using different heavy and light chains (each arm will have a different specificity). fab: fragment antigen binding; scfv: single chain fragment variable; vh: variable domain from the heavy chain. chimerization (murine variable domains linked to constant regions of human heavy and light chains), humanization (only hypervariable regions of murine origin), primatization (chimeric structure of human and primate origin) and the design of recombinant antibody fragments, such as fv (variable fragment), fab (antigen binding fragment), scfv (single chain variable fragment) and minibodies (artificial polypeptides with a structure based on the igv domain), are some of the methods that have been used over the last years to reduce antigenicity and maintain the binding affinity and specificity of the original ab. rituximab, a chimeric anti-cd mab, was the first mab approved by the fda for antitumor therapy. however, a year earlier, several mabs conjugated with radioactive elements were approved for in vivo tumor detection. every year since then, several mabs have been approved for therapy in the us and europe, and more than half of them are chimeric or humanized mabs (see table ). in addition to fully engineered antibodies, antibody fragments also have advantages compared to whole antibodies, especially in terms of the rate of solid tumor penetration. jainr [ ] determined that an intact igg molecule needed hours to move mm into a solid tumor, whereas a fab fragment reached the same distance in hours. while the expression of chimeric and humanized antibodies was carried out in eukaryotic hosts, such as mammalian or plant cells, bacteria have been the most widely used organism for the production of recombinant antibody fragments [ ] [ ] [ ] . however, despite numerous advantages, such as avoiding animal immunization and hybridoma production, their low cost and easier production [ ] , antibody fragments have shorter circulating half-lives compared to full-size antibodies, lack glycosylation and lack effector functions due to the absence of their fc region (unless added). thus, antibody therapies using incomplete antibodies have been relegated to those cases where rapid elimination of antibodies from the blood is required and to local therapy (e.g., macular degeneration). modified versions, such as pegylation of fragments (modification of a molecule by linking of one or more polyethylene glycol chains) [ ] to improve circulation half-life, glycosylation and fc region engineering are some of the recent approaches used by researchers to overcome these problems [ ] . in the mid s, thanks to the development of molecular biology techniques and microinjection and manipulation of embryonic cells, several groups created various transgenic mice models carrying human igs genes ( figure ). the introduction of human ig loci in these mice was carried out using various vectors, such as miniloci, yeast and human artificial chromosomes (yacs and hacs, respectively) and p vectors. transgenic mice can be immunized with almost any ag (including human tumor cells), and their spleens can be used to obtain hybridomas following the conventional protocol [ ] [ ] [ ] [ ] . moreover, mice can produce human abs of intermediate/high affinity because they can introduce mutations in their human igs transgenes through the mechanism of somatic hypermutation. fully human monoclonal antibodies show several advantages in human therapy, which include low or no immunogenicity, better interaction with human effector systems and patterns of glycosylation and a longer half-life in human serum. in recent years, many fully human mabs have been introduced into clinical trials and some of them have been approved by regulatory agencies (table ) . in addition to the use of transgenic mice to generate fully human mabs, other alternatives have been developed, such as the use of immunodeficient mice receiving human hematopoietic tissue, the use of chicken eggs with human ig coding genes inserted into embryonic cells and the generation of transgenic tobacco plants for producing human mabs. moreover, several groups are working on modifications of the basic antibody structure to generate monovalent and multispecific reagents that may have various therapeutic properties and even completely new structures. examples of these new reagents include antibody alternative protein scaffolds based on leucine-rich repeat molecules of lamprey variable lymphocyte receptors (vlrs), libraries of fibronectin domains and designed ankryin repeat proteins (darps) [ ] . with all these novel antibody formats, immunogenicity, stability and aggregation problems should be carefully considered. soon after mabs generation was reported in , the potential of mabs became clear and many companies showed interested in developing new reagents for diagnosis and designing new equipment, among other contributions. however, when it came to the field of human therapy, pharmaceutical companies did not initially show much interest in the development of monoclonal antibodies, although several research groups were showing promising results in preclinical and clinical studies. the reasons for their reluctance are many: . a number of pharmaceutical companies had experience with generating small compounds, most of them chemically synthesized, but not with generating large biological molecules produced by cells. moreover, sophisticated equipment and cell culturing under controlled conditions, with full quality assurance, are necessary for antibody production. . there was the perception by pharmaceutical companies that production of mabs was not going to yield sufficient profit. most companies preferred to concentrate their efforts on developing analogues of well-known drugs rather than on new products, while at the same time most clinicians opted for trials using combinations of known agents. this view took years to change. advances in mab engineering helped develop more effective mab drugs with high specificity, improved potency and stability and decreased immunogenicity, which helped change the companies' initial reluctance. . in terms of clinical trials, there were concerns about the cost of the trials (around times more expensive today than years ago), the time required for preclinical pharmacology and toxicology studies (which are much more regulated) and the difficulty in conducting early clinical trials. since new drugs can only be tested against advanced and usually heavily pretreated disease, it is unlikely that dramatic responses will occur with these patients. . the requirement for fetal calf serum in cell hybridoma cultures introduced another problem when mad cow disease was identified in the early s. the fda proposed a limit on materials used in some medical products in order to keep them free of the agent thought to responsible for mad cow disease (also known as bovine spongiform encephalopathy or bse), making it necessary to find alternatives, such as enriched media without serum. since , mabs have entered clinical studies for various diseases, with % of those currently in clinical development. some of these mabs are listed in table . the first mab approved for cancer therapy was rituximab (rituxan tm ), a chimeric antibody directed against cd , for non-hodgkin's lymphomas. since then, many others have reached the market, including those for the treatment of breast cancer (trastuzumab, herceptin ® ), acute myeloid leukemia (gemtuzumab ozogamicin, mylotarg tm ), chronic lymphocytic leukemia (alemtuzumab, campath- h ® ), colorectal tumor (cetuximab, erbitux tm ) and several types of cancer (bevacizumab, avastin tm ). companies such as genentech inc., amgen, bristol-myers-squibb, imclone systems and trion pharma represent only a portion of the pharmaceutical companies involved in the antibody market related to cancer therapy ( table ) . new developments have also occurred in the immunoconjugate field and many of them are currently being explored by the pharmaceutical industry. immunoconjugates include antibodies linked to cancer-killing agents such as drugs, cytokines, toxins and radioisotopes. the objective is for the antibody to act as a transporter for the cancer-killing agent, concentrating the agent directly in the cancer cell, with minimal damage to healthy cells. although conjugated antibodies showed toxicity in the past, more recent approaches under development appear to decrease unwanted side effects. pharmaceutical companies are developing immunoconjugates independently, forming partnerships with specialized players and even acquiring small biotech companies that are focused on the field of immunoconjugates. although the challenge of their potential immunogenicity requires special attention, there are several practical advantages to immunoconjugates over single antibodies. these include lower dosages, which may lead to lower treatment costs and fewer side effects; the reintroduction of antibodies that historically have shown low efficacy in isolation; the possibility of using bacteria or plant cells to produce immunoconjugates rather than using mammalian cell cultures (decreasing costs and complexity) and the large number of potential combinations (antibodies-cancer killing agents) that are possible. the advantages of immunoconjugates over single antibodies make them crucial players in new cancer therapy developments. although many researchers have worked on monoclonal antibodies and cancer (close to , reports on this subject can be found in pubmed (http://www.ncbi.nlm.nih.gov/pubmed) the therapeutic mab market moved much more slowly than initially expected, due mostly to the problems indicated above. this situation has changed in recent years and mabs are now the largest class of biological therapies under development, representing a multi-billion dollar worldwide market. as reported recently by scolnik [ ] , the mabs currently marketed in the us have a sales growth rate of % compared to less than % for small-molecule drugs. oncology and autoimmune diseases are the most successful indications for these drugs, with five mabs having sales in excess of $ b. thanks to basic research, researchers are identifying new biomarkers, which could be potential targets for mabs. there are currently numerous mabs at various developmental stages and it is expected that many of them will be available for clinical use in the near future. chemotherapy, radiation therapy and surgery are the most common types of cancer treatments available today. more recent treatments, which are at various stages of development, include angiogenesis inhibitor therapy, biological therapies (including interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy and nonspecific immunomodulating agents), bone marrow and peripheral blood stem cell transplantation, laser therapy, hyperthermia, photodynamic therapy and targeted cancer therapies [ ] . in the last two decades, a large number of nanoscale and nanostructure-based therapeutic and diagnostic agents have been developed, not only for cancer treatment but also for its prevention and diagnosis [ ] . targeted cancer, hyperthermia, photodynamic and gene therapies are just some of the cancer treatments that use engineered nanomaterials. these therapies can be used in isolation or in combination with other cancer treatments, thereby taking advantage of their ability to target tumors (actively or passively), to respond to physical or chemical stimulation (internal or external) and to deliver therapeutic genes to the cell nuclei. the main objective of nanomaterials in cancer treatment is to deliver a therapeutic moiety to tumor cells in a controlled manner (depending on the required pharmacokinetic) while minimizing side effects and preventing drug resistance. nanoscale and nanostructured materials may also be used in diagnosis to detect and prevent pathologies as soon as possible, ideally being able to sense cancer cells and associated biomarkers. compared to conventional therapies, nanoparticles show six clear advantages in cancer treatment and/or diagnosis: ( ) they can be synthesized in specific sizes and with surface characteristics to penetrate tumors by taking advantage of the enhanced permeation and retention effect (epr) (a mechanism known as passive targeting); ( ) they can be engineered to target tumor cells by surface functionalization with biomolecules that attach to tumor-specific cell markers (a mechanism known as active targeting); ( ) they can be engineered to penetrate cells and physiological barriers (e.g., blood-brain barrier, blood-retinal barrier); ( ) they can increase the plasma half-life of carried chemotherapeutic drugs, which are usually highly hydrophobic; ( ) they can protect a therapeutic payload from biological degradation; and ( ) they can be synthesized as multifunctional platforms for combined imaging and therapeutic applications (theragnostic nanoparticles). examples of various nanostructured materials with potential applications in oncology are shown in figure . the advantages of biocompatible nanomaterials have contributed to their significant expansion in cancer treatment. targeted therapies for oncology are predicted to reach a billion euro global market by [ ] . the total market for nanobiotechnology products reached as high as $ . billion in [ ] . table compiles some of the clinically approved nano-based therapeutics for cancer treatment and diagnosis. many other nanoscale or nanostructure-based therapeutic and diagnostic agents are currently in clinical trials at various stages of development. in , zhang et al. [ ] reported on clinical trials being conducted for nanoparticle-based therapeutics. a year later, ongoing clinical trials using nanoparticles for cancer were mentioned by bergin [ ] , and at present, there are more than clinical trials under development [ ] . this large number of commercial nano-based therapeutics for use in cancer treatment is also reflected in the exponential increase in scientific publications and patents involving nanomaterials in recent years. figure shows the evolution over the last decade in the number of published scientific papers and issued patents involving nano-based applications developed to fight cancer. the number of papers and patents involving traditional forms of therapy (chemotherapy, radiation therapy and surgery) grew linearly over the last decade. however, the use of the terms "nano-" and "cancer" has shown exponential growth over the past decade, demonstrating a major focus on nano-based tools applied to cancer treatment and diagnosis. recent advances in the use of nanoscale and nanostructured-based therapeutic agents in cancer treatment are reported below. nanoparticles are engineered to achieve cell targeting by using selective moieties (e.g., antibodies and their fragments, carbohydrates, peptides, nucleic acids), which binds to its corresponding antigen, cell surface carbohydrate or over-expressed receptor in tumor cells. the rapid cellular proliferation of these cells is also exploited by coupling the nanoparticles with different biological agents, such as folic acid. the rationale for coupling these carriers with folic acid is that the folate receptor is over-expressed in a broad range of tumor cell types, including solid and hematological malignancies [ ] . once it has reached the target, the cargo is released into the interior of the cell, and ideally, a signaling marker attached to the vector will aid the physician in visualizing the tumor. such a vector may also be grafted with a moiety (usually peg), which retards recognition by the reticuloendothelial system (res) to increase nanoparticle systemic circulation. in addition to recognition moieties, carried drugs and signaling elements attached to nanoparticles, numerous authors have also envisioned and designed vectors with additional functionalities, including cell-penetrating moieties, combinations of several drugs, combinations of drugs and genes, prodrugs (which become drugs upon biochemical modification by tumor cells), stimulus-sensitive agents that can be externally triggered and molecules for evaluating therapeutic efficacy. the more functionality added to the vector, the better the chances of reaching the target; however, its chances of being detected by the res also increase. therefore, currently marketed nanoparticles use passive targeting and active targeted nanoparticles are still being developed. examples of active targeted nanoparticles are reviewed elsewhere [ ] . targeted nanoparticle fabrication remains a challenge due to the multiple steps involved, which include biomaterial synthesis and assembly, targeting ligand coupling/insertion, drug loading, surface stabilization and final purification, which could cause batch-to-batch variations and, therefore, quality concerns. for this reason, single-step synthesis of targeted nanoparticles by self-assembling pre-functionalized biomaterials provides a simple and scalable manufacturing strategy [ ] . mass production is also a serious concern and continuous synthesis procedures are therefore still being sought. when using batch reactors to synthesize nanoparticles, several drawbacks usually appear, including: ( ) heterogeneous distribution of reactants and temperatures in the reactor; ( ) insufficient mixing; ( ) variations in the physicochemical characteristics of products from different batches; ( ) their inherent discontinuity; and ( ) the numerous post-synthesis purification steps that are usually required. in order to overcome these disadvantages, microfluidic reactors (e.g., micromechanized micromixers, capillaries, junctions) have been used in the continuous synthesis of nanoparticles to precisely control reaction temperatures and residence times, thereby rendering nanoparticles with narrow particle-size distributions. other continuous synthesis processes are usually preferred when synthesizing nanoparticles on a large scale (e.g., laser pyrolysis, arc discharge methods). another concern is the adaptive response of the immune system after repeated applications of nanoparticles. immunological memory, created from the primary response to a specific nanoparticle, provides an enhanced response to secondary encounters with the same type of nanoparticle. as an example, the recognition of pegylated liposomes by anti-peg antibodies has been reported to occur between to days after the first administration of peg-liposomes, leading to fast clearance from circulation [ ] . finally, one of the last major barriers to achieving the transition of targeted nanoparticle use into clinical practice is the complete understanding of potential toxicological properties of these materials, along with their exact pharmacodynamics and pharmacokinetics. in spite of these hurdles, many research groups are focusing their efforts on solving them. other groups are also directing their efforts towards designing more efficient targeted nanoparticles for cancer treatment in terms of structure, morphology, biocompatibility and surface functionalization. some of those advances will be described later in this document. novel targeted theragnostic nanoparticles have been synthesized and their bi-functionality demonstrated. among them are perfluorocarbon nanoemulsions, which are in clinical trials [ ] . quain et al. [ ] coupled pegylated gold nanoparticles to a single-chain variable fragment antibody, which recognized the epidermal growth factor receptor overexpressed in many types of malignant human tumors, and demonstrated the targeting capabilities of these vectors in nude mice bearing human head-and-neck tumors. the nanoparticles were also able to function as tags for spectroscopic detection with surface-enhanced raman spectroscopy. magnetic targeting has also been used as a physical method for targeting and visualizing tumors. effects of magnetic targeting on the extent and selectivity of nanoparticle accumulation in tumors of rats harboring orthotopic l-gliosarcomas were analyzed using magnetic resonance imaging (mri) [ ] . sun et al. also demonstrated the targeted drug release capabilities of iron oxide nanoparticles conjugated with a drug (methotrexate) and a targeting ligand, chlorotoxin, while monitoring tumor-cell specificity in vivo using mri [ ] . weng et al. demonstrated the targeted tumor cell internalization and imaging of multifunctional quantum dot-conjugated immunoliposomes, in vitro and in vivo [ ] . in this targeted delivery system, anti-her single chain fv fragments were attached to the end of peg chains located on the surface of liposomes. targeting via extracellular activation of the nanocarrier is a promising method for achieving active targeting using physiological stimuli present in the tumor environment. triggering mechanisms that only release the transported cargo of nanocarriers into the tumor environment take advantage of its acidic ph and uncontrolled enzyme production. a complete description of these systems is reported elsewhere [ ] . tumor targeting of prodrugs that become active once they reach tumor cells is another novel strategy for avoiding unwanted side effects of the drug, and it allows for the delivery of large doses of drugs. following this approach, dhar et al. [ ] synthesized pt(iv)-encapsulated prostate-specific membrane antigen targeted nanoparticles of poly(d,l-lactic-co-glycolic acid) (plga)-poly(ethylene glycol) (peg)-functionalized controlled release polymers. after reduction in the interior of the tumor cells, the prodrug becomes cisplatin, which cross-links on nuclear dna. photodynamic therapy (pdt) is a technology that uses a photosensitizer that is activated upon exposure to visible or near infrared (nir) light, and transfers energy to molecular oxygen, thereby generating reactive oxygen species (e.g., singlet oxygen, free radicals, peroxides). the subsequent oxidation of lipids, amino acids and proteins induces cell death. a complete review of photosensitizers is reported elsewhere [ ] . fda-approved photosensitizers absorb in the visible spectral regions below nm, where light penetrates only a few millimeters into the skin. pdt is therefore limited to treatment of certain types of skin cancer and its effectiveness for other tumors is not yet apparent [ , ] . pdt is usually performed as an outpatient procedure and may be repeated and used in combination with other therapies, such as surgery, radiation and chemotherapy [ ] . photosensitizers are susceptible to photobleaching under light irradiation, and have therefore been loaded within nanoparticles to avoid this drawback. most photosensitizers are also highly hydrophobic, so nanoparticles are being explored as carriers to increase their bioavailability. noble metal nanoparticles have proven very useful as agents in photodynamic therapy due to their enhanced absorption cross sections, which are four to five orders of magnitude larger than those offered by conventional photoabsorbing dyes [ ] . silica nanoparticles synthesized in the non-polar core of micelles have been used to entrap the water-insoluble photosensitizing anticancer drug -devinyl- -( hexyloxyethyl) pyropheophorbide. upon nir light irradiation, nanoparticles embedded in hela cells generate singlet oxygen, resulting in a reduction in the percentage of cell survival [ ] . many other photosensitizers have been embedded within inorganic nanoparticles for pdt, including meta-tetra(hydroxyphenyl)-chlorin (m-thpc) [ ] . a complete review of various nanoparticulate-based carriers for pdt is reported elsewhere [ ] . preclinical studies will determine the added translational value of pdt therapies using photosensitizers loaded into these novel nanoparticles prior to their use in clinical settings. hyperthermia, as an anticancer therapy, consists of heating a tumor to inhibit proliferation of cancer cells with the aim of destroying or rendering them more sensitive to the effects of conventional protocols of radiation and chemotherapy. in fact, hyperthermia is currently used as an adjunct therapy to radiotherapy and/or chemotherapy. when cells are heated beyond their normal temperature they can become sensitized to conventional therapeutic agents such as radiation and chemotherapy. when high temperatures are used, typically above °c, the heat causes irreparable damage and results in tumor cell death in a process known as thermal ablation. the success of local thermal ablation consists of destroying the entire tumor mass without damaging adjacent vital structures. this requirement is particularly important for patients with limited reserves of tissue function. hyperthermia treatments make use of microwaves, ultrasounds and radiofrequency, which can be focused and used locally to target the tumor. a significant advantage of thermal technology is that it is minimally invasive. mild heat increases blood flow in the tumor, allowing chemotherapy to exert greater effect on cancer cells. by depressing the metabolic activity of target cells, heat also reduces the oxygen demand in the tumor and tumor tissue oxygenation increases, which makes hyperthermia one of the most potent radiosensitizers available [ ] . results from clinical trials conducted under quality assurance guidelines have shown hyperthermia to be beneficial in the treatment of several types of solid tumors, including breast cancer, melanoma, sarcoma and locally advanced cervical cancer, with reports demonstrating improved overall survival, as compared to patients who only receive radiotherapy or chemotherapy [ ] [ ] [ ] . it is widely accepted that the benefits of hyperthermia will significantly increase with refinements in heating delivery technologies as well as in monitoring strategies that ensure optimal thermal dose coverage, resulting in advanced local tumor control and prolongation of overall survival. integration of hyperthermia with emerging imaging technologies, such as non-invasive mr-based thermometry, will help unveil the full potential of hyperthermia for treating cancer. nanotechnology may offer a window of opportunity to improve heat delivery. for example, highly focused ultrasound energy transfer to deep brain tumors may be difficult to achieve due to the skull's electromagnetic barrier. magnetic fluid hyperthermia (mfh) uses iron oxides as a heating source due to their excellent magnetic properties and good compatibility [ ] . depending on the route of administration, magnetically mediated hyperthermia can be classified into two main types: arterial embolization hyperthermia, where arterial supply is used to deliver magnetic particles into the tumor tissue, and direct intratumoral injection hyperthermia. magnetic nanoparticles for hyperthermia settings show the advantage of being able to achieve site-specific tumor targeting through the aid of an external magnetic field. magnetic nanoparticles can also be simultaneously traced using mri. these nanoparticles are then selectively heated by application of a high frequency alternating magnetic field. magnetic energy dissipation from the nanoparticles (brown and néel relaxations) induces heating, which produces cell death at temperatures above °c. significant antineoplastic effects of mfh treatment were initially observed in animal models of glioma [ ] and prostate cancer [ ] . consequently, phase i and ii clinical trials with thermotherapy using magnetic particles have been conducted to treat prostate carcinoma [ ] and glioblastoma multiforme [ , ] . it has been demonstrated that magnetic hyperthermia in conjunction with a reduced radiation dose leads to longer survival following diagnosis of first tumor recurrence compared to conventional therapies in the treatment of recurrent glioblastoma [ ] . limiting factors of magnetic hyperthermia have been reported, including patient discomfort at high magnetic field strengths as well as irregular intratumoral heat distribution even upon direct intratumoral injection [ ] . magnetoliposomes, i.e., magnetic nanoparticles encapsulated within liposomes, have been designed to achieve active targeting of tumor cells by electrostatic interaction before hyperthermia treatment [ ] . other active strategies, including antibody-functionalized magnetoliposomes, have been used in combination with hyperthermia, demonstrating effective targeting and cytotoxic responses when applying alternating magnetic fields in tumor-bearing mouse models [ ] . the harnessing of therapeutic effects of nanoparticle-driven hyperthermia will likely take advantage of the feasibility of using these vectors to load drugs or biological agents and trigger their release upon heating, in order to increase tumor control and disease-free survival. the use of magnetic hyperthermia to trigger drug release has also been demonstrated as feasible in combinatorial approaches for cancer treatment. purushotham et al. [ ] developed magnetic nanoparticles coated with a thermoresponsive polymer poly-n-isopropylacrylamide (pnipam). with these nanoparticles, simultaneous hyperthermia and drug release of therapeutically relevant quantities of doxorubicin at hyperthermia temperatures was achieved in vitro. in vivo targeting of those doxorubicin-loaded nanoparticles injected directly via the main hepatic artery to hepatocellular carcinoma in a rat model was followed by mri examination. nir-absorbing nanoparticles have the advantage of being able to absorb or scatter light, thus producing heat, which increases the temperature in the tissue where the nanoparticles have been embedded. this region of the electromagnetic spectrum is notable for minimal absorption by water and biological chromophores [ ] . therefore, nir light is preferable as a trigger in biomedical applications because it has maximal penetration of tissues due to their minimal absorbance at those wavelengths [ ] . hemoglobin and water, the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficient in the nir region (around - nm). nir light has been shown to travel at least cm through breast tissue and cm through skull/brain tissue and deep muscle using microwatt laser sources (fda class ), while light at higher power levels (fda class ) has been shown to penetrate through cm of muscle and neonatal skull/brain [ ] . the use of sio /au nanoparticles (nanoshells) as nir-absorbing tags is also considered for the photothermal ablation of solid tumors [ ] . a pilot study on patients with refractory head and neck cancer is currently being conducted [ ] . au/aus sulfide nir-absorbing nanoparticles ( - nm) provide higher absorption than nanoshells ( % absorption and % scattering for au/aus versus % absorption and % scattering for sio /au nanoshells) as well as potentially better tumor penetration [ ] . other nanoparticles used in nir include hollow gold nanoparticles, which are smaller than sio /au nanoshells thus giving them prolonged blood circulation half-life and increased chances of reaching the tumors [ ] . maltzahn et al. [ ] demonstrated that (peg)-protected gold nanorods exhibit superior spectral bandwidth, higher photothermal heat generation per gram of gold and longer circulation half-life when compared to gold nanoshells, as well as an approximately two-fold higher x-ray absorption than a clinical iodine contrast agent. nir-absorbing nanoparticles have also been functionalized with anti-her antibodies to achieve tumor targeting in medulloblastoma cells [ ] . hollow gold nanoparticles were loaded with an Į-melanocyte-stimulating hormone analog [ ] , a potent agonist of melanocortin type- receptor overexpressed in melanoma, demonstrating selective photothermal ablation of b /f melanoma. nanoshells have been loaded into cells of monocyte lineage, which acted as carriers. once incorporated into human breast tumors in nude mice, the photoinduced cell death of nanoparticle-loaded macrophages was able to induce the death of malignant cells in the tumor's hypoxic microenvironment [ ] . current studies are focused on engineering more efficient nir-absorbing nanomaterials and on their functionalization with targeting moieties. compared to currently available non-invasive procedures with capabilities of increasing the temperature of target tumors, the main drawbacks of magnetic and nir-absorbing nanoparticles arise from their necessarily invasive nature as well as from the relatively indiscriminate nature of the tissue damage. due to their efficient intracellular uptake, concerns regarding acute and long-term effects of inorganic nanoparticles accumulation and cytotoxicity are emerging in the biomedical research community [ ] [ ] [ ] . despite the increasing number of newly developed nanoparticles designed for hyperthermia applications, the number of studies addressing their toxicity is low [ ] . collected data indicate that size, crystallinity, shape and surface chemistry strongly influence the mechanism of inorganic nanoparticle internalization by cells, their biodistribution, metabolism and potential toxicity, highlighting the great importance of increasing understanding of healthy and tumor cell interactions with nanoparticles. it is expected that ongoing studies will help reconcile conflicting data and demonstrate the safety of inorganic particles to those reporting transient or acute in vivo toxicity. gene therapy aims to treat diseases by introducing dna, rna, small interfering rna and antisense oligonucleotides into specific target cells or tissues to restore missing functionality and to eradicate pathogenic dysfunction. the therapeutic gene material is delivered to specific target cells using efficient vectors that aim to sustain stable, regulated gene expression without creating unwanted side effects. viral carriers, organic cationic compounds, recombinant proteins and inorganic nanoparticles are the four kinds of carriers currently being explored for gene delivery applications [ , ] . all of them show advantages and disadvantages, but none of them fulfill the criteria for an ideal vector. indeed, viruses can be regarded as nanoparticles due to their dimensions, regular geometries and well-characterized surface properties. the most widely used viral vectors for gene transfer include adenoviruses (ad), which are the dominant gene delivery systems in clinical settings, adeno-associated viruses, herpes simplex- viruses, retroviruses and lentiviruses [ ] . viruses are very efficient carriers; however, some of them have limited dna cargo capacity, can cause immunogenicity and toxicity and their manufacture is rather expensive. in general, synthetic delivery systems prevent specific immune responses and may carry higher amounts of material, without strict limitations on the size of the genetic drugs. the concept of gene therapy was initially envisioned in the s, but due to the cumbersome nature of the testing required to design and produce effective and safe vectors, gene therapy systems were not fully developed until the early s. the first clinical trials were approved in , and during the s numerous vectors carrying various therapeutic genes were engineered, and their usefulness was tested in preclinical studies. due to a simplistic belief in the straightforward success of gene therapy, many of these viral vectors rapidly moved to clinical settings. although success could be demonstrated in some early clinical studies, even when conducted with far from perfect vectors, serious adverse effects and patient deaths led to rigorous regulation of gene therapy protocols for human use. the evolution of currently successful cancer strategies discussed in sections and also included significant failures and setbacks, which did not restrain investments in chemotherapy and immunological therapies. however, the pharmaceutical industry has not yet developed a single cancer gene therapy product, and so the development of genetic medicines has been left to academic institutions and small biotechnology companies. in addition, the drawbacks of clinical trials for gene therapy led to extended periods of severe cuts in public research funding. the fda has not yet approved a human gene therapy product for sale, although gene-related research is growing rapidly and many clinical trials are ongoing. most of these are in phase i or ii and are aimed at dose determination and toxicity assessment [ ] . due to the unknown safety profile of gene vectors, design and approval of human trials were facilitated for life-threatening diseases. approximately , trials have been conducted worldwide since , and more than two-thirds of them were conceived for cancer diseases. due to the complex nature of cancer, the numerous gene therapy approaches for fighting it include strategies for restoring mutant suppressor gene functions, inactivating oncogenes, expressing suicide genes and eliciting protective immune responses [ ] . oncolytic viruses have also been engineered that exploit tumor cells characteristics by replicating them in these target cells as a method for improving the dissemination of biological agents in solid tumors [ ] . for the delivery of therapeutic genes encoding proteins with cytotoxic or anti-angiogenic actions, transcriptional targeting using regulatable promoters has been explored as a way of restricting transgene expression to an optimal therapeutic window [ ] . to date, there are two gene therapy products available on the market for clinical use, both of which have been approved for cancer treatment in china. since china has been the only country in the world where gene therapy is licensed for practice. these products are adenoviral vectors marketed under the brand names gendicine tm and oncorine tm [ , ] . gendicine tm is a p -overexpressing, replication-incompetent ad for the treatment of head and neck squamous cell cancer in combination with radiotherapy. oncorine tm is an e b- k-gene-deleted oncolytic ad, similar to the discontinued onyx- [ ] . a few examples of viruses that have almost reached the market are given below. cerepro ® (sitimagene ceradenovec) is an adenoviral vector containing the herpes simplex virus thymidine kinase gene cdna under the control of a cytomegalovirus promoter, manufactured by ark therapeutics ltd., for the treatment of high-grade glioma with oral ganciclovir [ ] . cerepro ® demonstrated significant efficacy in a recent phase iii trial, but a further trial is still required before approval in order to provide a sufficient level of evidence of clinical benefit [ ] . similar to gendicine tm , advexin tm (contusugene ladenovec; ing ) was developed by introgen therapeutics inc. as a replication-impaired, adenoviral vector carrying the p tumor suppressor gene under the control of a constitutive viral promoter. numerous human cancers have abnormalities in some of the molecules associated with the p pathway, contributing to tumor resistance to a variety of conventional therapeutics. preclinical data has demonstrated increased amounts of p wild-type protein after transduction with advexin tm , and phase ii and iii trials were conducted in unresectable recurrent head and neck squamous cell carcinoma [ ] . responders to the adenovirus therapy had a characteristic p profile, with either low expression of mutated p or wild-type p inactivated by upregulation of inhibitors. genetic immunotherapy was conceived to deliver immune mediators as an efficient and safe approach that also prevents the need to produce and purify large amounts of recombinant proteins [ ] . tnferade tm , developed by genvec [ ] , is a second-generation adenovirus vector containing e , e and e deletions harboring a tnf-Į gene, functionally controlled by the radiation-inducible egr- promoter. tnferade tm was successfully tested in multicenter phase ii and iii randomized controlled trials in combination with chemoradiation in patients with locally advanced pancreatic cancer [ ] . despite initially encouraging results, genvec stopped the phase iii trial in march , as an interim analysis could not demonstrate relevant evidence of effectiveness. an example of a retroviral vector in cancer gene therapy is rexin-g£, currently in clinical trials for advanced pancreatic, metastatic breast cancer, osteosarcoma and soft tissue sarcoma [ ] . rexin-g£ is a replication-incompetent, collagen-targeted vector, encoding a dominant negative mutant of the human cyclin g gene, which makes it lethal to cancer cells [ , ] . impressive results were obtained in phase i and ii clinical trials, which demonstrated unprecedented tumor control, prolonged survival and clinical remissions in late-stage cancer patients [ ] . genomic and proteomic technologies are quickly evolving to detect specific molecular targets in patient tumor samples, fulfilling the promise of a personalized treatment approach. information collected from these emerging technologies will help engineer vectors that carry therapeutic genes specifically targeted to the specificities of individual tumor properties. it is now envisioned that future cancer gene therapies will use a combination of viral and non-viral vectors tailored to meet patientspecific tumor characteristics. consequently, many research groups have focused their efforts on the generation of synthetic carriers that incorporate features that mimic the biological mechanisms of viral gene delivery. the ideal synthetic vector would incorporate a polycationic sequence to condense nucleic acids and a coating to evade the reticuloendothelial system. it would exhibit colloidal stabilization properties to prevent accumulation in the lung capillaries, and would contain specific target-cell entry, endosomal escape and nuclear localization signals. the goal is to synthetically manufacture biodegradable vectors than can be administered systemically to reach micro metastases. these carriers were initially prepared from polymers, lipids and dendrimers [ ] . the first non-viral gene therapy trial was conducted in , on patients with advanced melanoma who received intratumor injection of dna-liposome complexes [ ] . the results demonstrated for the first time the safety and feasibility of cancer treatment by gene therapy protocols using non-biological carriers. cationic polymers have demonstrated superior gene transfer properties to those of polymers having anionic or neutral charge at physiological ph. however, most clinical trials have been conducted with carriers classified as safe [ ] , such as the nonamine polymers polyvinyl pyrrolidone and poly(lacid co-glycolic acid). allovectin- tm , a registered trademark of vical incorporated (san diego, ca, usa) is a promising cancer gene therapy product formulated with a cationic lipid system. allovectin- tm contains a bicistronic plasmid encoding human leukocyte antigen-b and beta- microglobulin. this plasmid allows the immune system to recognize metastatic melanoma lesions as foreign by incorporating a mhc class i complex into the tumor through direct injection, as demonstrated in phase i/ii trials [ ] . a phase iii trial is currently being conducted to compare the efficacy of allovectin- tm to conventional chemotherapy. encouraging results were also obtained in a recent phase i trial conducted on women with recurrent, chemotherapy-resistant ovarian cancer to assess the safety and tolerability of a plasmid carrying the human gene for interleukin- plasmid formulated with a synthetic lipopolymer, polyethylene glycol-polyethyleneimine-cholesterol [ ] . currently, numerous nanostructured systems are being developed and tested in preclinical studies. for example, self-assembled nanoparticles containing sirna, carrier dna, protamine and lipids, including polyethylene glycol and a ligand, anisamide, to target cancer cells were prepared and tested by li et al. [ ] . these authors demonstrated the high efficiency of these systems in delivering genetic material to xenograft tumors after intravenous administration in athymic nude mice. folate groups have also been linked to liposomes for sirna delivery, which resulted in significant suppression of xenograft growth in mice [ ] . folate-peg-polymeric nanoparticles have also been tested in vivo for suicide gene therapy applications, using ganciclovir as a prodrug [ ] . peg-modified gelatin-based nanocarriers have been used in vivo to deliver plasmid dna encoding for the soluble form of the extracellular domain of the vascular endothelial growth factor receptor- (vegf-r or sflt- ) in antiangiogenic therapy [ ] . upon intravenous administration, overexpressed sflt- was therapeutically active as shown by suppression of the xenograft tumor growth. nanoparticles also offer the ability to monitor the delivery of genetic material. tan et al. [ ] were able to synthesize chitosan-based nanoparticles encapsulating quantum dots coupled to sirna and demonstrate efficient silencing and transfection tracking. finally, inorganic nanoparticles are also under development, which, despite their low synthesis efficiency, have the significant advantage of low toxicity and easy functionalization [ ] . for example, magnetic liposomes have also been tested in magnetic hyperthermia settings to induce therapeutic tnf-α expression driven by the promoter of the stress-inducible gadd gene [ ] . the combined thermal and gene therapy treatment significantly arrested tumor growth in nude mice, which encouraged the refinement of this type of cancer gene therapy, which was then successfully tested in preclinical studies [ ] . after more than two decades of cancer gene therapy using biological vectors, preclinical studies yielded excellent results and clinical trials reported satisfactory results in terms of reporting mild or no long-term toxicity. however, a real breakthrough cannot be claimed in clinical therapy. the reasons for the different outcomes of preclinical and clinical trials include the inherent limitations of rodent models, which develop homogeneous tumors arising from clonal cell lines, while tumors found in clinical practice are composed of heterogeneous cell types. the therapies described in sections and also confronted similar limitations during their development. the main players in gene therapy, vectors and transgenes, will evolve to achieve the highest possible degree of specificity for targeting cancer cells. nanotechnology has already engineered powerful non-biological carriers of a variety of therapeutic genes that have demonstrated efficacy and safety in preclinical tests. since current knowledge of cancer cell biology is far from complete, ongoing and future clinical trials with these synthetic systems are expected to suffer similar drawbacks in terms of efficacy as those experienced with viral gene therapy systems. as we have seen from other therapies that have already been incorporated into the clinical routine of cancer treatment, the success of cancer gene therapies will be preceded by many failures, which will likely be due to a greater extent to our technological limitations than to flaws in their general concept. this review has tried to summarize the history and evolution of the most common types of cancer treatments available today, but also new therapies under study in the last years. in addition to surgery, chemotherapy, radiation therapy, hyperthermia, photodynamic therapy or immunotherapy, new therapies are now at different stages of development trying to decrease drug toxicity in health tissues and increase efficacy by targeting tumor angiogenesis, by exploring cell and gene therapy, or by using new nanostructures for diagnosis or therapeutic purposes. nanotechnology is offering new products, which either used alone, due to their intrinsic properties, or in combination with other biomolecules (anti-tumoral drugs, folic acid, albumin, antibodies, aptamers) could be used to target cancer cells. however, the history tells us that the fight against cancer is not an easy task. many types of cancers are able to resist to conventional therapies, and different combinations of drugs and therapies (e.g., surgery together with radiotherapy and chemotherapy) are usually the only way to destroy tumoral cells. this may be also true for the new therapies arriving now to the clinic. much more studies are required but these new ways of treatment are opening doors to hope for many patients waiting for a successful therapy. symposium on advances in pharmacology resulting from war research: therapeutic applications of chemical warfare agents nitrogen mustard therapy: use of methyl-bis (h-chloroethyl) amine hydrochloride and tris (h-chloroethyl) amine hydrochloride for hodgkin's disease, lymphosarcoma, leukemia, and certain allied and miscellaneous disorders 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predict p gene therapy efficacy in recurrent, squamous cell carcinoma of the head and neck improvement of different vaccine delivery systems for cancer therapy translation of the radio-and chemo-inducible tnferade vector to thetreatment of human cancers molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von willebrand factor inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin g construct noteworthy clinical case studies in cancer gene therapy: tumor-targeted rexin-g advances as an efficacious anti-cancer agent gene delivery with synthetic (non viral) carriers direct gene transfer with dna-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans handbook of pharmaceutical excipients a phase study of high-dose allovectin- in patients with advanced metastatic melanoma phase-i clinical trial of il- plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer tumor-targeted delivery of sirna by self-assembled nanoparticles folate-linked lipid-based nanoparticles for synthetic sirna delivery in kb tumor xenografts folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase antiangiogenic gene therapy with systemically administered sflt- plasmid dna in engineered gelatin-based nanovectors quantum-dot based nanoparticles for targeted silencing of her /neu gene via rna interference heat-inducible tnf-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy targeted hyperthermia using magnetite cationic liposomes and an alternating magnetic field in a mouse osteosarcoma model we greatly appreciate the support from grants mat - -c - from the ministerio de ciencia e innovación (ministry for science and innovation, spain), fundación mutua madrileña, ibercaja, fundación ramón areces and the ramón y cajal program. n.v. is supported by program i sns from the fondo de investigaciones sanitarias (healthcare research fund, spain). m.a. is especially indebted to the graduate students and researchers of ina for providing some of the images in figure . we also greatly appreciate the support from the inbiomed ( / , xunta de galicia), immunonet (soe /p /e , immunotherapy network, sudoe-feder) and hinamox ( ° eu program) projects. we thank josé me, r. esteban and a. esteban for their help in compiling data. cancers , the authors declare no conflict of interest. key: cord- -wjkbrfkn authors: schmidt, rüdiger; scheuermann, ernst-heinrich; viertel, achim; geiger, helmut; scharrer, inge title: antiphospholipidantikörpersyndrom date: journal: med klin (munich) doi: . /bf sha: doc_id: cord_uid: wjkbrfkn □ background: antiphospholipid antibodies comprise a family of auto-antibodies mainly characterized by the presence of the lupus anticoagulant (la) and anticardiolipin antibodies (aca). □ clinical appearance: the antiphospholipid antibody syndrome is defined by the appearance of frequent thromboses, repeated fetal losses and thrombocytopenia. other clinical manifestations associated with apa include migraine, chorea, hemolytic anemia, heart valve disease, budd-chiari syndrome, perpetual pancreatitic episodes, intestinal infarctions, malignant hypertension, livedo reticularis, pre-eclampsia, fetal growth retardation or catastrophic antiphospholipid syndrome. la and aca occur in a variety of clinical conditions (secondary antiphospholipid antibody syndrome, saps), including other autoimmune disorders, infectious diseases, neoplastic disorders, in association with the use of certain drugs or in otherwise healthy individuals (primary antiphospholipid antibody syndrome, paps). □ treatment: patients with thrombosis associated with apa should receive long-term anticoagulation therapy, whereas treatment of asymptomatic patients seems to be not indicated, because only approximately % of patients with apa may develop thrombotic complications. in patients with paps there is no evidence that the prophylactic administration of immunosuppressive drugs will prevent thromboembolic events. kfidiger schmidt i , ernst-heinrich scheuermann ~ , achim viertel , helmut geiger , lnge scharrer budd-chiari-syndrom, rezidivierende pankreaufiden, intestinale apoplexie, maligne hypertonie;livedo reticularis, pr~ieklampsie, fetate wachstumsverzsgenmgen oder das sogenannte,,katastrophale antiphospholipidantik rpersy ndrom,_ ira gegensatzzum ,prim~en antiphospholipidantik rpersyndrom'" rinden sich lupusantikoagulanziea und an¡ beim ,,se-kund~iren antiphospholipidantik/srpersyndrom" im t(ahmen ron autoimmun-erkrankutlget , von.neoplasfen und infektionen oder sind mit dem gebrauch bestimmter medikamente assoziiert. therapie: die behandlung asymptomatischer patienten ist nicht indiziert, da nur bis % der patienten mit positivem nachweis ron antiphospholipid-antik rpern thromboembolische komplikationen entwickeln. in letzteren f~uen umfabt die konservative therapie eine zeitlich nicht limifierte orale antikoagulation. eine zus~itzliche prophylaktische therapie mit irnmunsuppressiva fª bei patienten mit prim~irem antiphospholipidantik rpersyndrom zu keiner verminderung thromboembotischer komplikationen. [] clinical appearance: the antiphospholipid antibody syndrome is defined by the appearance offrequent thromboses, repeated fetal losses and thrombocytopeina. other clinical manifestations associated with apa include migraine, ch rea, hemolytic anemia, heart valve disease, budd-chiari syndrome, perpemal pancreatitic episodes, intestinal infarcfions, malignant hypertension, livedo reticularis, pre-eclampsia, fetal growth retardation or catastrophic anfiphos- [ ] ; dieses zeigt zudem eine fa-mili~ire h~iufung [ ] sowie eine assoziation mit hla dr. , dr , dqw und dr.w [ ] . je nach art des antiphospholipidan-tik rpers unterscheidet man ein lupusantikoagulanssyndrom (las) vom anticardiolipinantik rpersyndrom (acas) [ ] , welche jeweils prim~irer oder sekund~irer genese sein k nnen. ven se thrombosen pr igen ª gend das klinische bild beim prim iren lupusantikoagulanssyndrom [ ] , hingegen stehen beim sekun&iren, meist mit einem systemischen lupus erythematodes assoziierten lupusantikoagulanssyndrom eher herzklappenver~inderungen, erniedrigte komplement-c -spiegel sowie neutropenien im vordergrund [ ] . je nach organmanifestation kann das anticardiolipinan-photipid s mdome. la and aca occur in a variety o(ctinical conditions (secondar? antiphospholipid antibody syndrome, saps), including other autoi;nmunr disorders, infectious diseases, neoplastic disorders, in association with the use ofcertain dmgs or in ottaerwlse healthy indixdduals (primary andphospho-[ipid andbody syndrome, paps). ~itreamwnt: patients with thrombosis associated witt,, apa should receive long-term anticoagulation therapy, whereas r o f asymptomatic patients see¡ to be not indicated, bec'mse, onlv appr¢ %:. ,. ofpatients with:apa ma,,." d.evelop thrombotic r in patients with paps.there is no evidenc› that,the prophylactic admmistration ,qflmmunosuppressive drugs wiª prevent thromboembolic events zidenz schwankt zwischen % [ ] und -wohl realistischer - % [ ] . im gegensatz dazu betr igt die pr~ivalenz ron lupusantikoagulans in der normalbev kerung zirka bis %, wobei ª junge frauen betroflen sind [ ] . bei % der patienten mit systemischem lupus erythematodes k nnen anticardiolipinantik rper, bei % der f~ille lupusantikoagulans beobachtet werden. allerdings sind knapp die hkilfte aller patienten mit antiphos-pholipidantik rpern nicht an einem systemischen lupus erythematodes erkrankt. einem breiten erregerspektrum k nnen periinfekti s auftretende antiphos-pholipidantik rper assoziiert sein. vaarala et al. [ ] in der ersten gruppe scheint der nachweis eines lupusantikoagulans mit einem h/sheren thromboserisiko assoziiert zu sein ( % versus %) als beim nachweis eines anticardioli-pinantik rpers ( % versus %). das thromboserisiko steigt in diesem kollektiv linear mit der h he der anti-phospholipidantik rpertiter an [ ] . bei patienten ohne zeichen eines lupus erythematodes korreliert der nachweis von lupusantikoagulanzien, nicht jedoch der ron anticardioli-pinantik rpern mit dem auftreten ven ser thromboembolien [ ] . neurologie ganz im vordergrund stehen zentrale isch imien. das klinische spektrum reicht dabei von einzelnen umschriebenen herden bis hin zu einem multiinfarktsyndrom [ ] mit konsekutiver demenz [ ] . erneut erlaubt hier der nachweis von antiphospholipidanti-k rpern aussagen zur prognose: lassen sich im kahmen des ersten zentral-isch~mischen insultes antiphospholi-pidantik rper nachweisen, so liegt das rezidivrisiko f'ª einen schlaganfall bei , % pro jahr und fª eine transitorische isch~imische attacke bei , % [ ] . obwohl sich kein lestes verh~iltnis zwischen der antiphospholipidantik r-pertiterh he und dem isch~imierisiko aufzeigen l~iflt, so korreliert dennoch die antiphospholipidantik rpertiterh he direkt mit der schwere des neurologischen defizits [ ] . besonders bei ]ungen frauen sollten migr~ineartige zephalgien ah das vorliegen eines anti- [ ] . so liel sich zum beispiel bei knapp % der patienten mit prim~irem anti-phospholipidantik rpersyndrom zumindest eine beinvenenthrombose in der vorgeschichte evaluieren, wovon wiederum die h~ilfte der patienten zu-s~itzlich eine lungenembolie erlitten hatte [ ] . das auftreten einer pulmonalen hypertonie als folge rezidivierender lungenembolien ist bislang nur in wenigen einzelf• beschrieben worden [ ] . m glicherweise wird die inzidenz dieser komplikation unter-sch~itzt, da sturfelt et al. [ ] bei immerhin einem fª ihrer patienten einen erh hten pulmonalarteriendruck hatten nachweisen k nnen. zum vergleich: bei % der patienten mit sogenannter ,,idiopathischer pulmonaler hypertonie" sollen sich antiphospholi-pidantik rper rinden lassen [ ] . in einzelf• wurde die assoziation eines prim~iren antiphospholipidanti-k rpersyndroms mit einem ,,adult respiratory distress syndrome" (ards) beschrieben [ ] . bei schwerer pulmonaler hypertonie aufdem boden eines prim~iren antiphospholipidantik rpersyndroms er ffnet sich m glicherweise mit der langzeitgabe von iloprost ein erfolgversprechendes therapiekonzept [ ] . kardiologie unabh~ingig von einer pr~iexistenten koronaren herzerkrankung werden sowohl thrombosen der groben epikar-dialen gef~be [ ] als auch eine diffuse thrombotische mikroangiopathie [ , ] beobachtet. aus der synopsis mehrerer studien [ , , [ ] [ ] [ ] l~iflt sich eine infarktinzidenz von , % ableiten. demgegenª berichteten hamsten et al. [ ] von einem positiven anticardio-lipinantik rpemachweis bei einem fª jª infarktpa¡ w~ihrend einer nachbeobachtungsperiode von dreijahren kam es bei der h~fte der anticardiolipinantik rperpositiven patienten emeut zu arteriellen thrombosen. morton et al. [ ] beschrieben eine positive korrelation zwischen der anti-phospholipidantik rpertiterh he zum zeitpunkt einer aortokoronaren bypassoperation und der inzidenz von bypassverschlª zw f monate sp~iter. in-trakavit~ire thromben -ª rechtskardial angesiedelt -k nnen zu lebensbedrohlichen komplikationen anlai geben [ ] . obgleich nicht unwidersprochen [ ] , so rinden sich sowohl beim prim~iren [ , , ] [ ] . neben der orden antikonzeption repriisentieren antiphospholipidantik rper die zweit-h iurigste benigne ursache [ ] des budd-chiari-syndroms [ , ] . bei patienten mit fulminantem leberversagen lassen sich in der mehrzahl der fs le anticardiolipinantik rper der klasse iga nachweisen, wobei zwischen der anticardiolipinantik rpertiterh he und dem residualen leberparenchym eine inverse korrelation beobachtet wurde [ ] . ebenso signirikant soll die korrelation zwischen antiphospholipid-antik rpem und dem vorliegen einer alkoholhepatitis bzw. einer ~ithyltoxischen leberzirrhose sein [ ] . kontrollbedª erscheint der mithin ª befund zu sein, wonach sich bei patienten mit hepatitis c ira verlaufeiner ~-interferon-therapie unter anderem vermehrt antiphospholipi-dantik rper haben nachweisen lassen. ursache des sogenannten ,,katastrophalen antiphospholipidsyndroms", dessen letalit~it % ª gen soll [ , ] , ist nicht bekannt. pathophysiologisch handelt es sich beim katastrophalen antiphospholipidsyndrom um akut auffretende, diffuse organthrombosen [ , l. klinisch rinden sich die zeichen des multiorganversagens mit akutem nieren-und leberversagen, einem ,,adult respiratory distress syndrome", darmnekrosen, lactatacidose, thrombopenie und diversen neurologischen auff• insgesamt also ein bild, das von einem fulminant septischen schockgeschehen oder einer thrombotischen mikroangiopathie (morbus gasser, morbus moschcowitz) nur schwer zu unterscheiden ist. besonderes augenmerk sollte auf die m glichkeit eines postpartalen auftretens eines katastrophalen antiphospholipidsyndroms gerichtet werden [ ] . antiphospholipidantik rper lassen sich bei einem dfittel aller frauen mit rezidivierenden spontanaborten in der vorgeschichte nachweisen [ ] . knapp ein viertel aller patientinnen mit systemischem lupus erythematodes erleidet eine oder mehrere spontanaborte [ ] ; dieses p.,isiko steigt auf bis % [ ] , wenn sich antiphos-pholipidantik rper nachweisen lassen, und f:illt auf %, wenn sich antiphos-pholipidantik rper nicht rinden lassen. zwischen der antiphospholipid-antik rpertiterstufe -hier insbesondere dem anticardiolipinantik rpertiter [ ] -und der p,.ate an fehl-und frª sowie der h?iurigkeit und dem ausmafl fetaler wachstums-verz gerungen besteht eine lineare korrelation [ , ] . pathophysiologisch stehen rezidivierende plazentathrombosen mit nachfolgender relativer oder absoluter plazentainsuffizienz ganz ira vordergrund dieses geschehens [ , ] . bemerkenswert erscheint die beobachtung, wonach diaplazentar ª antiphospholi-pidantik rper in der lage zu sein scheinen, bereits beim f tus schwerste thrombotische komplikationen zu verursachen [ ] . als h~iurigste materne komplikation wird die eklampsie besch¡ [ ] . daneben rinden sich einzelbe¡ iiber die pe¡ assoziation mit elneto hellp-syndrom [ ] . auch die koinzidenz mit einem hiimolytisch-urlimischen syndrom [ ] oder mit einem katastrophalen anti- in etwa bis % ist mit dem primiiren bzw. dem sekundiiren antiphospholipid-antik rpersyndrom eine thrombozytopenie assoziiert [ , , , ] . in der gruppe der antiphospholipidantik rperpositiven patienten mit systemischem lupus erythematodes ist eine thrombozytopenie in % der f~ille mit einem anticardiolipmantik rper, in % der l::~ille hingegen mit dem lupusantikoagulans assoziiert; ohne den nachweis eines antiphospholipidanti-k rpers bleibt cine thrombozytopenie die ausnahme und liil t sich nur in )% der f~ille nachweisen. ob den antiphospholipidantik rpcrn eine alleinige r.olle in der atiologie der thrombozytopenie zukommt, erscheint fraglich und bedarf weiterer studien [ ] . selten kann eine assoziation mit einer hypoprothrombin~imie bestehen [ ] . auch soll das evans-syndrom eine weitere manifestation des antiphospholipidsyndrom darstellen [ ] . auch ohne hochdosierte cortisonmedikation kann es beim antiphospholipidsyndrom zur polytopen entwicklung avaskul~irer, thrombotischer knochennekrosen kommen; hª krosen stellen den pr~iferierten pfiidilektionsort dar [ , ] . nephrologie hiiurig ist mit dem verlust der betroffenen niere eine ein-oder beidseitige nierenvenenthrombose, eine typische komplikation des antiphospholipidsyndroms, verbunden. das ausmab, ob diffus oder fokal, sowie die geschwindigkeit des auftretens einer thrombotischen mikroangiopathie des renalen gef.'if bettes entscheiden ª den grad der niereninsuffizienz und erlauben progmostische aussagen. so rindet man im zusammenhang mit einem antiphospholipidsyndrom sowohl die entwicklung einer chronischen niereninsufrizienz als auch rapid progressive verlkiufe, die rasch in der terminalen, dialysepflichtigen niereninsuffizienz enden [ ] . in der differentialdiagnostik des nephrotischen syndroms wie in der der malignen hypertonie sollte nicht zuletzt an ein antiphospholipidsyndrom gedacht werden [ , ] . zwischcn • koagulans in diesem patientenkollektiv signifikant mit dem auftreten von fistelthrombosen korreliert zu sein [ ] . trotz ausreichender antikoagulation mub auch nach nierentransplantation mit rezidivthrombosen im transplantat und konsekutivem transplantatverlust gerechnet werden [ ] . zwischen dem nachweis von antiphospholipid-antik rpem und der zahl an ab-stobungsepisoden, der transplantatverlustrate bzw. der nierenfunktion ein jahr nach transplantation scheint hingegen kein zusammenhang zu bestehen [ ] . der isolierte nachweis von an¡ pholipidantik rpern stellt nach heutiger einschstzung keine indikation zur therapie dar, da nicht alle antiphospholipid-antik rperpositiven patienten thromboembolische komplikationen entwickeln und zudem die genauen pathogenetischen mechanismen der antiphospholipidsyndrome noch nicht bekannt sind [ ] . haben sich allerdin~ thromboembolische komplikationen ergeben, ~indert sich diese einsch~itzung. die konservative therapie um-fabt in diesen f~illen eine antikoagulation mil heparin bzw. cumarinderivaten und/oder eine hemmung der thrombozytenaggregation mit ass [ ], wobei die spontan verllingerte ptt ein ,,heparinmonitoring" nicht unerheblich erschweren kann [ ] . handelt es sich um ein sekund~res antiphospholipidsyndrom -l~bt sich zum beispiel eine ausl sende kollagenose diagnostizieren -, so stellt die immunsuppression sicherlich mit die therapie der ersten wahl dar, eine option, die auch dann gerechffertigt zu sein scheint, wenn eine alleinige antikoagulation zur vermeidung weiterer thromboembolischer komplikationen nicht ausreicht. an dieser stelle bedarf es allerdings des hinweises auf den zur zeit fehlenden konsens hinsichtlich einer behandlung mit steroiden und/oder zytotoxischen medikamenten, mil der hochdosierten gabe von immunglobulinen oder der indikation zur plasmapherese oder immunadsorption [ ] . reicht eine isolierte hemmung der thrombozytenfunktion oder die intermittierende gabe von (fraktioniertem oder unfraktioniertem) hepa-rin zur vermeidung weiterer thrombotischer kompiikationen nicht aus, verbleibt die orale antikoagulation mil cumarinderivaten (ziei-inr , bis , ) als mittel der wahl [ , ] . da die individuelle thrombosegef~ihrdung nicht mit der aktueuen antiphospholi-pidantik rpertiterstufe korreliert und weiterhin gezeigt werden konnte, dal das thromboserisiko unmittelbar nach absetzen sowohl der oralen antikoagulation [ ] als auch von ass [ ] steil nach oben steigt, sollte weder die orale antikoagulation noch die einnahme von ass einer zeitlichen begrenzung unterliegen. literatur. el al antiphospholipid antibodies and the anti-phospholipid syndrome in systemic lupus erythematosus antiphospholipid antibodies, haemostatic variables and thrombosis 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the lupus anticoagulant in male pafients, ja uber die entwicklung und den gegen-w~irtigen stand der serodiagnosfik gegenª syphilis heparin monitoring in acute thrombosis associated with antiphospholipid antibody syndrome immunologic and coagulation disordcrs in chlorpromazine-treated patients sneddon's syndrome. a long-term follow-up of pa¡ arch der-mato key: cord- -qevosik authors: selvarajah, suganya; sexton, nicole r.; kahle, kristen m.; fong, rachel h.; mattia, kimberly-anne; gardner, joy; lu, kai; liss, nathan m.; salvador, beatriz; tucker, david f.; barnes, trevor; mabila, manu; zhou, xiangdong; rossini, giada; rucker, joseph b.; sanders, david avram; suhrbier, andreas; sambri, vittorio; michault, alain; muench, marcus o.; doranz, benjamin j.; simmons, graham title: a neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e protects from disease date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: qevosik the mosquito-borne alphavirus, chikungunya virus (chikv), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to . million cases of acute and chronic rheumatic disease. there are currently no licensed vaccines for chikv and current anti-inflammatory drug treatment is often inadequate. here we describe the isolation and characterization of two human monoclonal antibodies, c and e , from chikv infected and recovered individuals. c was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact chikv vlps. shotgun mutagenesis alanine scanning of percent of the residues in the e and e glycoproteins of chikv envelope showed that the epitope bound by c included amino-acid in the acid-sensitive region (asr) of the chikv e glycoprotein. the asr is critical for the rearrangement of chikv e during fusion and viral entry into host cells, and we predict that c prevents these events from occurring. when used prophylactically in a chikv mouse model, c completely protected against chikv viremia and arthritis. we also observed that when administered therapeutically at or hours post-chikv challenge, c gave % protection in a pathogenic mouse model. given that targeting this novel neutralizing epitope in e can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against chikv. chikungunya virus (chikv) is a mosquito-borne alphavirus first isolated in tanzania in [ ] that has caused sporadic outbreaks of predominantly rheumatic disease every - years, primarily in africa and asia. the largest epidemic of chikv disease ever recorded took place during - , and involved an estimated . to . million cases and the first autochthonous chikv infections in europe (italy in and france in ) [ , ] . imported cases were also reported in nearly countries, including european countries, japan, and the usa. the epidemic was associated with the emergence of a new clade of viruses, which were efficiently transmitted by aedes albopictus, a mosquito vector that has seen a dramatic global expansion in its geographic distribution [ ] [ ] [ ] . chikv disease is characterized by acute and chronic polyarthritis/polyarthralgia, which is usually symmetric and often incapacitating and occasionally protracted [ ] [ ] [ ] [ ] [ ] . other symptoms, such as fever, rash, myalgia, and/or fatigue, are often also present during the acute phase. the recent epidemic was also associated with atypical and severe clinical forms of chikv disease and some fatalities, which appeared to be restricted to the very young and elderly patients with comorbidities [ , ] . chikv virions contain three major structural proteins: glycosylated el and e envelope (env) proteins embedded in the viral membrane, and a non-glycosylated nucleocapsid protein. based on similarity to other alphaviruses, e mediates receptor attachment, while e is a class ii viral fusion protein. a third glycoprotein, e , is associated with mature virions in some alphaviruses [ ] , but not others [ ] , while k protein, a membrane-associated peptide created by cleavage of the poly-protein to release e and e , is incorporated into particles at a low level [ , ] . the organization of alphavirus surface glycoproteins in virions has been defined using cryo-electron microscopy (cryo-ems) [ ] , while the atomic structure of chikv glycoproteins was recently solved by x-ray crystallography [ ] , both for mature particles and for immature p env precursor polyprotein prior to furin cleavage. copies each of three glycoproteins (e /e /e ) come together to form a protein coat with icosahedral symmetry and containing spikes [ ] . the folding, transport to the surface and function of these glycoproteins relies on their correct interactions with each other. e consists of three b-sheet domains, termed i, ii and iii, while e contains three immunoglobulin-like domains (a, b and c, with a being at the n-terminus). in the complex, domain b lies at the membrane distal end and contacts e , domain c is closest to the viral membrane and domain a is in the center. e interacts laterally with e all along domain ii, along with additional points of contact from other regions of e . e contains an internal fusion loop at the tip of domain ii, which in the mature structure exists as a b-hairpin lodged in a groove between domains a and b of e [ ] . e also plays a role in protecting the fusion loop from premature exposure. treatment of chikv rheumatic disease usually involves nonsteroidal anti-inflammatory drugs (nsaids) and/or simple analgesics, which can provide relief but is often inadequate [ ] . although a number of vaccine strategies have been, or are being, explored [ ] [ ] [ ] [ ] , there are currently no licensed human vaccines [ ] . nevertheless, it is clear that chikv neutralizing antibodies from infected humans or vaccinated monkeys can mediate protection prophylactically, or soon after exposure. polyclonal immunoglobulins derived from humans recovered from chikv infection, when passively transferred into neonatal and interferon a/b receptor deficient (ifnar / ) mice, protected these animals from chikv-induced viremia and mortality [ ] . purified total igg from monkeys immunized three times with a chikv viruslike-particle vaccine (containing e and e ) similarly protected ifnar / mice from chikv viremia and mortality [ ] . a recent study described two monoclonal antibodies (mabs), f and b , which were isolated from chikv infected individuals. these mabs specifically neutralized chikv and o'nyong'nyong virus (onnv, a virus closely related to chikv), but none of the other alphaviruses tested [ ] . the f and b mabs, when used in escape mutant studies were shown to recognize key residues in e (v ) and e (t ), respectively [ ] . the combination of f and b were also shown to significantly delay chikv-driven lethality in mice deficient in ifna/b and ifnc receptors, and mature b and t cells [ ] . similarly, a group of mouse-derived mabs, clustering close to the putative receptorbinding domain of e [ ] , were also found to be protective to various degrees in mouse models of chikv [ ] . additionally, an immunodominant linear epitope at the n-terminus of e is also a target for protective antibodies [ ] . herein, we describe the isolation and characterization of two human mabs, c and e , from patients that were infected with chikv and recovered. we also report the characterization of antibody binding epitopes using a library of alanine scanning mutants of chikv envelope covering residues (. %) of the chikv e and e glycoproteins. although the binding epitopes for c and e both mapped to chikv e , c was able to potently neutralize chikv, while e was not. the neutralizing epitope bound by c mapped to the acid-sensitive region (asr) that is critical for the rearrangement of chikv e during fusion and viral entry into host cells. purified human c antibody, when used prophylactically in an adult wild-type mouse model of chikv disease, completely protected against viremia and arthritis. written informed consent was obtained from recovered chik donors in italy and reunion, france and collection complied with relevant human subjects research protocols approved by the institutional review boards of the university of bologna and the centre hospitalier universitaire, respectively. animal work was conducted in accordance with good animal practice (nhmrc, australia), and was approved by the qimr animal ethics committee. additional murine studies were performed at blood systems research institute with approval of the institutional animal care and use committee at isis services llc (san carlos, ca) and following the recommendations of the national research council's institute of laboratory animal resources as published in their guide for the care and use of laboratory animals. chikv mab c variable chains were sequenced by mc labs (south san francisco, ca). for mammalian expression, c variable heavy (vh) and light (vl) chain cdnas were synthesized by genscript (piscataway, nj). the closest human germline signal sequences (ss), vh a and vkiii a , were used to ensure efficient processing and secretion. ss-vh cassettes were cloned into a pcaggs mammalian expression vector as ecori-nhei fragments, upstream of the human igg heavy chain constant region. ss-vl cassettes were cloned as ecori-bsiwi fragments upstream of the human kappa light chain constant region. chikv fab cap a.e variable heavy and light chain cdnas bearing human il- signal sequences were synthesized by genscript. il- ss-vh and il- ss-vl cassettes were cloned as mfei-nhei and mfei-bsiwi fragments upstream of their respective constant regions, as described above. chikv is characterized by acute and chronic polyarthritis/ polyarthralgia that can be debilitating and protracted. currently there are no fda-approved vaccines or specific antiviral treatments for chikv. we thus identified and characterized human monoclonal antibodies directed against chikv that could be utilized in prophylactic and immediate post-exposure settings. such patient derived monoclonal antibodies could also provide useful information on critical antigens and epitopes for development of future vaccines and other biologics. we describe here the identification of two monoclonal antibodies (c and e ) isolated from recovered patients. c potently inhibited chikv infection in cells and prevented viremia and arthritis in a mouse model of chikv disease. the epitope for this antibody includes an amino-acid residue in a key acidsensitive region of the e glycoprotein of chikv. rearrangement of this region following exposure to low ph is critical for uncovering portions of the companion e glycoprotein, required for successful entry of chikv into cells. we hypothesize that binding of antibodies to this region stabilizes the native complex and thus prevents such rearrangements. chikv wild-type envelope pseudovirion production chikv envelope (e /e /e ) in a pcaggs vector was used for pseudoparticle preparation as described previously [ ] . lentiviral pseudotypes were produced essentially as described [ ] by using mg of luciferase reporter plasmid, (pnl-luc, based on pnl - -r-e-) [ ] and mg of plasmid encoding viral envelope. virions were concentrated by ultracentrifuge concentration at , g in a sw rotor (beckman) through a % sucrose cushion for . h at uc. the pellets were resuspended overnight in hbss at uc.vsv-g and alphavirus envelopes expressing the rrv, sfv and sinv were used as controls for pseudovirion neutralization assay [ ] . chikv wild-type pseudovirus neutralization assay hek t cells were plated at cells/well in dmem (hyclone) containing additives and incubated at c in % co overnight. the following day, serial dilutions of antibody and virus pre-incubated for min were added to the hek t cells. a spin infection was performed at , g for min and cells incubated for an additional hours at uc. the antibody-virus mix was removed by aspiration and replaced with ml of prewarmed fresh media. the cells were incubated for hrs before samples were recovered for measurement of luciferase activity in the cell lysates as per manufacturers protocol (promega). chik wild-type virus production, plaque assay and % prnt assay chikv was obtained from atcc (atcc # vr- ), from a strain originally isolated in from the serum of a patient in east africa and expanded in suckling mice. replication competent chikv was grown in vero cells. vero cells ( . ) were plated in a -well (costar) plate overnight. serial dilution of the virus stock ( ml) was incubated with cells for hr at uc. one hour after incubation, an overlay of % agarose (life technologies) in dmem supplemented with % fbs was added to cells and incubated at uc for hrs. subsequently, wells were fixed with % formaldehyde and stained with . % crystal violet in methanol: ethanol. plaques were counted against a white background. vero cells ( . ) were plated in a -well (costar) plate overnight. serially diluted monoclonal antibodies were mixed with chik live virus diluted to pfu/ml and pre-incubated for an hour at uc. following this ml of the antibody-virus mixture was added to the confluent vero cell monolayer for an additional hour. subsequently, the virus was removed and an overlay of % agarose in dmem supplemented with % fbs was added and cells were incubated at uc for hrs. the plaques were stained and counted as described above. the prnt titer is calculated as the reciprocal of the serum dilution, where $ % reduction in the number of plaques is compared to the negative control in the presence of media and no mabs. the pbmcs for ebv transformed b cell isolation were obtained from two chikv infected and recovered individuals. b cells were isolated using the miltenyi macs switched memory b cell isolation kit ( - - ) according to the manufacturer's protocol. the cells were plated at cells per well in u-bottom plates. pbmc from unrelated donors were treated with mitomycin c and used as feeder cells at cells per well. the cells were cultured in rpmi supplemented with % fbs, iu/l il- (roche) and . mg/ml r peptide (invivogen) [ ] . filtered b - ebv supernatants (diluted in ) were added to each well and incubated for one week before being replaced with fresh media. ebv transformed b cell supernatants expressing chikv specific antibodies were screened for chikv pseudotype neutralization potential [ ] . the cells from the positive wells were clonally isolated by limiting dilution followed by expansion and cloning. an immune fab phage display library was constructed from peripheral blood donated by three chikv-infected individuals. all three individuals were infected in réunion island, france, during the outbreak. peripheral blood samples were drawn - years after infection and serum was analyzed for the presence of neutralizing antibodies using chikv reporter virus pseudotypes. total rna was prepared using tri-reagent (sigma) with standard protocols. rna was converted to cdna using super script first-strand synthesis system for rt-pcr (invitrogen) following the manufacturer's instructions. construction of the library was performed by genscript (piscataway, nj) as previously described [ ] . the final library was transformed into e.coli tg cells (invitrogen) using electroporation, and the quality of the library was assessed by sequence analysis of randomly picked clones. all biosensor studies were performed at uc using a fortebio octet red biosensor system (fortebio, menlo park, ca). chikv vlps were loaded onto amine-reactive biosensor tips (ar g) using an immobilized human antibody against chikv (e d . , a gift from dendritics, lyon, france). briefly, ar g tips were activated for minutes with a mixture of mm edc ( -ethyl- -( -dimethylaminopropyl)carbodiimide hydrochloride, sigma, st. louis, mo) and mm sulfo-nhs (n-hydroxysulfosuccinimide, sigma, st. louis, mo) in water. e d . diluted to mg/ml in mm sodium acetate, ph . , was allowed to react for minutes and then the tips were deactivated for minutes with m ethanolamine (sigma, st. louis, mo). after a brief rinse, chikv vlps diluted to mg/ml were loaded for minutes followed by a minute stabilization. tips were then transferred to pbs buffer supplemented with mg/ml bsa (pbs-b) for subsequent antibody binding studies. c was prepared as a twofold serial dilution (starting at mg/ml) plus buffer blanks. antibody association was measured for minutes followed by a minute dissociation in buffer. non-specific binding was assessed using sensor tips without vlps. data analysis was performed using octet data analysis v . (fortebio, menlo park, ca). binding kinetics were analyzed using a standard : binding model. a chikv env expression construct (s strain) with a cterminal v tag was subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library. primers were designed to mutate each residue within the e , k, and e regions of env to alanine, while alanine codons were mutated to serine. in total, chikv env mutants were generated ( . % coverage), sequence confirmed, and arrayed into -well plates. each env mutant was transfected into hek- t cells and allowed to express for hrs. cells were stained for h with human mabs c ( . mg/ml), e ( mg/ml), ckv ( . mg/ml, isolated from phage display library in identical manner to e ), e d . ( . mg/ml, a gift from dendritics), and rabbit polyclonal antibody ( : dilution, a gift from ibt bioservices) diluted in % ngs (sigma). mabs were detected using . mg/ml alexafluor -conjugated secondary antibody in % ngs (jackson immunoresearch laboratories) for h. mean cellular fluorescence was detected using the intellicyt high throughput flow cytometer (htfc, intellicyt). antibody reactivities against each mutant env clone were calculated relative to wild-type env protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type env-transfected controls. mutations were identified as critical to the epitope if they did not support reactivity of the test human mab, but did support reactivity of the other chikv antibodies. this counter-screen strategy facilitates the exclusion of env mutants that are misfolded or have an expression defect [ ] . critical amino acids required for antibody binding were visualized on chikv env crystal structures (monomer pdb id # n and trimer pdb id # xfc, [ ] ), to obtain d epitope maps. female c bl/ mice ( weeks old) were inoculated with chikv as described previously [ ] . briefly, mice were inoculated with chikv { log % cell culture infectivity dose (ccid )} in ml rpmi (supplemented with % fetal calf serum) by shallow subcutaneous injection into the top, towards the lateral side, of each hind foot in the metatarsal region, injecting toward the ankle. mice (n = mice per group) were injected with (i) pbs; (ii) purified c mab or (iii) purified control human mab at . mg/mouse by the intraperitoneal route one day (day ) prior to infection on day with chikv. in order to avoid stimulating non-specific immune responses that may interfere with chikv infection of adult mice [ ] , c and control antibodies with endotoxin levels below eu/mg were used. arthritis was monitored by measuring the height and width of the metatarsal area of the hind feet using digital calipers [ ] . the data is presented as a group average of the percentage increase in foot height width for each foot compared with the same foot on day . viremias were measured by collecting ml of blood from a tail vein into . -ml minicollect serum separation tubes (greiner bio-one gmbh, kremsmunster, austria). the tubes were spun at , g for . min on a bench-top microcentrifuge. serum was collected and viral titers were determined as described previously and expressed as ccid per ml [ ] . the ability of antibodies to protect against the lethal effects of chikv infection were evaluated in a murine model as previously described [ ] . all animal experiments were performed with approval of the institutional animal care and use committee at isis services llc (san carlos, ca). briefly, c bl/ j mice were purchased from jackson laboratories (sacramento, ca) and bred at bsri. breeder pairs were housed under specific-pathogen free conditions in micro-isolator cages (innovive inc., san diego, ca). mice were checked daily and the date when litters were first observed was considered day . on day , litters with their mothers were transferred to static disposable cages (innovive inc.) and transferred a bsl- facility for infection and treatment. neonatal c bl/ j mice were infected with pfu of chikv (s strain) intradermally in the ventral thorax. some mice were also intraperitoneally injected with c or control human igg/mouse in . ml phosphate-buffered saline (pbs) immediately prior to chikv infection. the control igg used in this experiment was purified igg from human serum (sigma-aldrich). results were analyzed using kaplan-meier survival curves using aable version . software (gigawiz ltd., co., ok). the significance of differences was determined using log-rank chisquare analysis with the results not adjusted for multiple comparisons. results are considered significant if p# . . the anti-chikv human monoclonal antibody c was isolated by ebv transformation of b cells from a chikv infected and recovered individual identified during a outbreak of chikv in northern italy [ ] . chikv pseudovirus [ ] neutralization was used as the primary screening assay for the selection of b cell clones and heavy and light chains were subsequently sequenced from the clones. separately, a fab fragment (e ) was isolated from a phage display library constructed from multiple chikv infected and recovered individuals from the - epidemic on la réunion as described in the materials and methods. a virus-like particle (vlp) binding assay, using vlps produced from chikv capsid and e /e /e envelope (env) glycoprotein expression was used as the primary screen for panning phage, followed by use of the chikv pseudovirus (hiv-backbone based, without chikv capsid) neutralization assay for downstream characterization. subsequently, the antibody heavy and light chains for c and e were sequenced and cloned into human full length igg vectors for protein production and evaluation. c and e were tested in neutralization assays performed in hek t cells using chikv pseudoviruses bearing an envelope from the prototypical west african, asian, and east/central/ south african (ecsa) chikv strain, s . the c and e igg antibodies neutralized chikv pseudoviruses at approximately . mg/ml and . mg/ml (ic ) respectively (figure ). pseudoparticles produced using envelopes derived from the lr opy- strain from the la reunion outbreak were similarly sensitive to neutralization, with ic values of . mg/ml and mg/ml for c and e respectively ( figure s ). similar neutralization was observed regardless of the cell type used (data not shown). neutralization was specific to chikv, with no binding of anti-chikv antibody c to intact chikv vlps was detected using the fotebio octetred biosensor. c mab ( . nm) binding to chikv vlps or a nonparticle surface control is used to show binding specificity of mab to intact chikv vlp. (b) c dose response curve for binding intact chikv vlps. raw data curves for antibody associating and dissociating from captured chikv vlps are shown in black and fitted curves are shown in red. data were fitted to a : binding model to determine association rate (k on ) and dissociation rate (k off ), and equilibrium binding affinity (k d ) was calculated. c binds chikv vlps with . nm apparent affinity. doi: . /journal.pntd. .g detectable cross-reactivity to pseudoviruses expressing other alphavirus envelopes from rrv, sfv and sinv, as well as vsv-g (figure ). the mab also neutralized chikv envelopes with a naturally occurring mutation at a critical site near the fusion loop in e (a v) that is associated with increased chikv infectivity for, and transmission by, the mosquito vector, aedes albopictus (c , ic . mg/ml; e , ic . mg/ml for s [a v]) ( figure ) [ ] . when tested in a replication competent chikv plaque reduction neutralization test (prnt) using the s strain, c exhibited a prnt value of approximately . mg/ml. a comparable level of neutralization was also observed with the lr opy- strain. in contrast to the weak neutralization observed with the chikv pseudovirus assay (figure ), e failed to neutralize replication competent chikv, even at concentrations up to mg/ml. similarly, little to no inhibition by e was noted utilizing vesicular stomatitis virus-based pseudotypes (rather than hiv-based) or in a cell-cell fusion assay, while c maintained similar neutralizing and inhibitory activity (data not shown). based on these findings, c can be categorized as a strongly neutralizing antibody, with similar potency to other human mabs [ ] , while e is a non-neutralizing, or weakly neutralizing, antibody of live virus. in order to determine how strongly each mab interacts with the native virion, intact chikv vlps were captured onto the surface of fortebio octet red biosensor tips (fortebio, menlo park, ca) and antibody binding to the immobilized particles was measured using biolayer interferometry (fortebio, menlo park, ca). whereas c bound to vlps with an apparent affinity of . nm (figure a & b) , e failed to recognize chikv envelope protein on intact vlps (data not shown), suggesting that the e epitope may be occluded in the native e /e conformation on virions. this finding is consistent with the inability of e to neutralize live chikv. c and e antibodies recognized envelope derived from chikv vlps under semi-native conditions (protein run in sds-page gels without reducing agent), suggesting that both c and e recognize conformation specific epitopes that are dependent on disulfide bonds ( figure s ) . chikv envelope proteins were immunostained with e antibody. clones with reactivity , % relative to wild-type chikv env were identified as critical for e binding. mutation of six individual e residues to alanine (y , f , v , g , t , and d ) significantly reduced e binding (red bars) but did not affect binding of c (green bar) or other control antibodies (gray bars). residues are numbered according to e in pdb entry # n [ ] . (b) critical binding residues for e (shown in green) were visualized on the chikv env crystal structure. the e , e , and e envelope protein subunits in the monomer (pdb entry # n ) are depicted in yellow, red, and blue, respectively and the fusion loop is shown in silver (left panel). in the side-view and top-down trimeric representations (center, and right panels, pdb entry # xfc), e is not in the structure. in the side view trimeric representation (center panel), the viral membrane is positioned at the bottom of the figure. doi: . /journal.pntd. .g mab epitope mapping using shotgun mutagenesis in order to identify residues in the binding epitope of c and e , the mabs were screened against a comprehensive chikv mutation library in which nearly every residue within the e , k, and e envelope subunits (encompassing amino-acid residues with . % coverage) were individually mutated to an alanine (alanines were mutated to serines). each clone was expressed in hek- t cells and assessed for c and e antibody binding using immunofluorescence staining. mean fluorescence was determined by high-throughput flow cytometry and antibody reactivity to each mutant was calculated relative to reactivity to wild-type (wt) chikv env. clones were identified as critical for binding if they had low reactivity to c or e but high reactivity to other chikv e -specific control antibodies (ckv , e d . , and rabbit polyclonal antibody, described in materials and methods). this counter-screen strategy facilitates the exclusion of env mutants that are globally or locally misfolded or that have an expression defect [ ] . residues identified in this way are the energetically critical contributors of an epitope, the so-called 'hot-spots' of mab binding [ , ] . six amino acids clustered within the e domain a were identified as critical for e binding. residues e -y , e -f , e -v , e -g , e -t , and e -d , when mutated to alanine, all reacted at less than % of wt reactivity when screened with e , but had high reactivity compared to three other antibodies (ckv , e d . and rabbit polyclonal antibody), suggesting that the mutant envelope proteins are expressed and properly folded ( figure a) . the e epitope appears to be partially occluded when visualized on the native trimer structure ( figure b ), which likely accounts for the poor neutralization exhibited by e . similar epitope mapping studies using shotgun mutagenesis alanine scanning identified residue e -a , located in the bconnector region between domains a and b of chikv e , as a chikv envelope proteins were immunostained with c antibody. clones with reactivity , % relative to wild-type chikv env were identified as critical for c binding. mutation of residue a in e to serine significantly reduced c binding (green bar) but did not affect binding of e (red bar) or other control antibodies (gray bars). residues are numbered according to e in pdb entry # n [ ] . (b) a s and a v pseudoviruses were tested for c inhibitory potency. the infectivity of the mutants compared to wt was tested (inset graph), indicating that the mutants did not hinder chikv env folding or function. average raw luminescence units are shown for each construct and an env-minus negative control. (c) the critical residue a (shown in green) was visualized on the chikv env crystal structure. the e , e , and e env protein subunits in the monomer (pdb entry # n ) are depicted in yellow, red, and blue, respectively and the fusion loop is shown in silver (left panel). in the side-view and top-down trimeric representations (center and right panels, pdb entry # xfc), e is not in the structure. in the side view trimeric representation (center panel), the viral membrane is positioned at the bottom of the figure. doi: . /journal.pntd. .g critical residue required for c recognition (figure ) . the e -a residue is solvent exposed and is predicted to be easily accessible when chikv env is in the native trimer conformation (figure ) . the e -a residue is in the acid-sensitive region (asr), sandwiched in a critical pocket between chikv e , e and e , as determined by the chikv envelope crystal structure [ , ] . interestingly, the asr, along with the e domain b, was also recently described for alphaviruses as being unstructured following acid ph triggering [ ] . in our study we found that residue e -a , when mutated to serine, reacted at % of wt reactivity against c but reacted at greater than % of wt reactivity against other anti-chikv antibodies, strongly suggesting that the e -a s mutant is properly folded and involved in the c /envelope binding interaction ( figure a ). other residues are also likely to be involved in the c epitope, but either contribute weakly to the interaction or their individual mutation to alanine does not sufficiently disrupt mab binding to be detected as 'critical'. using pseudovirions, no virus entry defects were observed with e -a s, further indicating that the mutant envelope is properly folded. to confirm the importance of this residue in c binding, infection experiments were conducted with wild type and mutant pseudovirions. e -a s pseudovirions were inefficiently neutralized by c , with a -fold increase in the c ic , demonstrating that this residue is required for potent c inhibition (figure ). in contrast, wild type e and e -a v, a naturally occurring variant [ ] , remained fully sensitive to c . to assess the potential protective activity of mab c in vivo, we used an adult ( week old) wild-type mouse model of chikv disease [ ] . mice received an intra-peritoneal injection of purified c igg ( . mg/mouse or approximately - mg/kg) the day before being infected with the reunion island isolate of chikv (lr -opy- ) [ ] . a control monoclonal antibody that did not recognize chikv (produced in the same fashion as c ) and pbs were used as negative controls. infected mice were monitored for viremia and foot swelling as described previously [ ] . in both control groups, chikv infection of -week old mice resulted in a - day viremia and increased foot swelling similar to that described previously in control animals [ ] . in contrast, in the same experiment, week old mice injected with c igg hours prior to exposure to virus, showed no detectable viremia or foot swelling ( figure ). these results demonstrate that the c antibody completely protected adult animals prophylactically against viremia and arthritic disease. in order to evaluate the therapeutic potential of c mab, we inoculated c bl/ j neonate mice with pfu of chikv and monitored the survival rate. mice infected with chikv survived for days, while mice given control human igg survived for days ( figure ; table ). in contrast % of the neonate mice injected with c at mg (, mg/kg) co-incident with infection survived (p# . compared to virus alone or human igg). we also observed that c ( mg/mouse or mg/kg), when administered therapeutically at hours and hours after chikv challenge, completely protected % of mice (table ) . therefore, c is a potent neutralizing antibody that can therapeutically protect wild-type neonate mice from a lethal dose of virus at and hours post chikv inoculation. protection in wild-type mice from virus infection in vivo by a monoclonal neutralizing antibody is thought to require close to the ic levels of antibodies in the serum [ , ] . however, there is very little known for chikungunya protection in mice with human anti-chikv mabs. we performed in vivo antibody titration experiments and dosed neonates immediately before chikv challenge via intraperitoneal injection with c mab at , , , , and . mg/mouse (or approximately equaling , , . , . , . and . mg/kg respectively). mice were fully protected with as little as mg/kg of c mab, while over % of mice survived after receiving . mg/kg of c mab concurrent with viral challenge (figure ). mice that were given . mg/kg of c mab succumbed to chikv-driven lethality similarly to control groups. our results demonstrate that with a potent neutralizing antibody such as c (ic of . mg/ml) we can protect % of mice with mg/kg of c mab and about half of all neonatal mice with only . mg/kg of c . this study describes the isolation and characterization of two human monoclonal antibodies, c and e , from chikv infected and recovered individuals. we previously developed a chikv pseudovirus assay [ ] that we found amenable in our current study for high-throughput screening and selection of b-cell clones expressing chikv neutralizing antibodies. c neutralizes both chikv pseudoviruses and replication-competent viruses with high potency. the e monoclonal antibody shows less dramatic neutralization of pseudovirus and does not neutralize live virus at the highest concentration tested ( mg/ml). this suggests that although chikv antibody selection can be carried out using the high-throughput pseudovirus assays for greater convenience, a live virus-prnt assay is the more reliable confirmatory assay for chikv neutralization. we also report the development of a novel, comprehensive chikv envelope site-directed mutation library in which nearly all of the residues of the full-length e /e chikv envelope protein were individually mutated to alanine in order to identify critical amino acids that are recognized by human mabs c and e . e recognized spatially proximal residues (y , f , v , g , t a and d ) in e domain a, however the nonneutralizing nature of the e antibody suggests that the residues are not easily accessible on the native chikv envelope on live virus, and indeed the epitope appears to be partially occluded when visualized on the native trimer crystal structure. the sitedirected mutagenesis mapping studies, confirmed by neutralization escape mutant studies, revealed that e -a is a critical all mice were infected with chikv at hour . data on chikv infected mice without antibody treatment and mice treated with control or c antibody at hour are also shown in figure . residue required for c mab recognition. interestingly, based on the crystal structure of the chikv envelope, the e -a residue is located in the asr of e that encompasses amino acids - and - [ ] . the asr in e , along with domain b, is a highly conserved functional region among alphaviruses and is involved in the conformational rearrangements triggered by acid ph that lead to the exposure of the fusion loop in e and finally results in membrane fusion [ , ] . it is possible that neutralizing antibodies such as c that bind to the asr region could fully or partially prevent the disordering and dissociation of e from e following ph triggering, thereby reducing fusion efficiency and chikv entry. residues within the asr have previously been reported to be critical for efficient particle formation and stability [ ] , highlighting the delicate conformational balance that this region brings to e . the critical e -a residue is highly conserved among different chikv strains and is represented in the 's west african isolates and s , as well as in the more recently circulating lr opy- strain. however, a previous study described a strain (ag ) isolated from uganda during a outbreak that has a valine at the e - position [ ] . mutating e -a to valine did not result in a loss of c potency, suggesting c should be active against most currently circulating strains of chikv and other strains that arise in the future with that particular amino-acid. the fact that e proteins bearing the aliphatic, hydrophobic amino-acids alanine or valine did not prevent c neutralization, while e with a polar serine residue at position escaped neutralization, suggests that c forms a critical hydrophobic interaction with that sidechain position. neutralizing antibodies have been shown to be critical for recovery from alphavirus infections and a number of neutralizing epitopes have been characterized, albeit only a handful for chikv. of particular note, antibody r /r is specific to sinv and has been previously documented to have an escape mutant at position k n (equivalent to chikv residue e -t ) in the asr of sinv e glycoprotein [ ] . the isolation and characterization of additional chikv mabs should offer insight into the proportion of antibodies elicited against this particular epitope in chikv infected individuals and the timing at which they appear. for example, an elegant study recently described that a predominant proportion of the very early response to chikv envelope is composed of igg antibodies directed against the nterminal sequence in e (e ep ) [ ] . there is very little known concerning chikungunya protection with human monoclonal antibodies. in order to elucidate whether strong in vitro c neutralization would translate to protection in vivo, we used the c antibody in an adult wild-type mouse model of chikv disease. in contrast to control mice, mice pretreated with c antibody had no detectable chikv viremia or arthritis. in a previous study, human igg purified from the pooled plasma of chikv recovered individuals was titrated in a pathogenic neonate mouse model of chikungunya (similar to one of the models used in our current study), and was shown to fully protect only when present at a concentration of - mg per mouse [ ] . another recent study described two monoclonal antibodies ( f and b ) that were protective in vivo in ifndeficient mice. these two antibodies protected % of the mice at , . mg/kg ( mg per mouse) [ ] . however, the antibodies failed to protect mice at , . mg/kg ( mg per mouse) and were only able to delay chikv-driven lethality. in the in vivo antibody titration experiments described here we demonstrate that % of the mice were protected with mg/kg of c mab (in vitro ic of . mg/ml), while over % of mice were protected with only . mg/kg of c . the data with c is consistent with what has been determined for in vivo protection from viral infection by neutralizing antibodies [ ] , while c at . mg/kg could also protect over half of animals from chikvdriven lethality. in order to evaluate the therapeutic potential of c mab we inoculated wild-type (c bl/ j) neonate mice with a robust dose of chikv ( pfu) and monitored the survival rate. we observed that c mab ( mg/mouse), when administered therapeutically at and hours after chikv infection, completely protected % of mice. this report demonstrates prophylactic and therapeutic protection against viremia in vivo by a neutralizing antibody that targets the acid-sensitive region in chikv e . although passive antibodies likely would not be utilized for protection from chikv on a regular basis, one can envisage a scenario where a potent antibody like c can be manufactured and used for protecting highly susceptible individuals such as pregnant women, infants and older individuals during a chikv epidemic, or for protecting travelers or military inadvertently exposed to the virus. while, the isolation and epitope characterization of c antibody and demonstration of its potent neutralization in vitro and in vivo are invaluable to future studies aimed at identification of neutralization epitopes in order to produce effective antigens for vaccines, we hypothesize that the epitope recognized by the c antibody is also an important region to target for antibody-based intervention in future anti-chikv strategies. recent studies have demonstrated that the use of combinations of mabs is advantageous for in vivo protection by limiting development of resistance [ ] and different mechanisms of viral spread [ ] . given that c is directed against a region of chikv e not targeted by other mabs, it will be particularly useful as a partner in such combinations. figure s human mabs c and e neutralization. neutralization of pseudovirus bearing chikv lr -opy- strain (orange lines) chikv s strain (green lines) and vsv-g control (magenta lines) envelope by (a) c or (b) e mabs. antibody concentration is shown in the x-axis. the results are expressed as the percentage of no antibody control and represent mean of triplicate wells, and is representative of three experiments. (tiff) figure s western blot of reduced and non-reduced chikv vlps using c and e mabs. antibodies ( ug/ml) were tested for reactivity against ug chikv vlps that were treated with dtt/heat (+) or not ( ) . hrp signal was detected using luminescence by adding a : ratio of femto supersignal. (tiff) text s supporting methods. (docx) an epidemic of virus disease in southern province, tanganyika territory, in - . i. clinical features infection with chikungunya virus in italy: an outbreak in a temperate region chikungunya virus, southeastern france chikungunya, an epidemic arbovirosis how did chikungunya reach the indian ocean? chikungunya epidemic in india: a major public-health disaster persistent chronic inflammation and infection by chikungunya arthritogenic alphavirus in spite of a robust host immune response arthritogenic alphaviruses-an overview systemic involvements and fatalities during chikungunya epidemic in india formation of the semliki forest virus membrane glycoprotein complexes in the infected cell biochemical studies of the maturation of the small sindbis virus glycoprotein e the sindbis virus k protein can be detected in virions and is acylated with fatty acids fate of the k membrane protein of semliki forest virus during virus assembly mapping the structure and function of the e and e 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of sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein e the authors acknowledge bridget puffer for assistance in phage display library construction, simona jusyte for assistance with phage display library panning, surabhi srinivasan and nick distasio for assistance with epitope mapping, joe couto and silveria rodriguez for dna plasmid engineering and construction, and dr. marina fomin for mouse breeding. the authors would like to thank james robinson (tulane university) for advice and reagents for b-cell cloning. we also thank dendritics for kindly providing an anti-chikv monoclonal antibody and ibt bioservices for provision of the rabbit anti-chikv polyclonal antibody. key: cord- -ke az nf authors: schlake, thomas; thess, andreas; thran, moritz; jordan, ingo title: mrna as novel technology for passive immunotherapy date: - - journal: cell mol life sci doi: . /s - - - sha: doc_id: cord_uid: ke az nf while active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. in either case, administration or expression of recombinant proteins plays a fundamental role. mrna prepared by in vitro transcription (ivt) is increasingly appreciated as a drug substance for delivery of recombinant proteins. with its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mrna combines several advantages. for example, compared to transfected dna, mrna harbors inherent safety features. it is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. compared to the administration of recombinant proteins produced in bioreactors, mrna allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. based on great progress in understanding and manipulating mrna properties, efficacy data in various models have now demonstrated that ivt mrna constitutes a potent and flexible platform technology. starting with an introduction into passive immunotherapy, this review summarizes the current status of ivt mrna technology and its application to such immunological interventions. our bodies are continuously exposed to molecules that may indicate disease or parasitic invasion. the immune system is responsible for the detection and clearance of such molecules and for control of the underlying causes. immediate discrimination between self and foreign upon first exposure is mediated by innate immunity. signals that induce innate immunity are called pathogen-associated and damageassociated molecular patterns (pamps and damps) [ , ] . the innate immune system is characterized by induction of generic defenses against a broad spectrum of infectious agents and is mainly aimed at clearance of tissue damage at the site of infection and interruption of further pathogen replication. responses against specific targets following prolonged or repeated exposures are processed by the adaptive immune system [ ] . important effectors of adaptive immunity are highly specialized cells: b cells secrete antibodies against soluble or cell-associated antigens [ ] . cytotoxic cd + t cells (ctls) recognize and kill infected or neoplastic cells [ ] . regulatory cd + t cells augment b cell maturation or inhibit auto-reactive immune cells [ ] . dendritic cells (dcs) process and present antigen for the transition from innate to adaptive immunity [ ] . some b and t cells progress towards persisting memory cells that react faster and with greater affinity to the foreign molecules upon re-exposure. compared to innate immunity, adaptive immunity usually requires weeks (as opposed to minutes) until an effective response against novel targets is achieved. advantages of the adaptive immune system include the ability to launch specific immune responses also against antigens of endogenous (not only microbial) origin that may be associated with degenerative or neoplastic disease. manipulation of the immune system is an important component of many prophylactic and therapeutic applications against infectious, degenerative and neoplastic diseases. the diverse repertoire of methods can be roughly divided into four approaches: active immunization (vaccination) prepares adaptive memory responses, usually prior to first exposure, and constitutes one of the most efficacious and cost-effective medical interventions. immunization with antigens generally only leads to the induction of antibody production, whereas for instance inoculation with attenuated viruses also elicits cytotoxic effector cells [ ] . in addition, active immunization can be accomplished by adoptive cell transfer. in a typical setting, dendritic cells are loaded with tumor antigens ex vivo and subsequently infused into patients to induce an adoptive immune response against cancer cells [ ] . in contrast, passive immunotherapy circumvents the initial steps required for immune responses to be launched and directs the immune system efficiently to the desired medical targets. for cellular approaches, ctls are equipped with recombinant receptors ex vivo. these cells are designed to attack neoplastic cells that express the cognate tumorassociated antigen immediately after infusion [ ] . passive immunization by administration of processed antibodies derived from human or animal donors is a well-established emergency procedure for treatment of snake-bite envenomation or post exposure prophylaxis against, for example, rabies [ ] . the advantage of passive immunization is that protective antibodies can be provided in a very short time. application of recombinant antibodies further expands the number of available targets and is increasingly important for augmentation of conventional therapies against cancer [ , ] . passive immunotherapies either require or can exploit modern nucleic acid-based methods, among which mrna is the latest technology. the present review is dedicated to provide an overview on mrna in passive immunotherapy after introducing the immunological approach as well as mrna technology as such. it is well known that protection raised by most of today's licensed vaccines is primarily antibody-dependent (fig. a ) [ , ] . basically, this explains the long and successful history of passive immunotherapy (fig. b) . the protective capacity of serum against bacterial toxins was discovered in the early s [ ] . the avoidance or control of infection by such passive immunization is based on the transfer of serum and later polyclonal immune globulin (= antibody) preparations from convalescent or vaccinated humans or animals [ , ] . prior to the discovery of antibiotics, serum was the only antidote for bacterial diseases [ ] . thus, following successful passive immunization against diphtheria toxin [ ] , a whole plethora of serum or immune globulin therapies for viral and bacterial diseases as well as to neutralize snake toxins was developed [ ] . clinical benefits of serum and immune globulin therapy were demonstrated for viral diseases such as influenza, measles, and polio and bacterial infections with meningococcus or pneumococcus [ ] [ ] [ ] . with the advent of antibiotics, the use of serum or polyclonal immune globulin preparations as antibacterial agent was largely discontinued in late . however, such therapies retained a niche as treatment for venoms, toxins, and certain viral infections. in the second half of the twentieth century, polyclonal antibody preparations were developed and in part licensed for the prophylaxis and treatment of hepatitis a and b, cytomegalovirus, varicella-zoster virus, vaccinia virus, rabies, respiratory syncytial virus, west nile virus, and various hemorrhagic fevers [ ] . for instance, passive immunization against the argentine hemorrhagic fever shows beneficial effects when applied within week after the emergence of symptoms, and post-exposure treatment with human or equine immunoglobulins are recommended for rabies [ ] [ ] [ ] . moreover, botulism is treated with equine antitoxin [ , ] . such immune globulin preparations of non-human origin are particularly prone to elicit an immune response that obstructs their therapeutic use or efficacy [ ] . further disadvantages or difficulties associated with the use of serum or polyclonal globulin preparations are the often high content of non-neutralizing antibodies, batch-to-batch variations, and in case of human sources the availability of appropriate immune donors [ , ] . in , groundbreaking work described the production of monoclonal antibodies (mab) by immortalization of b cells [ ] . the resulting hybridoma technology was then rapidly exploited for clinical use, for instance, to produce a mab to cd for preventing organ rejection [ ] . recombinant technologies further expanded the available therapies based on mabs. in vitro antibody selection technologies like phage or ribosome display were developed to enable the generation of highly specific human mabs out of libraries that may even be naïve for the specific antigens [ ] [ ] [ ] [ ] [ ] [ ] schematic illustration comparing active immunization and passive antibody immunotherapies. a during active immunization triggered by natural infection or vaccination, antigenic patterns are presented by antigen-presenting cells in the lymph nodes. this leads to t cell mediated activation of antigen-specific b cells. as a consequence, b lymphocytes differentiate into plasma cells which produce and secrete antigen-specific antibodies that bind to cognate structures, finally leading to their clearance. b instead of being produced by plasma b cells, antibodies can be manufactured recombinantly and administered for instance by subcutaneous injection for passive immunization. after injection, antibodies enter circulation by diffusion and act like endogenous antibodies. c for dna-based passive immunization, dna is often packaged in nanoparticles, e.g., virus capsids, which for instance can be injected intramuscularly. after uptake by muscle cells, dna is released into the cytosol. for transcription into mrna, the dna has to enter the cell nucleus first. mrna is then translated into antibodies which are secreted to bind their cognate targets a high throughput technique to amplify and clone antibody genes from single human b cells was described [ , ] . although recombinant mab technology is exploiting a large variety of different antibody formats (fig. ) , the prevalent type is still a full-size antibody of the igg class. in addition to the variable domains essential for antigen binding, they contain constant domains including the fc-region. the latter is important for antibody function and can mediate antibody-dependent cellular cytotoxicity (adcc), complement-dependent cytotoxicity (cdc), and antibodydependent cellular phagocytosis (adcp) [ ] . furthermore, binding to the neonatal fc receptor (fcrn) plays a role in controlling the antibody half-life which is - days for human igg [ ] [ ] [ ] . fcrn rescues bound antibody from degradation by transporting it back to the cell surface where it is released into the extracellular space [ , ] . specific mutations in the fc region that increased the affinity to fcrn have been shown to prolong antibody half-life up to fivefold [ ] . in addition to the impact of fcrn binding, half-life also benefits from the large size of iggs. it obstructs antibody clearance by the kidney as well as metabolization by cytochrome p [ , ] . the downside of the large size is the correspondingly low access to and penetration of tissue which can affect therapeutic efficacy [ ] . full-size antibodies are often posttranslationally glycosylated, modulating fc function. although aglycosylated iggs can be produced in bacteria [ ] , modern production processes usually take advantage of the cellular machinery for advanced posttranslational maturation and secretion of eukaryotic cells [ ] [ ] [ ] [ ] . the vast majority of approved therapeutic antibodies are currently produced in mammalian cells [ , ] . production processes have been optimized especially for the predominant production system, the continuous chinese hamster ovary (cho) cell line. cho cells secrete antibodies with negligible non-human glycoforms [ ] and are also amenable to glycoengineering [ ] . differences in glycoforms depend on the production system and affect distribution and stability of the antibody, fc effector function and immunogenicity in recipients [ ] . since traditional expression hosts such as e. coli do not allow efficient production of full-size antibodies, smaller proteins consisting of fragments derived from the variable domains were developed as promising alternatives. such single-chain variable fragments (scfv) and various derivatives thereof preserve antigen binding while facilitating manufacturing (fig. b, c) [ ] . another type of antibody fragment is derived from camelids or cartilaginous fish. these animals produce single-domain antibodies devoid of light chains (fig. e) [ , ] . since antigens are recognized by a heavychain-only v h domain (v h h) in camelids [ ] , the variable v h h fragment can be easily engineered into nanobodies that offer additional advantages such as improved heat and ph stability [ ] . moreover, they can also be assembled into v h h-based neutralizing agents (vnas) (fig. e) [ ] . various studies demonstrated that multivalent formats were more effective than monovalent single-domain antibodies [ , ] . notably, all formats based on antibody fragments can be relatively efficiently produced with less expensive bacterial expression systems, typically employing e. coli [ , ] . the antibody fragments produced in this system are often targeted to the oxidative environment of the periplasm using specific signal peptides to foster disulfide bond formation and proper folding [ , ] . moreover, enhanced expression of chaperones and cytoplasmic oxidases has been demonstrated to increase the yield of antibody fragments [ , ] . small antibody fragments were also the basis for developing the concept of bispecific antibodies more than years ago. initially, single chain antibodies with a different binding specificity were fused to the c-terminal ends of heavy chains of iggs [ ] . generation of first bispecific igg molecules benefited from the knob-into-hole technology [ ] . today, many different bispecific antibody formats combining two different antigen binding domains in one molecule are available ( fig. d ) [ ] [ ] [ ] [ ] . among them, bispecific diabodies (bi-(scfv) ) and bite antibodies are prominent examples [ , ] . in general, bispecific antibodies can be deployed to target therapeutic substances such as toxins, radionuclides, and drugs as well as effector cells like ctls to the site of cognate antigen expression [ ] . associated with their small size, many formats using antibody fragments are cleared by renal elimination [ , ] . moreover, in the absence of an fc region, recycling by the fcrn rescue mechanism cannot take place [ ] . as a consequence, these formats usually reveal short plasma half-lives [ ] . for instance, bi-(scfv) antibodies have a serum half-life of less than h which usually requires continuous infusion [ ] . in case of the bite blinatumomab, the antibody is usually administered daily due to its short halflife [ ] . possible strategies to extend serum half-lives are site-specific pegylation and fusion to an fc region [ , ] . however, the latter approach would negate various advantages of antibody fragments including their better and faster tissue penetration [ , ] . it has been shown that small single-domain antibodies could even cross the blood-brain barrier [ ] . in case of an anti-rabies antibody, this allowed partial rescue of mice challenged with virus injection into the brain in contrast to full-size immunoglobulins [ , ] . today, monoclonal antibodies play an important role in the therapeutic armamentarium. dozens of antibodies have been licensed to treat cancer, rheumatoid arthritis, multiple sclerosis, psoriasis, allergy, systemic lupus, and other diseases. in addition, mabs have shown promise in protecting against various microorganisms, viruses, and fungal infections as well as in treating neurodegenerative diseases [ ] [ ] [ ] [ ] . however, mabs for infectious disease indications are still scarce among licensed products. palivizumab for respiratory syncytial virus prophylaxis in high-risk infants was the first antiviral mab approved by the fda [ ] . since then, antibodies against anthrax and rabies in india became available. in addition, bezlotoxumab binding to clostridium difficile toxin is used to prevent recurrent bacterial infections [ ] . mabs for treating cancer are still the largest group of licensed products. here, rituximab, directed against the transmembrane protein cd on the surface of b lymphocytes, was the first mab in clinical use [ ] . however, as for many other mabs in antitumor therapy, high doses are needed to obtain clinical efficacy [ ] . a successful example for a bispecific antibody is the first-in-class bite against cd /cd , blinatumomab, which is approved for the treatment of acute lymphoblastic leukemia [ ] [ ] [ ] . in addition to the administration of immunoglobulins, passive immunity can also be conferred by transferring functionalized immune cells. adoptive transfer of ctls was shown to be a potent therapeutic means to treat both viral infections and cancers [ ] [ ] [ ] . to this end, t cells can be equipped with an additional t-cell receptor (tcr) or a chimeric antigen receptor (car) [ ] . while cars are limited to the binding of surface antigens, tcrs recognize mhc-presented peptides derived also from intracellular proteins. the first successful clinical trial with an engineered tcr on ctls demonstrating tumor regression was reported in [ ] . subsequently, passive immunotherapies with tcr-engineered t cells became an important approach for antitumor treatments [ ] . efficient targeting and killing of cancer cells expressing the respective antigen in patients with various forms of cancer including metastatic melanoma, synovial sarcoma, and colorectal carcinoma have been demonstrated [ , [ ] [ ] [ ] . a possible problem specific to the use of tcr-engineered t cells is the presence of an endogenous tcr. mispairing of endogenous and introduced α-and β-chains may create new specificities with potential reactivity to host molecules [ , ] . to avoid such mispairing, the use of γ/δ t cells has been suggested, since γ/δ-chains do not pair with α-or β-chains [ ] [ ] [ ] . alternatively, gene editing is now being explored to disable endogenous tcr expression [ , ] . the concept of cars was developed in [ ] and then refined using a scfv fragment to obtain antibody-like receptor specificity without the need to transfer multiple genes [ ] . first generation cars consisted of an antigenspecific scfv, fused to a transmembrane and intracellular cd ζ tcr signaling domain, conferring transient activation and cytotoxicity to t cells [ ] . upon target binding through the scfv domain, the engineered t cell is activated in an mhc-independent manner [ ] . subsequent generations of cars were improved with respect to cytotoxicity and persistence by including additional co-stimulatory domains such as cd , ox- or - bb [ , [ ] [ ] [ ] . t cells engineered with such cars targeting specific tumor antigens are remarkably successful in treating hematological malignancies like leukemia and lymphoma [ , ] . for instance, cd -directed car t cells repeatedly revealed complete and durable remissions in patients with b-cell acute lymphoblastic leukemia (b-all) [ ] [ ] [ ] . in contrast, car t cell therapies face various challenges for solid tumors [ ] . at present, the most common techniques for generating tcr-or car-engineered t cells utilize viral gene transduction with retro-or lentiviral vectors [ ] . however, permanent expression of the transgenic receptor mediated by this efficient technology can be disadvantageous in case of therapy-related severe toxicities due to accidental cross reactivity [ ] [ ] [ ] . on-target/off-tissue and off-target toxicities by engineered t lymphocytes attacking healthy host cells as well as cytokine release syndrome are feared side-effects which were repeatedly reported when virus vector transduced cells were applied [ ] [ ] [ ] [ ] . another concern of using retroviral vectors is the potential to induce insertional mutagenesis and genotoxicity in effector cells [ ] [ ] [ ] [ ] . hence, more precise cell manipulations are currently under investigation. among them, gene editing of primary human t cells was recently demonstrated to be an efficient approach [ ] . while t cell engineering for adoptive transfer inevitably requires the use of nucleic acids encoding a target-specific receptor, antibody immunotherapy can deploy recombinant proteins. however, as pointed out above, maintaining therapeutically effective levels may require frequent administrations dependent on clearance and indication [ ] . thus, dna-mediated antibody expression directly in the body may represent an attractive alternative to administration of recombinant proteins (fig. c ). both, plasmids as well as viral vectors have been used for passive immunization. although efficient in small animal models, strong expression of recombinant genes using unformulated dna does not scale well to larger animals (including primates) [ ] . consequently, application of recombinant adeno-associated viruses (aavs) is currently the preferred method for transduction of the antibody gene of interest [ ] . early work reached single digit µg/ml serum titers [ ] . later studies with advanced vector designs reported expression levels in the high µg/ml or even in the single digit mg/ml range [ ] [ ] [ ] . much work has been done in the field of hiv prophylaxis. here, a single intramuscular injection of recombinant aav was demonstrated to elicit peak antibody titers above µg/ml in mice [ ] . treated mice revealed substantial anti-hiv antibody levels for more than a year and were protected from hiv- challenge. although aav usually maintains an episomal state, this vector still harbors an inherent risk of insertional mutagenesis. in patients with hepatocellular carcinomas integration of aav into known cancer genes was observed [ ] . moreover, aav-based immunotherapy faces various issues regarding immunogenicity [ ] [ ] [ ] . a substantial percentage of the population has already been in contact with the used virus and consequently shows pre-existing immunity limiting the efficacy of treatment [ , ] . the induction of anti-viral responses during immunotherapy may have similar consequences if a single virus serotype is used in repeated treatments [ ] . pre-existing or induced immunity could lead to clearance of the viral vector and/or aavtransduced cells. finally, gene delivery by aav has been reported to induce immune responses against the encoded protein. even when using an endogenous gene such as erythropoietin (epo), some macaques receiving epo-encoding aav intramuscularly developed severe autoimmunity against the protein [ ] . when non-human primates were treated with aav vectors expressing antibody or antibody-like proteins, titers dropped rapidly in some animals [ ] . one reason is the possibility of inducing anti-idiotype antibodies which requires immune suppression to obtain sustained expression [ ] . further studies indicated that primates may be more prone to develop a robust t cell response to the aavencoded protein compared to mice [ ] . in summary, the risks of insertional mutagenesis and genotoxicity, long-lasting expression without control in case of adverse events, and various potential issues regarding immunogenicity associated with viral vectors that may limit efficacy emphasize the high demand for other vectors for passive immunotherapy than dna. how mrna offers a viable option to meet the demands is reviewed in the following sections. the cellular machinery uses mrna as a transient carrier of genetic information for the synthesis of proteins. based on this fundamental biological concept administering exogenous mrna represents an alternative to dna-mediated protein delivery in vitro and in vivo. using mrna instead of dna as therapeutic substance is attractive due to the absent risk of insertional mutagenesis. moreover, efficient expression is even obtained in non-dividing cells, since mrna does not require a nuclear phase for activity. compared to the delivery of proteins and peptides, mrna may prolong the availability of effector molecules, however, not as much as dna. in contrast to the latter, mrna therapy, therefore, has to cope with the short half-life in vivo of exogenously delivered mrna as for instance indicated by mrna-mediated vegf expression in myocardium returning to baseline within h [ ] . while this may be a therapeutic disadvantage in various instances, it can be considered advantageous from a safety perspective, particularly in case of adverse events. mrna was first employed for the expression of a protein of interest in the early s when rna preparations were microinjected into xenopus oocytes and synthesis of the encoded protein was demonstrated [ , ] . in , the group of inder verma presented a reliable method to efficiently introduce rna into a variety of cells using a cationic lipid [ ] . almost at the same time, mrna-mediated protein expression in vivo was demonstrated after direct injection into mouse muscle [ ] . much of the early work on a potential therapeutic use of mrna focused on the development of active vaccination approaches, in part since low amounts of antigen suffice due to the amplifying nature of the immune response. subcutaneous injection of liposomeencapsulated, antigen-encoding mrna was the first example of eliciting an antigen-specific ctl response in mice [ ] . gene gun delivery of mrna into mouse epidermis provided the first evidence of an antigen-specific antibody response [ ] . in addition, mrna turned out to be a potent means to load dendritic cells with antigens to convert them to tailored antigen-presenting cells in vitro and in vivo [ ] . later, a new vaccination protocol was introduced which elicited a complete adaptive immune response consisting of antigenspecific antibodies and t cells with lytic activity without requiring any transfection reagents, special equipment or heterologous boost [ ] . from a retrospective view, this event marked the starting point of the commercial development of mrna vaccines. in the minimal structure, mrna contains a protein-encoding open reading frame (orf) flanked at the ′-and ′-end by two elements essential for the function of eukaryotic mrna: the "cap", a -methyl-guanosine residue (m g) bound to the ′-end of the rna via a ′- ′ triphosphate bond, and, with the exception of histone mrnas, a poly(a) tail at the ′-end [ ] [ ] [ ] . synthetic mrna is transcribed in vitro from a plasmid dna template that contains at least a bacteriophage promoter, the orf, and a unique restriction site for linearization of the plasmid to ensure defined termination of transcription. typically, the template also contains a poly(d[a/t]) sequence transcribed into poly(a). alternatively, the poly(a) tail can be generated by enzymatic in vitro polyadenylation of transcribed rna. the cap may be incorporated into the transcript during transcription by including an m gpppg dinucleotide as a structural homolog of the endogenous cap structure in the transcription reaction. different options for how to derive ivt mrna are summarized in fig. . various elements of an mrna molecule contribute to the level and duration of expression of the encoded protein. the cap structure is required for efficient translation and stabilizes mrna towards exonucleolytic decay [ ] [ ] [ ] . various structures have been repeatedly used for ivt mrna (fig. ) . the basic m gpppg cap analog is incorporated in both orientations into the rna by the bacteriophage rna polymerase [ ] . however, reverse incorporation of the cap analog results in mrna molecules that lack the m methylation at the cap and are not recognized by the translational machinery [ ] . substitution of the hydroxyl group in c ′ or c ′ position of the m g with a methoxy group prevents reverse incorporation of the cap analog by inhibiting elongation at the m g. the dinucleotide is, therefore, called 'antireverse cap analog' or arca [ , ] . arca-capped mrna revealed increased as well as prolonged protein expression in cultured cells and enhanced reporter protein expression in mouse dendritic cells up to -fold [ , ] . for further optimization of the cap structure, modifications were introduced within the triphosphate linkage to inhibit decapping. substitution of a non-bridging oxygen in the β-phosphate moiety of an arca by sulfur results in the β-s-arca dinucleotide. while β-s-arca maintains recognition by the translational machinery, protein expression from mrna capped with β-s-arca was extended in hc cells and immature dcs, but not in mature dcs [ , ] . enzymatic capping represents an alternative to the cotranscriptional approach, avoiding a n -unmethylated cap as does arca. to this end, mrna is transcribed without cap analog and subsequently capped using the vaccinia virus capping complex [ ] . this complex with triphosphatase, guanylyltransferase and (guanine- )-methyltransferase activity adds a natural cap to the ′-triphosphate of an rna molecule. such a cap structure can be further converted into a cap group by o-methylation of the ribose of the cap-proximal nucleotide using the vaccinia virus ′o-methyltransferase [ ] . cap (and cap harboring a further o-methyl-ribose at the subsequent nucleotide) structures are typical of eukaryotic mrna and are recognized less by cytosolic rna sensors of the innate immune system, thereby rendering the mrna less immunostimulatory. for instance, rig-i is activated by cap but not cap mrna and ifit binds cap mrna more tightly than cap molecules [ , ] . such sensor-mediated immune stimulation may in turn negatively impact mrna translation [ ] . it is interesting to note that since recently a new cap analog, cleancap, is available which enables the cotranscriptional incorporation of a cap structure into ivt mrna. like the cap structure at the ′-end, details of the poly(a) tail at the ′-end influence translation and stability of mrna [ , ] . while numerous studies demonstrated a positive effect of a poly(a) tail and some correlation of effectiveness and length of the element, details of observations were remarkably variable. the majority of studies indicate an enhancement of translation when extending the poly(a) length from approximately - to - nucleotides or by tail extension using enzymatic polyadenylation [ , [ ] [ ] [ ] . however, effects were mostly moderate, but reached a maximum of a -fold increase in one particular setting. in contrast, another study reported an optimum of approximately nucleotides; protein expression declined with further increasing poly(a) length [ ] . in addition to poly(a) length, one report suggests a positive role of using a type iis restriction enzyme for dna template linearization to obtain a free poly(a) end rather than one extended with unrelated nucleotides [ ] . further elements that can affect mrna translation and/or half-life are untranslated region (utr) sequences flanking fig. schematic representation of different cap structures. a the typical ′ cap of eukaryotic mrnas. a guanosine is methylated at position and linked to the first nucleotide of the mrna by an unusual ′ to ′ triphosphate bridge. depending on the degree of methylation of the first two bases of the mrna, the full ′ terminal structure is referred to as cap , cap or cap . the cleancap™ analog, a trinucleotide introducing a cap structure during ivt, is indicated in blue. b a plain cap analog (orange) is incorporated in two orientations during ivt. c inverse orientation can be avoided using anti-reverse cap analogs (arcas, highlighted in orange). such analogs are characterized by the presence of a methoxy group at either c ′ or c ′ of m g. to improve resistance to decapping, a phosphorothioate was positioned in the ′- ′ bridge of arca (β-s-arca) the orf sequence. trans-acting regulatory rna-binding proteins (rbps) interact with distinct rna sequence elements, thereby affecting ribosome recruitment and transit [ ] . for instance, β globin ′-and ′-utrs, duplicating the β globin ′-utr, the ′-utr of tobacco etch virus, and a structure of the ′-utr of human heat shock protein all enhanced mrna translation in mammalian cells [ , , , ] . according to a very recent survey of a combinatorial utr library, ′-utr sequences appear to be most critical for protein expression [ ] . in contrast, ′-utrs seem to be the key driver for mrna half-life as exemplified by the stabilizing effects of the α globin ′-utr as well as a duplication of the β globin ′-utr [ , ] . codon usage of the orf sequence also affects translation efficacy in many species. although in humans codon usage bias does not correlate with trna levels and gene expression [ , ] , still increased protein expression from mrna upon codon usage optimization has been reported. for instance, codon usage adaptation of hiv- gag improved protein yield from mrna approximately . -fold in a human t lymphocyte cell line [ ] . a more pronounced increase in protein expression as a result of codon optimization was reported upon transfection of mrna encoding angiotensinconverting enzyme into a and hepg cells [ ] . however, alternatively to a direct effect of codon usage, the enhancement may be an indirect result of the use of modified nucleotides, the content of which was altered by codon optimization. furthermore, coding sequence engineering exploiting more advanced concepts like codon optimality may prove valuable in designing therapeutic mrnas of high efficacy. according to recent insights, codon usage may also affect fidelity of translation or the stability of transcripts [ ] . in addition, orf as well as utr sequences can have an effect on immunostimulation and thus on translational activity. initially, researchers applied mrna containing only the four unmodified bases a, u, c, and g [ ] [ ] [ ] ] . in this context, for instance, u-rich sequences, as well as several rna structural features were described as immunostimulatory due to interactions with various rna sensors such as toll-like receptors (tlr), rig-i, and protein kinase r (pkr) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as a consequence, development of mrna therapies was hampered by the immunogenicity of in vitro transcribed mrna. the consideration of mrna for therapeutic purposes gained momentum by the finding that incorporation of modified bases into in vitro transcribed mrna reduced immunostimulation of such preparations. various modified bases found in natural rnas suppressed recognition by tlrs in vitro [ ] . among these, particularly pseudouridine increased translation and stability of mrna [ ] . in addition to the effect on tlr binding, replacement of uridine by pseudouridine affected binding to and activation of further sensors such as pkr and ′- ′-oligoadenylate synthetase, contributing to higher and longer protein expression [ , ] . however, the effects of mrna modifications on translation, immunostimulation and resulting protein expression appear to be variable, for instance dependent on the type of target cells. in vitro testing of various modifications and combinations thereof revealed decreased protein yield for any of them in a context dependent manner, while most of them reduced immunostimulation in raw . macrophages [ ] . as found in vitro, pseudouridine modification of mrnas reduced immunostimulation and increased level and duration of protein expression after intravenous (iv) or intraperitoneal administration of formulated mrna in mice [ , ] . in contrast, another study on systemic administration of nanoparticle-complexed mrna concluded that neither immunostimulation nor protein expression benefited from pseudouridine modification [ ] . after intradermal or intramuscular injection of formulated mrna, only n -methyl-pseudouridine, but not pseudouridine, substantially enhanced expression [ ] . in line, a different study applying lipid nanoparticle (lnp)-formulated mrna intradermally found that replacement of uridine with n -methylpseudouridine resulted in much increased and longer lasting protein expression [ ] . notably, recent studies revealed that also endogenous eukaryotic mrna harbors various modified nucleotides [ ] [ ] [ ] [ ] . however, the total level of modification is rather low which is in strong contrast to the usually % replacement of an unmodified nucleotide in ivt mrna. moreover, heavy nucleotide modification appears to interfere with the function of translation-enhancing rna elements such as utrs and internal ribosomal entry sites (ireses) [ ] . at the time when modified nucleotides were introduced to minimize immunostimulation of ivt mrna, the importance of stringent purification of such preparations was recognized. chromatographic purification, particularly hplc, can separate mrna according to size, thereby removing smaller or larger by-products such as abortive transcripts, mrna from traces of non-linearized dna template or double-stranded rna (fig. ) [ , ] . such purification enhanced protein yield, most probably by enriching functional transcripts and depleting contaminants causing detrimental immunostimulation [ , ] . the latter is corroborated by the finding that stringent purification reduced the beneficial effect of chemical modification or even made it dispensable, particularly in combination with specific sequence-engineering of the mrna [ , ] . importantly, the specifics of mrna formulation are another layer that can influence activation of the innate immune system by masking mrna from recognition by sensors, particularly tlrs. for example, immune responses after mrna administration into the central nervous system were effectively suppressed by the use of a nanomicelle formulation compared to naked mrna [ ] . after in vivo administration of mrna was proven to be feasible, the concept of using mrna as a basis for therapeutics was pursued almost immediately. the very first report on a therapeutic effect with exogenous mrna was already published in and described a temporary reversion of diabetes insipidus in rats by intrahypothalamic injection of vasopressin mrna [ ] . thereafter, it took almost two decades until further studies started to demonstrate the broad potential of mrna-based protein therapies. meanwhile, there is a plethora of publications on a huge variety of indications comprising anemia [ , ] , hemophilia [ , ] , myocardial infarction [ , ] , cancer [ , ] , lung disease such as surfactant b deficiency and asthma [ ] [ ] [ ] , metabolic disorders [ ] [ ] [ ] [ ] [ ] , fibrosis [ ] , skeletal degeneration [ ] , tendon impairment [ ] , and neurological disorders such as sensory nerve dysfunction, friedreich's ataxia and alzheimer's disease [ ] [ ] [ ] . whereas evidence for the therapeutic potential of mrna is mostly restricted to mouse models, first data in swine indicate that mrna-based protein therapies are feasible also in large animals [ , ] . in view of the various indications, it is hardly surprising that this diversity goes along with different routes of administration and various formulations. only very few studies looking at local administration used uncomplexed and thus unprotected mrna [ , , , ] . the majority of investigations built on lipid-based formulations with a clear tendency to the application of lnps [ , , [ ] [ ] [ ] ] . most if not all groups purified their ivt mrna before in vivo administration. while some simply precipitated the mrna [ , , ] , most used commercial purification kits. only a few researchers applied hplc purification [ , , ] . with respect to the mrna, the vast majority of studies used long poly(a) tails of at least nucleotides. likewise, there is a clear prevalence to chemically modified mrna, although various examples suggest that this is not mandatory [ , , , ] . while most mrnas harbored -methyl-cytosine and/or pseudouridine initially [ , , ] , there appears to be a trend towards the use of n -methyl-pseudouridine at present [ , ] . regarding the cap structure, almost all early studies cotranscriptionally generated cap mrnas using arca [ , , ] . however, since about years, research groups prefer to apply mrnas with a cap ′-end [ , , ] . initial attempts to apply mrna to passive immunotherapy focused on cellular approaches for various reasons. adoptive transfer of ctls equipped with either an additional tcr or a car had shown great promise in cancers and viral infections. in contrast to typical scenarios of antibody therapy, receptor expression usually requires much lower protein levels. furthermore, t cells are loaded with receptor-encoding nucleic acid (dna or mrna) ex vivo. hence, passive cellular immunotherapy does not require sophisticated and highly efficient formulations for in vivo delivery but can build on the armamentarium of cell transfection methods. previous work on active cellular vaccination with antigen-presenting cells that had been transfected with antigen-encoding mrna revealed electroporation as easy and efficient means to load cells [ ] . comparison to passive cell pulsing and lipofection demonstrated that electroporation was also most efficient for transfection of t lymphocytes [ ] . rna electroporation had up to % efficiency without eliciting any critical toxicity [ ] . onset of transgene expression was very rapid and lasted about days [ ] . receptor transfer into t cells by mrna electroporation has now been well established for many years [ , ] . moreover, gmpcompliant protocols for manufacturing receptor-expressing retention time t cell preparations via mrna electroporation are available today [ , ] . electroporation of human t lymphocytes with various antigen-specific tcrs redirected them to recognize cancer cells in an mhc-dependent manner in vitro [ ] . mrnamediated tcr expression conferred in vitro cytotoxicity to t cells for at least h [ ] . the lytic efficacy of such cells was comparable to retrovirally transduced lymphocytes [ , ] . likewise, transfection of car-encoding mrnas generated cells that were lytically active in vitro. using an optimized ivt mrna for a cd -specific car, surface expression and cytotoxic function were detectable for up to days [ ] . to avoid as many manipulation steps as possible in generating t cells for adoptive transfer, it was demonstrated that human peripheral blood lymphocytes instead of purified t lymphocytes could be used as well to elicit strong cytotoxicity in vitro upon electroporation of a car mrna [ ] . currently, most car approaches deploy αβ t cells. however, γδ t lymphocytes are an attractive target as well due to their antitumor effector function which is not mhc restricted and does not require co-stimulation. accordingly, mrna-mediated tcr and car expression in such cells was investigated very recently and shown to kill target cells in an antigen-specific manner [ ] . to the best of our knowledge, there is so far just one study that started to systematically analyze the role of different mrna elements for receptor expression in t cells. to this end, the group of carl june built on previous findings in dendritic cells which revealed the superiority of a duplicated β globin ′-utr over a single copy of the same element and of a long ( nt) over a short ( - nt) poly(a) tail [ ] . with respect to receptor expression, as enhanced expression compared to as [ ] . a tandem repeat of the β globin ′-utr had also a beneficial effect, particularly in combination with a long poly(a). in contrast, the vegf translational enhancer as ′-utr element had even detrimental consequences. the authors speculated that this may be due to reduced capping efficacy but did not provide data corroborating their hypothesis. however, they demonstrated the important role of the cap structure. co-transcriptional cap using arca and an enzymatically generated cap structure were equivalent and outperformed the basic cap analog as well as an enzymatic cap . besides expression level, capping also appeared to have an effect on the persistence of expression. modification of the orf sequence by removing all internal orfs had no effect on receptor production. t lymphocytes transfected with tcr-or car-encoding mrna proved to be functional also in vivo. robust antitumor effects were observed in various preclinical models [ , ] . they were mrna-specific, since mock-transfected t cells had no or very little unspecific impact. although mrna-mediated receptor expression is transient, a single injection of car t cells against cd was sufficient to prolong survival of mice [ ] . using peripheral blood lymphocytes instead of purified t cells for car mrna transfection (see above) also enabled a strong antitumor response in vivo although those cells could not persist long-term in vitro [ ] . very recently it was shown that mrna cannot only be used to drive receptor expression, but can support the generation of t cells for adoptive immunotherapy. by expressing a chimeric membrane protein against cd , cells could be efficiently stimulated and expanded in vitro [ ] . after transfection with an mrna for an anti-cd bite, those cells mediated sustained reduction in tumor burden upon intraperitoneal injection. based on encouraging preclinical data, adoptive t cell therapies using mrna were already subjected to first clinical testing. in a phase trial on solid tumors addressing the safety and feasibility of using such cells, car transfected lymphocytes migrated to tumor sites after iv injection [ ] . in addition, the study appeared to provide initial evidence of antitumor activity. due to the transient nature of mrna expression, subjects received repeated infusions of t cells. this led to an anaphylactic response in one patient who developed antibodies specific to the scfv domain of the car [ ] . however, this could be a consequence of the murine origin of this domain. in another trial, mrnatransfected car t cells were injected intratumorally in metastatic breast cancer patients [ ] . treatment was well tolerated and elicited an inflammatory response within tumors. as discussed above, the use of viral vectors for adoptive t cell therapy has potential safety issues. in various studies, authors ascribed toxicities particularly to the persistence of receptor-expressing t cells. regarding such concerns, mrna-mediated tcr or car expression offers at least two advantages. first, mrna does not integrate into a cell's genome, thus excluding genotoxicity. second, due to its transient nature, any potential toxicities accompanying treatment are temporary as well [ ] . however, the increased level of safety has a substantial drawback. apparently, clinical efficacy correlates with long-term persistence of receptor-engineered t cells [ , ] . as a consequence, mrna-transfected cells are expected to have limited antitumor activity because of rapidly declining receptor expression. substantial mrna translation only lasts about day which translates into efficacious receptor levels on the cell surface for several days [ , ] . importantly, clinical studies demonstrated that iv infused t lymphocytes reached tumor sites only - days after administration [ , ] . as a consequence, intratumoral or intraarterial administration was suggested to counteract the delayed cell arrival. the problem of transience of mrna is further enhanced by the well-known ligand-induced receptor internalization. upon target recognition, tcrs as well as cars are rapidly internalized, a mechanism which is important for proper signal transduction [ , ] . this explains why lentiviral vectors generated a more robust treatment effect than mrna [ ] . mrna transfection can give rise to high receptor expression, equaling lentiviral vectors [ ] . however, mrna was only similarly effective during the first hours after electroporation. later, contact to target cells strongly down-regulated receptor on the cell surface while lentiviral expression remained constant [ ] . in comparison to a single transfer of cells with retroviral car expression, an mrna-encoded receptor required three consecutive lymphocyte infusions to obtain a comparable antitumor effect [ ] . these observations and considerations may be put into perspective at least in part by the finding that transferred t cells can become tolerized rather rapidly, thereby losing their ability to function in the tumor microenvironment [ ] . thus, frequent injection of t cells may be desired even for viral vector transduced t cells. notably, mrna was also considered to be of use in settings that require long-term expression for therapeutic efficacy and preclude repeated cell administration. the greater safety of mrna-transfected t cells may make the initial testing of novel antigens and receptors with unknown on-target/off-tissue toxicity less hazardous [ ] . passive immunization with antibodies often requires considerable amounts of polypeptide to obtain therapeutically active concentrations after systemic administration. this poses a substantial challenge to the broad applicability of any nucleic acid mediated passive immunization strategy. thus, compared to the very first attempts regarding in vivo protein expression with mrna in the s, the optimization of ivt mrna to enhance and extend expression is a prerequisite for many mrna-based passive immunotherapies. as reviewed above, great progress towards this goal was made during the last almost three decades. another potential hurdle for passive mrna immunization is related to delivery. to obtain high levels of in vivo antibody expression, the mrna should be targeted to as many "producer" cells as possible which in turn should be transfected with high efficiency. to this end, the mrna which is prone to degradation by rnases should also be protected against these ubiquitous nucleases in an appropriate manner. moreover, a viable mrna complexation reagent needs to be well tolerated. notably, various commercial transfection reagents can be used to formulate mrna and suffice research purposes. among them, transit has been repeatedly used for in vivo studies [ , , ] . with respect to potential therapeutic applications, the class of lipid nanoparticles (lnps) became the most widely deployed means of complexation [ , , [ ] [ ] [ ] . after iv delivery, lnps mainly route to the liver on the basis of an apolipoprotein e (apoe)-dependent mechanism [ ] . however, such nanoparticles were demonstrated to be also applicable to intramuscular and subcutaneous administration [ ] . advances in mrna and formulation technology led to a couple of recent intriguing studies as to passive immunization with mrna (fig. ) . while the group of drew weissman described successful passive mrna immunization for prophylaxis of viral infections [ ] , stadler et al. demonstrated the applicability of mrna-mediated antibody expression for cancer immunotherapy [ ] . the feasibility of using mrna for such indications was confirmed by thran et al. who applied different mrnaencoded antibody formats to diverse biological threats, viruses, toxins, and tumors [ ] . finally, sabnis and colleagues presented first antibody expression data in nonhuman primates (nhps) in a publication dealing with the development of novel lnp formulations [ ] . schematic illustration of mrna-mediated passive antibody immunotherapy. for in vivo administration, mrna is usually formulated in nanoparticles which for instance can be administered by iv injection. for many formulations, liver is the main target organ. upon uptake of nanoparticles by hepatocytes and release of the mrna into the cytosol, it is translated into antibodies that are typically secreted into circulation and finally bind their cognate antigens mrna design and formulation fundamental designs of antibody-encoding mrnas reveal only a few common features but several differences (table ) . among the latter, the exclusive use of chemically unmodified nucleotides by thran et al. as in a previous publication from the same group [ ] may be the most prominent one, since it contrasts to all other reports. other differences are much more diverse among these studies. hence, they do not provide an unequivocal guidance for future work, but at least commonalities may be taken as a recommendation. although obviously not mandatory, mrna with cap structure was clearly preferred. in addition, all mrnas harbored a poly(a) tail. the use of bipartite poly(a) elements by some groups may be owed to the experience that maintenance of long poly(d[a/t]) vector sequences is challenging and strongly dependent on bacterial strains [ ] . beyond these common rna elements, the publications suggest that mrna should be subjected to further optimizations to exploit its full potential for antibody expression. however, different strategies appear to be applicable, but little is known about the interchangeability of individual elements. finally, chromatographic purification of ivt mrna appears to be generally recommended as well (table ) . whether pardi et al. actually used fplc as stated throughout their report instead of hplc applied by other groups is not fully clear, since they referred to earlier publications describing the use of hplc [ , ] . pardi et al. encoded a well-known, broadly neutralizing antibody against hiv- , vrc [ ] . to this end, heavy and light chains of the full igg antibody were represented on separate mrna molecules. for delivery, heavy and light chain mrnas were mixed in a molar ratio of : . likewise, thran et al. used separate mrnas to encode heavy and light chain of various full igg antibodies [ ] . titration of heavy and light chain mrna found a molar ratio of approximately . : to be optimal for co-delivery. neither report provides a rationale for encoding chains on separate molecules. in principle, a bicistronic construct separating heavy and light chain by an ires sequence or an mrna for a polypeptide where a a sequence between heavy and light chain would lead to separate antibody chains by ribosome skipping could have been used [ ] . for pardi et al. the observation that modified nucleotides can hamper the function of ires elements may have affected the selection [ ] . sabnis et al. also worked with a full igg antibody, directed against influenza a, but did not provide any details on how heavy and light chain were represented [ ] . in contrast, stadler et al. chose bite antibodies directed against tcr-associated cd and one of three different tumor-associated antigens (taas) [ ] . they displayed the bites as fab(scfv) or scfv molecules but focused on the latter format. their findings on single-chain antibodies are complemented by thran et al. whose work covers single domain-derived vnas in addition to igg antibodies [ ] . for iv administration of antibodyencoding mrna all but the group of ugur sahin used lnp formulations which, however, may differ from each other in composition ( table ). the latter team exploited transit but switched the route of administration which had been intraperitoneal in previous studies [ , ] . as with lnps, nanoparticles were shown to mainly target the liver upon iv injection [ ] . drew weissman's group administered µg of vrc encoding mrna in most in vivo studies [ ] . this corresponded to doses between and . mg/kg due to differences in mouse weight among experiments. antibody serum titers h after administration, the earliest time of analysis, ranged between approximately and µg/ml in various mouse strains. obviously, slight differences in dosage, as well as the respective strain contributed to varying peak levels. notably, increasing the administered mrna dose in steps of two enhanced serum titers by more than twofold with each step. moreover, µg of mrna in lnps generated higher serum titers than µg of recombinant vrc protein. the kinetics of antibody serum titers revealed an accelerated decline after about a week in balb/c mice. the kinetics in nsg mice appeared to be basically the same, since the level at week after single administration was largely the same as in balb/c at this time. the observed kinetics may also explain why weekly injections of mrna-lnps in nsg mice did not show additive effects on serum titers at the times of analyses. since measurements were conducted days after each treatment, antibodies from the preceding injection probably dropped to background levels within this -week period as observed in balb/c animals. such accelerated decline of protein titers after a few days is often indicative of the induction of an anti-drug antibody (ada) response [ ] . the likelihood of such a response may be particularly high for the reported experiments, since the authors expressed a human antibody in mice. the emergence of adas cannot be fully ruled out because animals were not analyzed accordingly. however, the apparently similar kinetics in immunocompromised nsg mice which are unable to develop adas suggests a different explanation for the pharmacokinetics. possibly, the mrna continues to express antibody for a few days which would inevitably lead to a seemingly extended antibody serum half-life during that period. only after expression ceases, the actual shorter antibody half-life becomes evident. while this could also easily explain the serum profile of repeated treatment of nsg mice, it remains hypothetical due to the lack of respective analyses. stadler et al. first characterized their mrna in vitro demonstrating expression and secretion of functional antibodies [ ] . in a pbmc-mediated killing assay, mrna-derived bite antibodies targeted ctls to tumor cells via binding to cd on pbmcs and to the cognate taa on tumor cells, thereby inducing t cell activation and tumor cell lysis. these antibodies were equally potent as the corresponding recombinant protein. in immunodeficient nsg mice, antibody plasma levels peaked within h, but rapidly declined by more than % within the next h. subsequently, the decrease of bite titers became much slower. the authors did not provide an explanation of this striking kinetics. a pharmacokinetic analysis in non-tumor-bearing mice could have elucidated whether the initial kinetics reflects the trapping of antibody in the engrafted tumor until saturation of binding sites. bite plasma levels above background for a few days were in accordance with the sustained ex vivo cytotoxicity of plasma from mrna-treated mice. in contrast to the antibody plasma kinetics, cytotoxicity showed a steady and slow decline during the observation period. . µg of mrna were already sufficient to obtain strong plasma activity in the ex vivo killing assay. µg of mrna (approx. . mg/kg) were comparable to - µg of recombinant antibody with respect to peak plasma concentrations that were in the range of . µg/ml in nsg mice. as opposed to this modified and hplc-purified mrna, antibody plasma levels were almost undetectable with mrna preparations without modification and chromatographic purification. plasma titers declined much faster for recombinant protein compared to mrna, thereby demonstrating the substantial impact of mrna on bite pharmacokinetics. consequently, only mrna was able to maintain a sustained cytotoxic activity of plasma by weekly administrations. the various mrna-encoded antibodies of thran et al. included vrc which had been used in drew weissman's work [ , ] . however, analyses were limited to in vitro characterization, preventing a direct comparison between studies. as observed for bites, igg and vna antibodies produced from mrna in vitro revealed potencies comparable to that of the respective recombinant proteins. iv administration of µg of unmodified mrna (approx. mg/kg) gave rise to antibody serum titers between and µg/ ml in immunocompetent mice. this contrasts strongly with the finding of stadler et al. who found unmodified mrna to be basically inactive. differences in purification and mrna design may be responsible for this striking discrepancy. similar to the weissman work, thran and colleagues observed a disproportionate increase of antibody serum titers with elevated mrna doses. onset of antibody expression was rather rapid, being already substantial h after injection and reaching peak levels after approximately h. this confirms findings on other mrna-mediated protein therapies showing that mrna starts accumulating in hepatocytes within minutes after administration and leads to substantial protein levels within a couple of hours [ , ] . serum half-life of igg antibodies appeared to be in the range of week and thus slightly longer compared to pardi et al. [ ] . as in the latter study, one of two iggs showed an accelerated decline after about week, however, only in approximately half of the animals. here, the expedited clearance could be assigned to the development of an ada response against the mrna-encoded antibody. importantly, this response was antibody-dependent and not intimately linked with the use of mrna. as expected, vnas revealed a much shorter serum half-life of about - days. compared to published kinetics data on recombinant vnas, mrna appeared to contribute to extended antibody availability during the first days after administration as it has been observed for bite-expressing mrna by ugur sahin's group. however, the lack of a headto-head comparison hampers a detailed analysis. while previous in vivo studies on mrna-mediated antibody expression were limited to mice, sabnis et al. presented expression results in nhps using a proprietary lnp formulation [ ] . a . mg/kg dose of mrna gave rise to antibody serum titers of about µg/ml h after iv administration which is at least at the lower end of the range of efficacy observed in mouse studies. however, data on a different protein suggest that efficacy of the formulation may be slightly lower in nhp than in mouse. whereas a . mg/ kg dose induced protein levels of approximately µg/ml in mice, a . mg/kg dose generated protein titers between and ng/ml in nhps. all mouse studies on mrna-encoded antibodies investigated their therapeutic efficacy. pardi et al. used two different humanized mouse models to demonstrate that mrnaderived vrc protects from hiv- challenge [ ] . mrna encoding a reporter protein was utilized as control. mrna-lnps were administered h prior to challenge with one of two hiv- isolates. in the authors' first model, a vrc mrna dose of . mg/kg was ineffective, but . mg/kg already reduced viral rna copies in the plasma to undetectable levels as assessed by quantitative real-time polymerase chain reaction (qrt-pcr). the latter dose is well below the - mg/kg doses that are typically used for prophylactic immunization with recombinant antibody in humanized mice to reach therapeutic concentrations [ , ] . however, the authors did not titrate the dose of recombinant vrc but used a mg/kg dose as control which was sufficient to completely eradicate viral rna copies in the plasma. mrna efficacy could be also demonstrated in the second mouse challenge model. to show in vivo efficacy of mrna-mediated bite expression, stadler et al. implanted tumor cells subcutaneously in immunodeficient nsg mice [ ] . about week before mrna treatment, human pbmcs were engrafted into these animals. µg of bite mrna (approx. . mg/ kg) given three times with an interval of week could eliminate tumors entirely. in contrast, tumors progressed in control animals that received mrna encoding a reporter protein. the recombinant bite required three injections per week and a total of ten injections of - µg each to obtain a comparable antitumor effect as with bite mrna. the need for a more frequent administration corroborated the previous finding that mrna substantially improved antibody plasma half-life. due to the diversity of antibodies included in their study, thran et al. utilized various disease models for demonstrating therapeutic efficacy [ ] . in contrast to all other studies, the authors applied mrna encoding irrelevant antibodies instead of a reporter protein as control. a µg dose (approx. mg/kg) of antibody mrna could protect mice from challenges with either rabies virus or botulinum toxin. in the intoxication model, mrna was proven to be equally protective as recombinant antibody. however, mice received approximately . mg/kg of recombinant vna compared to approximately mg/kg of mrna. based on protein expression levels from mrna dose titrations, lower doses than mg/kg may still confer full protection but this remains hypothetical, since the authors did not conduct an mrna dose titration in their challenge model. notably, mrna was effective in pre-as well as in post-exposure settings. the latter is important for some indications of passive immunization and confirms the aforementioned rapid onset of antibody expression. the post-exposure scenario for botulinum toxin requires very rapid availability of neutralizing antibodies. whereas recombinant protein can act immediately after administration, mrna needs more time to provide the antibody. hence, it may well be that in such instances higher doses of mrna than of protein are required, not for obtaining the same peak level but for reaching meaningful titers in a timely manner. in a further model, thran et al. evaluated their mrna approach with respect to anti-tumor efficacy. using a disseminated tumor model for rituximab, they showed efficient tumor growth control with injections of µg (approx. . mg/kg) of rituximab mrna twice a week. higher doses ( µg, approx. mg/kg) of recombinant rituximab were less potent. this finding is reminiscent of results of drew weissman's group and contrasts those of ugur sahin and colleagues who required similar doses of mrna and recombinant protein (but less frequent dosing with mrna) to obtain equivalent therapeutic effects [ , ] . notably, the difference among studies may be related to the use of igg antibodies on the one hand and a scfv protein on the other hand. moreover, the irrelevant antibody control used by thran et al. appeared to have a slight unspecific anti-tumor effect. it may have contributed to the superiority of mrna compared to recombinant protein regarding dosing. amongst other explanations, the potential unspecific effect may be due to an mrna-lnp-independent response to repeated treatment or may be the consequence of a weak and transient cytokine response observed after mrna-lnp administration. however, the phenomenon was not investigated further. in line with previous reports, pardi et al. confirmed the importance of highly purified ivt mrna. in combination with lnps, only modification with n -methyl-pseudouridine plus chromatographic purification was sufficient to avoid cytokine release by innate immune activation [ ] . for this analysis, however, the authors deployed an mrna encoding a different protein than the vrc antibody which had been used for in vivo expression and efficacy experiments. tolerability of mrna-lnps was also addressed by repeated mrna treatments. translation of vrc mrna was not compromised over time, but the analysis was conducted in immunodeficient nsg mice. to overcome this caveat, the authors complemented their study by repetitive treatment of immune competent balb/c mice. to this end, they switched to an endogenous protein, since human vrc may be recognized as foreign and thus elicit an immune response. again, mrna injections did not lose efficacy over time. however, the authors also changed the formulation (transit instead of lnp) as well as the route of administration (intraperitoneal instead of iv) compared to the use of vrc mrna. hence, evidence for immune silence and overall tolerability of vrc mrna in lnps is just circumstantial yet. stadler et al. did not observe any liver toxicity upon treatment with mrna in transit according to liver enzyme analyses [ ] . moreover, bite mrna administration did not elevate murine cytokines such as ifnα and tnfα above background in plasma. likewise, analysis of systemic human cytokine release from engrafted pbmcs did not show any unspecific t cell activation. as opposed to modified and hplc-purified mrna, preparations without nucleotide modification and chromatographic purification induced detectable levels of murine cytokines. similar to the weissman group, the authors also assessed the tolerability of formulated mrna by repeated injections. administrations did not lose efficacy over time, but as in the corresponding weissman experiment immunodeficient nsg mice were used. using chemically unmodified mrna formulated in lnps, thran et al. did not observe any liver toxicity in histopathological analyses [ ] . only a few animals developed an ada response which was dependent on encoded antibody and was, thus, no intrinsic consequence of treatment with mrna-lnp. in addition, treatment appeared to elicit a transient weak cytokine release which, however, neither suppressed antibody expression nor induced adverse effects. since there are ample differences among studies on mrna-mediated antibody expression and no detailed analyses of the issue, the role of mrna, lnp, and/or encoded antibody/protein in cytokine induction remains elusive. an earlier study on erythropoietin showing the absence of any appreciable immunostimulation suggests that the use of chemically unmodified instead of modified mrna is not the decisive parameter [ ] . quite a few in vivo studies provided compelling evidence for the principle feasibility of mrna-based immunotherapies. as discussed above, challenges and open questions regarding adoptive t cell transfer are less related to the mrna and its formulation or transfection but more of fundamental character. in contrast to ex vivo loading of cells, mrnamediated antibody expression is strongly affected by body size. thus, while there are now convincing efficacy data in diverse small rodent models, the translation to larger animals and finally humans has still to be demonstrated. first data suggest that substantial expression can be obtained in small nhps. however, the utilized lnps appeared to lose some efficacy when switching from mouse to nhp. hence, the development of human therapies may perhaps require further advancements of the mrna technology as well as primatespecific formulations with improved efficacy. in addition, tolerability of formulations has to be analyzed further and in more depth in the future. for instance, repeated dosing of nanoparticles can induce complement activation-related pseudoallergy (carpa) [ ] . however, this can in principle be counteracted by optimization towards better biocompatibility. in case of lnps, fast degradation was shown to be particularly important [ , , ] . while antibodies for cancer treatment were initially developed for iv administration, there is a trend towards subcutaneous injections today. for instance, rituximab was initially formulated for iv infusion which is typically administered over a period of . - h [ ] . this treatment schedule poses a substantial burden to patients as well as the healthcare system. thus, a formulation which reduces the time and required resources would be advantageous. to meet these goals by subcutaneous administration, the antibody solution was concentrated -fold [ , ] . since this volume was still too large for subcutaneous injection, rituximab was co-formulated with human hyaluronidase which limits swelling and associated pain by increasing the dispersion and absorption of co-administered substances [ , ] . now, median administration time for rituximab using the subcutaneous route is min. as a consequence, antibody immunotherapy with mrna does not only require competitive efficacy and costs but also routes of administration to become a viable alternative to recombinant proteins. although other routes than iv have been shown to be possible for mrna, there are still a few open questions to be addressed by future studies. where are the advantages of using mrna for antibody immunotherapies? compared to dna it may be primarily the safety aspect. concerning recombinant proteins various points matter. as reviewed above, mrna provides benefits as to the pharmacokinetics when short-lived antibodies such as scfv, (bi)-scfv or vna are used. moreover, solving the challenges of antibody cocktails may be easier using mrna. different mrna sequences are much more similar with respect to their physicochemical characteristics than different proteins are. hence, producing a cocktail may be less demanding for mrna compared to protein. however, co-delivery and thus co-expression implicates the risk of antibody chimerism and thus requires specific solutions such as knob-into-hole concepts [ ] . last but not least, while proteins are difficult to deliver directly through the cell membrane [ ] , mrna-mediated protein expression makes a large number of potential intracellular targets accessible to antibody immunotherapy. particularly single-chain and single-domain formats are amenable to functional expression in the cytosol and thus suited as intrabodies, since they are less dependent on disulfide bond formation [ , ] . the value of targeting intracellular proteins has already been demonstrated by various studies. a bispecific scfv could restore p function in mutant p colon cancer cells and trapping ccr in the er via an intrabody reduced hiv cell entry [ , ] . support for the potential of intrabodies as therapeutics also comes from further work in the field of oncology or neurodegenerative diseases [ ] [ ] [ ] . although it has been recently demonstrated that even a full antibody can be 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cytokine release syndrome preventing and exploiting the oncogenic potential of integrating gene vectors gene therapy. safer and virus-free? lmo -associated clonal t cell proliferation in two patients after gene therapy for scid-x genotoxicity of retroviral integration in hematopoietic cells generation of knock-in primary human t cells using cas ribonucleoproteins pharmacokinetics and immunogenicity of broadly neutralizing hiv monoclonal antibodies in macaques state of play and clinical prospects of antibody gene transfer vector-mediated antibody gene transfer for infectious diseases generation of neutralizing activity against human immunodeficiency virus type in serum by antibody gene transfer antibody-based protection against hiv infection by vectored immunoprophylaxis broad protection against influenza infection by vectored immunoprophylaxis in mice stable antibody expression at therapeutic levels using the a peptide recurrent aav -related insertional mutagenesis in human hepatocellular carcinomas general considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination pre-existing immunity against ad vectors: humoral, cellular, and innate response, what's important? hum vaccines immunother promise and problems associated with the use of recombinant aav for the delivery of anti-hiv antibodies preexisting anti-adeno-associated virus antibodies as a challenge in aav gene therapy in situ production of therapeutic monoclonal antibodies erythropoietin gene therapy leads to autoimmune anemia in macaques vector-mediated gene transfer engenders long-lived neutralizing activity and protection against siv infection in monkeys broadly neutralizing human immunodeficiency virus type antibody gene transfer protects nonhuman primates from mucosal simian-human immunodeficiency virus infection adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific t cell responses modified mrna directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction use of frog eggs and oocytes for the study of messenger rna and its translation in living cells translation of encephalomyocarditis viral rna in oocytes of xenopus laevis cationic liposome-mediated rna transfection direct gene transfer into mouse muscle in vivo induction of virusspecific cytotoxic t lymphocytes in vivo by liposomeentrapped mrna gene gun delivery of mrna in situ results in efficient transgene expression and genetic immunization dendritic cells pulsed with rna are potent antigen-presenting cells in vitro and in vivo in vivo application of rna leads to induction of specific cytotoxic t lymphocytes and antibodies ′-terminal cap structure in eucaryotic messenger ribonucleic acids how the messenger got its tail: addition of poly(a) in the nucleus formation of the ′ end of histone mrna the cap and poly(a) tail function synergistically to regulate mrna translational efficiency the enzymes and control of eukaryotic mrna turnover concerted action of poly(a) nucleases and decapping enzyme in mammalian mrna turnover reverse ′ caps in rnas made in vitro by phage rna polymerases synthesis and properties of mrnas containing the novel "anti-reverse" cap analogs -methyl( ′-o-methyl)gpppg and -methyl ( ′-deoxy)gpppg novel "anti-reverse" cap analogs with superior translational properties effective delivery with enhanced translational activity synergistically accelerates mrna-based transfection mrna transfection of dendritic cells: synergistic effect of arca mrna capping with poly(a) chains in cis and in trans for a high protein expression level phosphorothioate cap analogs stabilize mrna and increase translational efficiency in mammalian cells phosphorothioate cap analogs increase stability and translational efficiency of rna vaccines in immature dendritic cells and induce superior immune responses in vivo modification of the ′ end of mrna. association of rna triphosphatase with the rna guanylyltransferase-rna (guanine- -)methyltransferase complex from vaccinia virus cap-specific mrna (nucleoside-o ′-)-methyltransferase and poly(a) polymerase stimulatory activities of vaccinia virus are mediated by a single protein a conserved histidine in the rna sensor rig-i controls immune tolerance to n - ′o-methylated self rna inhibition of translation by ifit family members is determined by their ability to interact selectively with the ′-terminal regions of cap -, cap -and ′ppp-mrnas viral stressinducible protein p inhibits translation by blocking the interaction of eif with the ternary complex eif .gtp.met-trnai mrna poly(a) tail, a ′ enhancer of translational initiation modification of antigen-encoding rna increases stability, translational efficacy, and t-cell stimulatory capacity of dendritic cells multiple injections of electroporated autologous t cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor mrna with a < -nt poly(a) tail imparted by the poly(a)-limiting element is translated as efficiently in vivo as long poly(a) mrna optimized transfection of mrna transcribed from a d(a/t) tail-containing vector trans-acting translational regulatory rna binding proteins increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mrna encoding erythropoietin an element within the ′ untranslated region of human hsp mrna which acts as a general enhancer of mrna translation optimization of mrna untranslated regions for improved expression of therapeutic mrna an mrna stability complex functions with poly(a)-binding protein to stabilize mrna in vitro codon usage and trna genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with cgdinucleotide usage as assessed by multivariate analysis evolution of synonymous codon usage in metazoans quantitative effect of suboptimal codon usage on translational efficiency of mrna encoding hiv- gag in intact t cells translation of angiotensin-converting enzyme upon liver-and lung-targeted delivery of optimized chemically modified mrna codon optimality, bias and usage in translation and mrna decay reversal of diabetes insipidus in brattleboro rats: intrahypothalamic injection of vasopressin mrna species-specific recognition of single-stranded rna via toll-like receptor and recognition of double-stranded rna and activation of nf-kappab by toll-like receptor innate antiviral responses by means of tlr -mediated recognition of single-stranded rna ′-triphosphate rna is the ligand for rig-i differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing ′-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection ′-triphosphate-dependent activation of pkr by rnas with short stem-loops suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability incorporation of pseudouridine into mrna enhances translation by diminishing pkr activation nucleoside modifications in rna limit activation of ′- ′-oligoadenylate synthetase and increase resistance to cleavage by rnase l screening of mrna chemical modification to maximize protein expression with reduced immunogenicity efficacy and immunogenicity of unmodified and pseudouridine-modified mrna delivered systemically with lipid nanoparticles in vivo n( )-methylpseudouridine-incorporated mrna outperforms pseudouridine-incorporated mrna by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice nucleoside-modified mrna vaccines induce potent t follicular helper and germinal center b cell responses chemical and structural effects of base modifications in messenger rna chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome a mettl -mettl complex mediates mammalian nuclear rna n -adenosine methylation transcriptome-wide mapping reveals reversible and dynamic n( )-methyladenosine methylome sequence-engineered mrna without chemical nucleoside modifications enables an effective protein therapy in large animals messenger rna-based vaccines generating the optimal mrna for therapy: hplc purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mrna spontaneous cellular uptake of exogenous messenger rna in vivo is nucleic acid-specific, saturable and ion dependent in vivo messenger rna introduction into the central nervous system using polyplex nanomicelle therapeutic efficacy in a hemophilia b model using a biosynthetic mrna liver depot system systemic delivery of factor ix messenger rna for protein replacement therapy biocompatible, purified vegf-a mrna improves cardiac function after intracardiac injection week post-myocardial infarction in swine systemic delivery of modified mrna encoding herpes simplex virus thymidine kinase for targeted cancer gene therapy exploring cytotoxic mrnas as a novel class of anti-cancer biotherapeutics expression of therapeutic proteins after delivery of chemically modified mrna in mice in vivo genome editing using nuclease-encoding mrna corrects sp-b deficiency modified foxp mrna protects against asthma through an il- -dependent mechanism systemic messenger rna therapy as a treatment for methylmalonic acidemia targeted mrna therapy for ornithine transcarbamylase deficiency g pc mrna therapy positively regulates fasting blood glucose and decreases liver abnormalities in a mouse model of glycogen storage disease a quantitative systems pharmacology model of hugt a -modrna encoding for the ugt a enzyme to treat crigler-najjar syndrome type mrna treatment produces sustained expression of enzymatically active human adamts in mice chemically modified rna induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats tendon healing induced by chemically modified mrnas treatment of neurological disorders by introducing mrna in vivo using polyplex nanomicelles intrathecal delivery of frataxin mrna encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for friedreich's ataxia messenger rna-based therapeutics for brain diseases: an animal study for augmenting clearance of beta-amyloid by intracerebral administration of neprilysin mrna loaded in polyplex nanomicelles efficient genetic modification of murine dendritic cells by electroporation with mrna highly efficient gene delivery by mrna electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mrna and to electroporation of plasmid cdna for tumor antigen loading of dendritic cells high-efficiency transfection of primary human and mouse t lymphocytes using rna electroporation a new way to generate cytolytic tumor-specific t cells: electroporation of rna coding for a t cell receptor into t lymphocytes a gmp-compliant protocol to expand and transfect cancer patient t cells with mrna encoding a tumor-specific chimeric antigen receptor rna-transfection of gamma/delta t cells with a chimeric antigen receptor or an alpha/beta t-cell receptor: a safer alternative to genetically engineered alpha/beta t cells for the immunotherapy of melanoma transfer of mrna encoding recombinant immunoreceptors reprograms cd + and cd + t cells for use in the adoptive immunotherapy of cancer treatment of advanced leukemia in mice with mrna engineered t cells adoptive immunotherapy using human peripheral blood lymphocytes transferred with rna encoding her- /neu-specific chimeric immune receptor in ovarian cancer xenograft model novel t cells with improved in vivo anti-tumor activity generated by rna electroporation mesothelin-specific chimeric antigen receptor mrna-engineered t cells induce anti-tumor activity in solid malignancies t cells expressing chimeric antigen receptors can cause anaphylaxis in humans ) safety and efficacy of intratumoral injections of chimeric antigen receptor (car) t cells in metastatic breast cancer nonviral rna transfection to transiently modify t cells with chimeric antigen receptors for adoptive therapy adoptive t cell therapy for cancer in the clinic adoptive cell transfer: a clinical path to effective cancer immunotherapy survival and tumor localization of adoptively transferred melan-a-specific t cells in melanoma patients phase i trial of adoptive immunotherapy with cytolytic t lymphocytes immunized against a tyrosinase epitope signal transduction and endocytosis: close encounters of many kinds endosomes: a legitimate platform for the signaling train redirecting t cells to ewing's sarcoma family of tumors by a chimeric nkg d receptor expressed by lentiviral transduction or mrna transfection immunological quality and performance of tumor vessel-targeting car-t cells prepared by mrna-ep for clinical research interleukin- rescues tolerant cd + t cells for use in adoptive immunotherapy of established tumors elimination of large tumors in mice by mrna-encoded bispecific antibodies structure, activity and uptake mechanism of sirna-lipid nanoparticles with an asymmetric ionizable lipid expression kinetics of nucleoside-modified mrna delivered in lipid nanoparticles to mice by various routes administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv- challenge ) mrna mediates passive vaccination against infectious agents, toxins, and tumors a novel amino lipid series for mrna delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates hplc purification of in vitro transcribed long rna comparison of ires and f a-based locus-specific multicistronic expression in stable mouse lines hiv therapy by a combination of broadly neutralizing antibodies in humanized mice passive immunization with a human monoclonal antibody protects hu-pbl-scid mice against challenge by primary isolates of hiv- complement activation-related pseudoallergy: a stress reaction in blood triggered by nanomedicines and biologicals phase iii safety study of rituximab administered as a -minute infusion in patients with previously untreated diffuse large b-cell and follicular lymphoma subcutaneous administration of rituximab (mabthera) and trastuzumab (herceptin) using hyaluronidase non-clinical pharmacokinetic/pharmacodynamic and early clinical studies supporting development of a novel subcutaneous formulation for the monoclonal antibody rituximab a recombinant human enzyme for enhanced interstitial transport of therapeutics delivery of antibodies to the cytosol: debunking the myths intracellular and cell surface displayed single-chain diabodies phenotypic lentivirus screens to identify functional single domain antibodies construction and expression of a bispecific single-chain antibody that penetrates mutant p colon cancer cells and binds p barbas cf rd ( ) functional deletion of the ccr receptor by intracellular immunization produces cells that are refractory to ccr -dependent hiv- infection and cell fusion a novel intracellular antibody against the e oncoprotein impairs growth of human papillomavirus -positive tumor cells in mouse models a human singlechain fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein trapping prion protein in the endoplasmic reticulum impairs prpc maturation and prevents prpsc accumulation a cell-penetrating whole molecule antibody targeting intracellular hbx suppresses hepatitis b virus via trim -dependent pathway delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment acknowledgements we thank mariola fotin-mleczek for discussion on the manuscript. we are grateful to nigel horscroft and michael stolz for critical reading of the review. we also thank bettina danker for her graphical illustrations. finally, we apologize to those authors whose work was not cited owing to space limitations. key: cord- -e zhq g authors: yang, danlin; frego, lee; lasaro, marcio; truncali, kristopher; kroe-barrett, rachel; singh, sanjaya title: efficient qualitative and quantitative determination of antigen-induced immune responses date: - - journal: j biol chem doi: . /jbc.m . sha: doc_id: cord_uid: e zhq g to determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. here, we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. the application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. our workflow consists of the following two steps: ) the use of surface plasmon resonance to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and ) the recovery of serum iggs using an automated small scale purification system, followed by the determination of their epitope diversity using hydrogen deuterium exchange coupled with mass spectrometry. we showed that these methods were sensitive enough to detect antigen-specific iggs in the nanogram/μl range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. we also showed that this workflow can guide the selection of an animal that produces high affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. we postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens. one hundred years after the "magic bullet" proposal by paul ehrlich and decades after the establishment of hybridoma technology for producing monoclonal antibodies (mabs) by köhler ( ) and others ( , ) , antibodies have finally become very attractive therapeutic modalities. the high potency of antibodies and the ability to generate a seemingly unlimited diversity of specificities have led to antibody-based therapies that target many different illnesses and diseases ( - ) . the majority of therapeutic antibodies currently available on the market are of mouse origin. despite the proven success of animal immunizations for over decades, the generation of mabs for therapeutic purposes is often a labor-intensive and time-consuming process that requires months of work from the initial immunization to the identification of specific hybridoma antibodies. although other approaches, such as phage display ( ) and human memory b-cell immortalization and cloning ( ) , have been developed for the derivation of fully human mabs that do not require animal immunization, these methods have shown limited success due to the challenges associated with antibodies of non-immune origins ( ) . to date, the production of mabs from immunized rodents remains the most highly used platform for generating high quality therapeutic biologics, because of the natural in vivo recombination and affinity maturation of antibodies that occur during the humoral immune response in immunized animals ( - ) . generating an effective immune response is not only fundamental for therapeutic antibody discovery, but is also crucial for vaccine development in combating infectious disease. both begin with the host immune response elicited against the foreign antigen/vaccine after immunization. while animal immunization for therapeutic antibody generation requires a mechanism-driven strategy for obtaining antibodies that can demonstrate the therapeutic mode of action prior to hybridoma fusion and/or b-cell recovery efforts, vaccine development emphasizes the stimulation of protective immune responses by the host's immune system that are of sufficient strength and quality to sustain efficient and long term protection ( , ) . the development of an optimal immune response to meet either goal is challenging due to the sophisticated mechanisms that control immune responses ( ) . the process often involves multiple critical considerations, such as the selection of candidate antigens, choice of adjuvant, antigen/ vaccine design, dosage, frequency of application, duration of immunization, injection protocols, etc. ( - ) . immune responses are typically monitored by measuring antibody titers (the relative concentration of antigen-specific antibodies) in the blood samples collected from the immunized hosts over the course of immunization. while many commercial kits and automated systems are available, elisa remains the most widely used method for antibody titer measurements due to its simplicity and low cost. however, these conventional assays can only partially evaluate immune responses ( , ) , as they cannot accurately reveal data about the binding affinity, specificity, and epitope diversity of polyclonal antibodies, all of which are important for an effective immunization campaign that will lead to the generation of high quality antibody responses with distinct specificities. to increase the efficiency of our therapeutic antibody generation process to achieve long term success, we developed alternative methods for assessing the quality of polyclonal antibodies in immune sera, surface plasmon resonance (spr) and hydrogen deuterium exchange (hdx) coupled with mass spectrometry. we propose that a workflow integrating these sensitive methods into the antibody generation platform can help "engineer" the immune response by providing information on the effectiveness of various immunization methods and by selecting the most appropriate animals for antibody recovery. access to this information increases the probability of success in recovering antibodies that meet affinity and/or epitope requirements. in this report, we describe the following: the method development and establishment of the detection limits of our techniques, a proof-of-concept study using sera from il- immunized mice, the creation of a workflow named quality of antibody response (qar), and finally the implementation of this workflow for a therapeutic anti-b-cell activating factor (baff) antibody generation campaign to guide the selection of optimal animal donors for hybridoma generation. we demonstrate how these sensitive methods can effectively differentiate the antibody responses among various animals, thereby contributing to the successful recovery of functional antibodies. beyond its use in therapeutic antibody discovery, this qar workflow can also be applied to advance vaccine development toward the goal of developing robust vaccines. serum igg purification efficiency-to enable analysis by mass spectrometry, serum iggs were purified using the phynexus automated purification system (table ) . to ensure that the purification process achieved maximal igg recovery and optimal igg quality, sds-page and auc were used. we used naive serum to investigate the number of elution cycles required for full retrieval of the bound iggs; sds-page was used to track the presence of iggs in the samples from each of consecutive elution steps. we found that iggs could no longer be detected beyond the th elution cycle, suggesting that four elution steps were sufficient (data not shown). thus, we used four elution steps throughout this study. non-reducing sds-page analysis of the material from each of the purification steps showed that the proplus (protein a/g) resin effectively captured iggs from the mouse sera (fig. a) . although the remaining flow-through and washing step solutions contained multiple bands, the eluted solutions showed a single band whose size corresponded to the typical igg molecular mass of ϳ kda. this result was confirmed using reducing sds-page, which revealed the presence of two bands with molecular weights corresponding to those of the heavy and light chains. the igg bands from the four sequential elution cycles gradually decreased in signal intensity as expected. the pooled eluate, whose total collected volume was ϳ ml, was concentrated, and its purity and homogeneity were evaluated using auc. sedimentation velocity analysis showed that the sample contained % monomeric iggs (fig. b) . detection limit of antigen-specific iggs in serum using spr-the concentration-dependent binding of igg to human il- was demonstrated by spr using samples containing naive serum with various amounts of dl added. because of the high concentration of endogenous iggs, the serum samples were diluted -fold prior to protein a/g capture. the total iggs, which included both endogenous iggs and dl , were captured at a density of ϳ ru. as expected, the binding response decreased with decreasing dl concentrations in the serum (fig. ) . the affinity of dl was determined to be ϳ (Ϯ ) pm, and saturation was achieved at nm human il- . there was no difference in the maximal response level when and nm human il- solutions were injected over identical igg-containing surfaces (data not shown). the concentration limit at which antigen-specific iggs were detected in serum was determined to be ng/l, as signals Ͻ ru are considered to be background noise in this system. using equations and , described under "experimental procedures," the percentage of dl antigen-specific iggs among the total iggs was calculated for each reconstituted sample. as shown in table , the lower limit of ng/l corresponded to a concentration that was equivalent to ϳ % of the total iggs being human il- -specific dl . step-wise deposition to row e-h detection limit of antigen-specific igg in serum using hdx-lc/ms-to determine the igg-binding epitope on the antigen, the purified iggs were subjected to hdx coupled with lc/ms. this method compares deuterium exchange levels at different regions of the antigen in the presence and absence of antibody and identifies those that are protected by the iggs. first, a mock experiment using different concentrations of human il- in the absence of iggs was performed to determine the optimal concentration of protein that was required to ensure full sequence coverage of the protein. the results revealed that a concentration of m yielded the highest sequence coverage ( %) (fig. ) . thus, this concentration was used in the subsequent experiments using igg samples. the epitope mapping experiment was performed by incubating the purified iggs with human il- as described under "experimental procedures." deuterium uptake was compared in samples containing human il- in the absence and pres-ence of dl . because hydrogen has a mass of . da and deuterium has a mass of . da, the replacement of hydrogen in a protein with deuterium induces a shift in mass of ϳ da. because antibody binding inhibits deuterium exchange in the epitope regions, peptides exhibiting shifts in mass are associated with antibody epitopes. as shown in fig. a , a large shift in mass in comparison with the control was observed in the peptide encompassing residues - in human il- , indicating that this region was protected by dl . therefore, the shift in mass of this peptide was monitored in the limit of detection study, in which a fixed amount of human il- was incubated with samples containing titrated concentrations of purified dl . the endogenous iggs purified from naive serum were used as a negative control to establish the background signal. the results shown in fig. b indicated that the observed shift in mass corresponding to the reduced deuterium incorporation in human il- was igg concentration-dependent, as denotes the apparent corrected sedimentation coefficient calculated using the buffer's density and viscosity. the % peak represents igg monomers. expected. the limit of detection was determined to be ng/l, indicating that this was the minimum igg concentration required to yield a detectable amount of deuterium protection. proof-of-concept study using sera from immunized mice-a total of nine ϳ -l serum samples from mice of various strains that were immunized with human il- were evaluated using our spr and hdx-lc/ms methods. each of the serum samples had previously been characterized as a "binder" by a single point elisa analysis. as shown in the results summary (table ), our analysis showed that the serum samples contained different percentages of human il- -specific antibodies, and exhibited significantly different average apparent affinities (fig. ). among the nine serum samples, four (samples a-c and e) showed no human il- binding at the highest tested concentration ( nm). of the other five samples that showed binding, the apparent affinities ranged from single/ double digit picomolars to single digit nanomolars, as shown by the spr binding data. the difference in affinities was influenced by variations in both k a and k d values. although serum samples d and f-h exhibited faster on-rates and slower off-rates, and each bar below the sequence represents an individual peptide. the residues in green indicate sequences covered by the identified peptides, and those in black represent uncovered regions. the residue in red (n) in the identified peptides was found to be highly deamidated. hence lower k d values or higher affinity interactions than those of serum i, serum i exhibited - -fold higher amounts of human il- -specific iggs than the others, based on its larger experimental r max values. in addition to observing differences in the antigen-specific apparent affinities and quantities of polyclonal iggs among the nine serum samples, we found that the il- binding epitopes among the serum samples were also different. as shown in fig. , the iggs from serum h (panel c) protected the region encompassing residues - from deuterium exchange, indicating that this serum sample contained iggs that were specific to this region. similarly, the iggs from serum i (fig. , panel d) also showed protection on the same region as that of serum h, with a relatively less extensive mass shift that was likely due to weaker affinity to human il- . in contrast, the iggs from serum c (fig. , panel b) showed no protection at this region, as the exchange observed there was equivalent to that of the control, containing no iggs (panel a), suggesting that either this serum sample contained no iggs targeting this region or that the level of iggs present was below the detection limit of the method ( ng/l). the results obtained using both spr and hdx lc/ms were consistent, in that the non-binders showed no binding of any region in human il- . for those samples that exhibited epitope protection, a diverse range of epitopes were identified. as depicted in fig. , antibody responses directed toward various helix and loop regions of the human il- protein were detected. of the amino acids in human il- , were shown to be protected, indicating that % of the sequence elicited antibody responses. while serum g exhibited the most diverse responses, serum f, which contained antibodies that were exclusively directed against the c terminus of the protein (table ) , exhibited the least diverse response. integration of the workflow process into our antibody generation platform-given the success of our methods in differentiating the antibody responses among different immunized animals, we integrated the qar workflow into our antibody generation platform to help guide decision-making in future immunization campaigns. the flowchart shown in fig. indicates the time frames of the qar workflow components. approximately weeks after the immunization of animals with antigen or antigen/adjuvant mixtures, after the induction of an effective antibody response, serum samples are evaluated by elisa to determine the presence and titer of antibodies. if the figure . characterization of binding interactions between mouse serum iggs and human il- . the binding interactions were monitored over -min association periods followed by -min dissociation periods. the binding response curves were obtained by measuring the binding of human il- to iggs captured on the biosensor surface that were titrated by -fold dilutions as follows: nm (light blue); nm (dark blue); nm (green); . nm (purple); and . nm (orange). kinetic fit was performed on serum samples d and f-i using the : langmuir kinetic binding model in the proteon analysis software, as illustrated by the black lines that overlay the response curves. titer results are positive, the serum samples are directly analyzed by spr to determine their apparent binding affinities and to quantify the percentage of antigen-specific iggs. at the same time, the remaining volume of the serum samples (ϳ l) is subjected to igg purification using the phynexus automated purification system. while the spr method can measure six serum samples at a time (six parallel sample injections based on the ϫ multiplex configuration) with a protocol that typically includes multiple binding cycles, the phynexus mea system can purify samples at a time with an estimated run time of . h. multiple mea systems can be used in parallel to analyze more than samples simultaneously. taking into account the post-purification sample preparation time (enrichment and buffer-exchange steps), purified igg samples can be ready for epitope mapping within h. meanwhile, analysis of the spr data can provide early information on the affinity and quantity of the antibodies. after sample and reagent preparation, the hdx lc/ms experiment is performed overnight using an automated protocol that accurately controls both the deuterium exchange and quenching reaction times for each sample. deuterium exchange, involving the sequential analysis of three time points, is performed and completed by the next day. because of the large amount of data collected, the ms data analysis requires about a half-day of data processing using two inhouse-derived proprietary software applications. thus, a comprehensive data package can be generated within days that provides useful feedback for guiding the immunization approach. the workflow can be applied at any point during the immunization campaign to track the development of antibody responses. implementation of the qar workflow to guide antibody recovery-the qar workflow was integrated into the baff antibody generation campaign, and its performance was evaluated. similar to the anti-il- sera analysis, the anti-baff sera analysis (table ) revealed differences in antibody quality among the serum samples. in addition to analyzing antibody binding to the human antigen that was used for immunization, we also analyzed antibody binding to the cynomolgus (cyno) monkey protein using spr. even though the human and cyno-baff proteins share % sequence identity, serum samples from mice p - and p - exhibited k d values for cyno-baff that were five times those for human baff, indic- ative of weaker interactions. we analyzed the binding epitopes of the various antibodies, focusing on the antibody responses to the regions in human baff that are known to be important for receptor interactions. our in-house analysis indicated that regions encompassing residues - , - , - , and - are important for function (data not shown). therefore, we focused our attention on iggs that were found to target these regions. among the serum samples, only five (p - , p - , p - , p - , and p - ) showed binding responses toward at least one of the prioritized epitope regions. although four of these samples exhibited similar double digit picomolar or better affinities and less than ϳ -fold shifts in binding to cyno-baff, the serum sample from mouse p - contained the highest amounts (Ͼ %) of human baff and cyno-baff-specific iggs. this mouse was subsequently selected for hybridoma fusion, and analysis of the subcloned monoclonal antibodies (table ) revealed that they exhibited low picomolar ec binding to human baff. in addition, blockade of receptor binding was confirmed, indicating that the selection of mice based on the epitope mapping of serum iggs is an effective strategy. to increase the probability of success in recovering criteriameeting functional antibodies from immunization campaigns, we explored the feasibility of applying methods that are typically used later in the antibody identification process, namely spr and mass spectrometry, to characterize the antibodies in sera from immunized mice. the profiling of immune responses using the standard elisa method to measure the effectiveness of immunization and vaccination has been widely applied and reported ( ) ( ) ( ) ( ) . here we used spr instead, because it has several advantages over the traditional elisa method. spr is a label-free technique that can be performed in real time, but most importantly, it can provide highly precise measurements for determining association and dissociation rate constants ( ) ( ) ( ) , which cannot be achieved by elisa. the application of spr for analyzing complex biological samples has been demonstrated in several reports ( - ) , although its use for these applications is still rare compared with other immunoassays. another advantage of using spr for both quantitative and qualitative evaluation of iggs in serum is the ease of including a "purification" step in which iggs are captured by protein a/g onto the sensor surface. protein a/g has affinity for mouse igg isotypes , a, b, and . therefore, the use of this reagent ensures that the antigen affinities of all the antibodies in the mouse serum will be effectively evaluated and that the resulting binding signal is highly specific for the iggs. furthermore, the amount of antigen-specific iggs present in the serum can be quantified through calculations from the r max values. according to our results using reconstituted samples, the detection limit for high affinity, low picomolar binding antibodies in serum using spr was ϳ ng/l. epitope mapping to elucidate both the specificity of the antibody responses and their diversities is crucial for obtaining antibodies with a desirable mechanism of action ( , ) . traditionally, the antigen-antibody contact surfaces have been determined by methods such as high resolution x-ray crystallography and site-directed mutagenesis ( , ) . however, these methods are not applicable for our purpose, because our samples contain antibody mixtures. in addition, the amount of material available for analysis and the time frame for acquiring epitope information are both limited. in search of a suitable technique, we initially explored peptide microarray ( , ) , in which a library of linear synthetic peptides is used to screen for igg-binding epitopes. samples containing mabs with known binding epitopes, as well as sera from both naive animals and animals immunized with human il- , were tested using these methods. however, we were unable to identify specific epitopes for either the mab or the antibodies present in the anti-il- sera (data not shown). regardless, these findings provided two important insights. first, we learned that the binding epitopes of the control mabs were conformational, rather than linear, and second, we learned that the presence of serum proteins probably interfered with the methods. therefore, we considered alternative techniques that could facilitate the identification of native protein conformational epitopes in solution with high resolution. ultimately, we selected hdx coupled with lc/ms due to its low material consumption, relatively high throughput via robotic automation, and high sensitivity. the combination of hydrogen deuterium exchange with mass spectrometry has proven to be increasingly powerful for characterizing protein conformations, structural dynamics, and protein-protein interactions ( ) . even though hdx is an established method and had successfully been applied in-house to map epitopes of purified mabs at high concentrations, several challenges exist for the evaluation of iggs in serum. therefore, additional method development was required to improve the feasibility of this approach. one major challenge is the matrix effects from serum, which could produce significant interference if injected directly into the instrument. to address this issue, we developed a small scale affinity purification protocol using the phynexus system to purify iggs from the serum using protein a/g columns. this platform was selected because of the availability of system automation and its relatively high throughput, enabling the purification of serum samples simultaneously. the purification scheme was optimized to achieve maximal igg recovery and purity, and our results showed that highly pure iggs could be obtained from serum samples. the second chal- lenge involved the sensitivity of the method for detecting antibody epitopes from a mixture of iggs, each of which is present at a different concentration. to improve the sensitivity, several modifications were made in the sample preparation, including the use of concentrators to increase the igg concentration and ph adjustment via buffer exchange to further optimize the deuterium exchange efficiency. with the incorporation of these additional sample-processing steps, it was important to determine that the iggs were recovered at sufficient concentrations for signal detection. an experiment in which a high affinity mab with known binding epitopes was evaluated at different concentrations showed that the sensitivity of the method was ϳ ng of antigen-specific igg per l of serum. this high sensitivity can facilitate the detection of less abundant iggs exhibiting unique epitopes, maximizing the diversity of antibodies that can be recovered. after establishing the highly sensitive spr and hdx lc/ms methods for detecting antigen-specific antibodies in serum, we tested their performance using sera from immunized mice. nine serum samples collected from various strains of mice, which were immunized with human il- using different protocols, were analyzed with these methods in a "proof-of-concept" study. these samples were previously classified as binders by single point elisa analysis. as anticipated, the results of these analyses allowed us to differentiate the antibody responses among the immunized mice with respect to apparent antigen affinity, amount of antigen-specific activity, and epitope diversity. to inhibit il- function, the development of high affinity anti-il- antibodies that target different epitopes of the il- protein, resulting in either the disruption of il- and il- receptor binding or the disruption of il- and il- ␣-receptor binding, is required ( ) . our analysis of the nine different anti-il- mouse sera revealed important binding affinity and epitope information that allowed us to differentiate the quality of the antibody responses. although five of the serum samples were found to contain antibodies that bound to the surface of il- that interacts with the il- receptor (epitope - ), two sera (d and g) contained additional antibodies that bound to the surface of il- that interacts with the il- ␣-receptor (epitopes - (d) and epitopes - and - (g)). to maximize the recovery of functional antibodies, serum g therefore would be the best choice for subsequent antibody generation, as it contained high affinity antibodies that could target both mechanisms of action. although hybridoma fusions were not performed to assess the recovery of functional monoclonal antibodies targeting human il- , they were performed to track and assess the antibodies generated for the baff program (see below). during development of the qar workflow, a top priority was to ensure that the time frame for generating the data package was short enough to enable timely decision-making. as reflected by our selected methods, throughput was an essential consideration. according to our assessment, the complete data package can be compiled within business days, due in large part to the automation of our platform methods (multiplex spr array, tip column-based walk away serum purification, and liquid-handling hdx reaction control), minimizing the need for extensive manual work. furthermore, with the implementation of in-house ms software programs for automating the hdx data analysis (beyond the scope of this paper), the entire workflow process can be efficiently executed and can support additional iterations if necessary. as shown in tables and , we used the qar workflow to guide the development of therapeutic anti-baff antibodies, and we demonstrated that the data package contributed to the successful retrieval of functional anti-baff antibodies in a timely manner. we focused on the identification of immunized animals that produced high affinity antibodies that targeted well defined epitopes for use in hybridoma fusions. the comprehensive data package generated again confirmed that our methods can provide clear differentiation among the antibody responses in various animals. the finding that our methods consistently provided in-depth and timely information for directing the development of high quality antibodies suggests that they will have widespread utility in guiding therapeutic antibody production in the future. in summary, we have demonstrated that a combination of sensitive methods can be integrated into the antibody generation platform to help identify animal hosts exhibiting high affinity antibody responses and maximal epitope diversities. the combination of spr and hdx lc/ms enables the comprehensive quantitative and qualitative evaluation of antibodies present in serum samples. while spr analysis provides antibody affinity information and antigen-specific antibody quantification, ms analysis provides information about the structural interactions between serum antibodies and their antigens. thus, these are complementary methods that reveal unique information that cannot be obtained using conventional methods. the proposed workflow process can also be executed at a speed that the biopharmaceutical industry demands to discover novel epitopes for new therapeutic modes of action. in addition, the qar workflow can be useful in guiding the development of robust vaccines. the described methods can be used to monitor the development of novel immunogens, vaccine preparations, or delivery systems, at the early stages of development, to help predict their efficacy and safety. to our knowledge, this is the first report that demonstrates how the described methods can be implemented to produce in-depth information that distinguishes antibody responses and guides the production of therapeutic antibodies. we intend to follow up with additional studies to demonstrate the utility of this workflow in future therapeutic antibody and vaccine discovery programs. recombinant human il- , derived from escherichia coli, was purchased from r&d systems (minneapolis, mn) . mouse anti-il- mab clone dl , derived from a previous campaign, was produced by transient expression in cho cells and purified using mabselect sure-based affinity chromatography (ge healthcare). the purified mab was formulated in mm sodium acetate, ph . , buffer. to determine the detection limits of our methods, eight -l naive mouse serum samples containing different antibody concentrations ranging from g/l to . ng/l were prepared by serial dilution. recombinant human baff with a his tag and its receptor taci-human-fc chimeric protein were purchased from r&d systems. anti-baff mab supernatants were produced using hybridomas generated using spleen cells from mouse p - , followed by cloning and subcloning using limiting dilution. antibody isotypes were determined using the mouse isostrip tm monoclonal antibody isotyping kit (santa cruz biotechnology, dallas, tx). naive mouse pooled serum ( . m filtered) from mixed strains was purchased from bioreclamationivt (westbury, ny). serum samples from nine mice of different strains (balb/c, c bl/ , and swiss webster) immunized with human il- were stored at Ϫ °c and used for the proof-ofconcept analysis. mice of various mhc haplotypes were immunized twice with human baff. serum samples were obtained a week after the second immunization and stored at Ϫ °c. after - days, the mice were boosted with baff, and the spleens were harvested for hybridoma fusion using standard techniques. hybridoma screening was carried out with luminex beadbased assays using human baff. positive candidates were cloned by limiting dilution. to measure the ec value of the mab-baff interactions, magnetic luminex beads coated with human baff were incubated with a dilution series of the mabs for h at °c. after the mab-baff-bound beads were washed, they were incubated with a biotinylated anti-mouse fc-specific antibody ( g/ml) for h and then incubated with streptavidin-phycoerythrin ( g/ml) for another min for signal detection. the anti-baff hybridoma subclones were further characterized by their ability to block baff-taci interactions (ic ). in this assay, the baff-coated beads were first incubated with samples of diluted mabs for h at °c. taci-human fc ( ng/ml) was then added, and the mixtures were incubated for h, followed by the addition of biotinylated anti-human fc-specific antibody ( g/ml) for h. taci binding was then detected after a -min incubation with streptavidin-phycoerythrin ( g/ml). phynexus automation system and setup-mouse antibodies were purified in a fully automated -channel phynexus mea purification system (phynexus, inc.) using phytip columns packed with l of proplus resin (part no. ptr - ) in -ml disposable pipette tips. the proplus resin is a protein a-derived ligand that has attributes of both protein a and g, with binding specificities for both mouse igg and igg a isotypes. mouse serum samples were diluted in l of pbs and then transferred into the individual wells of a -deep well plate. in addition to the sample plate, two -well reagent plates containing the washing, elution, and neutralization buffer for each of the purification steps were also prepared. although the sample plate was placed in position on the mea instrument platform and kept at room temperature, the reagent plates were placed in positions and on top of a chiller to maintain the temperature at ϳ - °c. a number of phytip columns corresponding to the number of samples were transferred into row of an empty -well pipette tip box located at position . similarly, the same number of standard -ml rainin lts pipette tips were transferred into row of the same box for use in the last neutralization step, to transfer the basic solution to the eluent. after the pre-packed phytip columns and reagent plates were loaded onto the mea instrument platform, the mouse antibodies were isolated using a method programmed with phynexus mea software (version . . ) consisting of the following steps: ) pre-wash and equilibration of the resin with pbs; ) capture of iggs from the diluted -l mouse serum samples with the proplus affinity resins; ) washes with pbs and pbs plus m nacl to remove other serum proteins; ) elution of bound iggs from the resins with mm sodium acetate, ph . , three times, followed by one elution with mm sodium acetate, ph . , to ensure the full removal of any remaining bound iggs from the resins; and ) neutralization using % (by volume) of mm sodium acetate, ph . , to bring the final formulation to mm sodium acetate, ph . . the details of the purification steps with the respective operating configurations included in the protocols are listed in table . in table , one cycle represents one intake and one expulsion of solution through the proplus resin at a speed defined by the flow rate. the flow rate for capturing was maintained at . ml/min to ensure sufficient contact time between the serum sample and the resins. "delay" is the delay time between each intake and expulsion step to allow for contact with the solution. the position axis is the distance from the tip to the bottom of the plate; these values were pre-set using the settings recommended by the manufacturer. detection of iggs by sds-page-for protein detection, the samples were analyzed under reducing and non-reducing conditions using nupage - % bistris gels in nupage mops sds running buffer with the novex xcell surelock mini-cell system (invitrogen) at v for min. the gels were stained using instant blue coomassie-based solution (expedeon, san diego). images of the gels were obtained using a canoscan f scanner. quality evaluation of the purified iggs by analytical ultracentrifugation (auc)-the quality of the purified iggs was evaluated by sedimentation velocity experiments using absorbance optics on a beckman xli analytical ultracentrifuge (beckman coulter, inc.). the concentrated iggs from the elution pools were diluted in mm sodium acetate, ph . , to . mg/ml, and then l of each was loaded into the sample chamber, whereas buffer was loaded into the reference chamber of an auc cell that was assembled with standard doublesector centerpieces and quartz windows. the experiment was conducted at °c using an an ti four-hole rotor spinning at , rpm. the sedimentation process was monitored by collecting absorbance data at nm using the xl-i operating software. the collected data were analyzed using the continuous c(s) distribution model in the sedfit software (version . c) to provide the distribution of sedimentation coefficients. the s values obtained with the c(s) distribution in sodium ace-tate buffer were converted to s , w value with sednterp (version . ) using the measured density and viscosity of the buffer. were performed at °c using a proteon xpr instrument (bio-rad). after pre-conditioning, a glm chip docked into the machine with sds, naoh, and nacl across six channels in both directions, and protein a/g was amine-coupled onto the chip surface using a standard coupling protocol with the following steps. ) the six individual flow channels were activated in parallel by injecting a freshly mixed solution of . m n( -dimethylaminopropyl)-nЈ-ethylcarbodiimide in . m n-hydroxysuccinimide. ) protein a/g prepared at g/ml in sodium acetate, ph . , was immobilized on the activated channels. ) the excess reactive esters were deactivated with m ethanolamine. each step was performed at a flow rate of l/min for min. protein a/g at a high surface density ( response units (ru)) was coupled onto the chip surface to capture the serum iggs and characterize the binding affinities. characterization of serum igg-antigen binding kinetics-a small volume of each serum sample ( l) was diluted -fold into the pbs/tween/edta running buffer. six diluted samples were then injected simultaneously over the six available vertical channels at a flow rate of l/min, during which the iggs were captured by the protein a/g. the capture time was monitored to achieve a high antibody surface density ( - ru). because of the high abundance of mouse iggs in the serum samples, a capture time of ϳ - s was sufficient to reach the targeted density level. following a blank buffer injection of s over the six individual igg surfaces, five titrated concentrations of antigen in -fold dilutions were simultaneously injected in the horizontal direction. the binding interactions were monitored over a -min association period and a -min dissociation period using a high flow rate of l/min. the last channel was injected with pbs/tween/edta for reference subtraction. the surfaces were regenerated with two -s pulses of glycine, ph . , at l/min in both horizontal and vertical directions to allow the capture of different serum iggs for kinetic binding measurements. data analysis-the collected sensorgram data were processed by reference subtraction using inter-spots in addition to double-referencing with the in-line buffer blank. the integrated proteon manager software version . . . was used to fit the data with the langmuir model describing a : binding stoichiometry. k a is the association rate constant for the antibody-antigen binding; k d is the dissociation rate constant of the antibody-target complex, and k d is the equilibrium dissociation constant, defined by the k d /k a ratio. in addition to determining the apparent binding affinity from the fitted binding curves, the experimental binding capacity of the surface, r max , was also derived. a comparison of the experimental r max and theoretical r max provides the percentage of iggs that are specific to the antigen among the total iggs present in the serum (equation ). to obtain the theoretical r max , equation was used. % of antigen-specific iggs ϭ experimental r max theoretical r max ϫ where mw analyte is the molecular weight of the antigen; mw ligand is the molecular weight of iggs captured on the sensor surface, and n is the binding stoichiometry of the reaction. the immobilized ligand level is the level of captured serum iggs, which includes both endogenous iggs and antigen-specific iggs. sample preparation-to prepare samples for epitope mapping analysis by mass spectrometry, a diluted sample (ϳ ml) of each purified mouse igg was concentrated using a , molecular weight cutoff centrifugal filter device (millipore, billerica, ma). the loaded filter devices were spun in a swinging bucket rotor at ϫ g for min at °c. concentrates of ϳ l were collected and subsequently buffer-exchanged with pbs using zeba spin desalting columns (thermofisher scientific) to maintain a physiological ph for the binding interactions and to ensure consistent deuterium exchange. to determine the minimal amount of antigen needed for full sequence coverage, different amounts of antigen ( -, -, and -l aliquots) were diluted in pbs to a total volume of l. after analysis, the lowest amount of antigen that yielded the highest sequence coverage was selected and that amount was then directly added to the purified antibody sample to yield a total volume of l. deuterium exchange-using the h/d-x pal tm robotic system (leap technologies, inc.), samples containing iggs incubated with human il- were added to a d o-containing buffer, and the reactions were subsequently quenched at consistent times and temperatures using an automated sample run list. two separate sample compartments were used for each experiment. one compartment was kept at °c for d o labeling (deuterium exchange), and the other was maintained at °c for reaction quenching. after the samples were prepared, they were transferred into chromacol screen top vials (thermo-fisher scientific) and stored in individual positions within the °c sample compartments. a work list was written using hdx-director (version . . . ) with the following steps: ) dilution and mixing of l of sample with l of deuterium exchange buffer ( mm nah po in d o, ph . ), and ) incubation of the mixture at °c for various time periods ( , , and s), during which the exchange reaction took place. at the end of each incubation period, l of each mixture was transferred to a vial at °c containing l of quench solution ( m guanidine hydrochloride, . m tris( -carboxyethyl)phosphine hydrochloride) and mixed thoroughly. finally, l of each quenched reaction mixture was injected onto a poroszyme immobilized pepsin column (life technologies, inc.) for min, where the proteins were digested and subsequently desalted on an acquity uplc beh c vanguard pre-column (waters technologies) for an additional min prior to injection into a beh c column (waters technologies) for lc reverse phase separation. lc/ms system setup-after the digested peptides were injected onto the c column inside the column/valve °c temperature-controlled compartment, a gradient solvent system consisting of precooled mobile phase a ( . % formic acid, % hplc water, % acetonitrile) and mobile phase b ( . % formic acid, % hplc water, % acetonitrile) was used. the c column was first equilibrated at % b during the -min on-column pepsin digest and then the gradient was moved to % b during the -min peptide wash on the vanguard precolumn. the chromatographic separation was performed at °c at a flow rate of l/min by applying the gradient of mobile phase b from % at min to % at min to % at . - . min and finally to % at - min. after chromatographic separation, the sample entered the orbitrap fusion tm mass spectrometer operated in positive electrospray ionization mode. the employed method included activated types of collision-induced dissociation and electron transfer dissociation when identifying control peptides, using a resolution of , , a minimum signal of , an isolation width of . , and a normalized collision energy of . v. the s-lens radiofrequency (rf) level was set at %. for peptide identification, the data were collected in profile mode for the full ms scan and in centroid mode for the collision-induced dissociation and electron transfer dissociation ms/ms scans. the data were collected over a mass range of - da. for deuterated samples, no ms/ms data were collected. data processing-the collected raw lc-ms/ms fragmentation data from the pepsin digest were analyzed using various software tools for peptide identification. the fragmentation data of the control sample (antigen in the absence of iggs) were analyzed using proteome discover . (thermo scientific) and pmi byonic (protein metrics, inc.) and compared with the given sequence to generate a list of peptides and retention times. raw lc-ms/ms data files were preprocessed and converted to ascii format using proprietary in-house software. the identified peptides were then matched and summarized. the epitopes were determined by calculating the differences in average mass shifting induced by the protection of the region from deuterium labeling. author contributions-d. y. wrote the first draft of the manuscript. d. y. and l. f. developed various methods, performed the experiments, and analyzed the data. m. l. evaluated the peptide microarray and facilitated the implementation of the methods. k. t. provided hdx lc/ms experimental support and software for data analysis. r. k. provided technical guidance and oversight throughout the entire process. s. s. conceptualized the process and provided oversight. all authors discussed the results and implications and commented on the manuscript at all stages. derivation and diversification of monoclonal antibodies paul ehrlich's magic bullet concept: years of progress monoclonal antibodies-a proven and rapidly expanding therapeutic modality for human diseases therapeutic antibodies for autoimmunity and inflammation monoclonal antibodies: versatile platforms for cancer immunotherapy marketed therapeutic antibodies compendium phage display technology for human monoclonal antibodies an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery the molecular and cellular basis of affinity maturation in the antibody response clonal selection and learning in the antibody system generation of memory b cells inside and outside germinal centers translating innate immunity into immunological memory: implications for vaccine development therapeutic antibodies, vaccines and antibodyomes review of polyclonal antibody production procedures in mammals and poultry critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations immunisation strategies for antibody production challenges and responses in human vaccine development development and characterization of a standardized elisa including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines enzyme immunoassay and enzymelinked immunosorbent assay solid phase enzyme-linked immunosorbent assay (elisa) for anti-sheep erythrocyte antibody in mouse serum rapid antibody responses by low-dose, single-step, dendritic cell-targeted immunization quality and kinetics of the antibody response in mice after three different low-dose influenza virus vaccination strategies immunization with a vaccine combining herpes simplex virus (hsv- ) glycoprotein c (gc) and gd subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of hsv- dna in guinea pigs compared to immunization with gd alone surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress comparison of the results obtained by elisa and surface plasmon resonance for the determination of antibody affinity real-time, labelfree monitoring of tumor antigen and serum antibody interactions serodiagnostic comparison between two methods, elisa and surface plasmon resonance for the detection of antibodies of classical swine fever comparison of surface plasmon resonance imaging and enzyme-linked immunosorbent assay for the detection of antibodies against iridovirus in rock bream (oplegnathus fasciatus) determination of the mechanism of action of anti-fast antibody by epitope mapping and homology modeling epitope mapping for monoclonal antibody reveals the activation mechanism for ␣v␤ integrin threedimensional structure of an antigen-antibody complex at . a resolution site-directed mutagenesis in epitope mapping evaluation of pepscan analyses for epitope mapping of anti-muc monoclonal antibodies-a comparative study and review of five antibodies. anticancer res antibody signatures defined by high content peptide microarray analysis applications of hydrogen/deuterium exchange ms from to molecular and structural basis of cytokine receptor pleiotropy in the interleukin- / system solution structure of human il- and implication for receptor binding acknowledgments-we thank the antibody generation group for the animal immunizations and zhong-fu huang for helping with purification instrumentation. key: cord- - vo o w authors: matis, louis a.; rollins, scott a. title: complement-specific antibodies: designing novel anti-inflammatories date: journal: nat med doi: . /nm - sha: doc_id: cord_uid: vo o w nan / . molec. bini. , - ( ) . . barbas, c. f., amberg, w., somincsits ·-----··---------, the immunoglobulin variable gene repertoire generates enormous diversity through both combinatorial and somatic mutational mechanisms. consequently, it has the capacity to produce high-affinity, exquisitely specific antibodies to a vast number of potential antigenic targets. also, the constant domains of antibody molecules can harness effector mechanisms of the humoral and cellular immune systems in vivo'. thus, monoclonal antibodies hold great promise for application to a wide range of diagnostic and therapeutic clinical settings, as evidenced by the current clinical use of monoclonal antibody-derived products in transplantation', myo-nature mt:djcine, volume , number , august cardial revascularization' and tumour imaging'. recent advances in the molecular engineering of immunoglobulin genes have further broadened the potential utility of monoclonal antibody-based therapeutics. for example, fab fragments, as well as covalently linked single-chain fragments consisting of the heavy and light chain variable regions (scfvs), have been derived that retain the specificity and affinity of the original intact antibody'-'. these smaller molecules display distinct properties, such as reduced serum halflife and enhanced tissue penetration, which may be particularly useful for cer-tain clinical situations such as tumour imaging and therapy, or for the treatment of acute inflammation. in addition, they are amenable to modifications, such as covalent linkage to genes encoding toxins, enzymes or cytokines, or to the incorporation of dual specificity or catalytic function, which can impart novel functional properties to these monoclonal antibody-derived molecules .. ._ •. further, advances in combinatorial library and phage display technology may allow the recapitulation in vitro of the natural processes of selection and repertoire maturation to yield high-affinity, functionally useful antibodies without the requirement for immunization and hybridoma production'. • chronic application of monoclonal antibody therapy requires 'humanization' to prevent a host immune response from developing. current methods to accomplish this include complementaritydetermining region (cdr) grafting, or replacement of the hypervariable loops of a human antibody with those of the murine monoclonal antibody of desired specificity, selection by phage display", or potentially using humab mice, in which endogenous ig loci have been inactivated by mutation and then replaced by large segments of human immunoglobulin genomic loci '". complement proteins represent an attractive target for the development of monoclonal antibody-based antiinflammatory therapeutics. the complement system is composed of more than serum proteins that interact in a precise series of enzymatic cleavage and membrane binding events leading to the generation of products with immunoprotective, immunoregulatory, and proinflammatory properties • complement can be activated through either of two distinct enzymatic cascades, referred to as the classical and alternative pathways (fig. ) . the classical pathway is generally initiated by the interaction of c q with antibody/ antigen complexes, whereas the alternative pathway is initiated by deposition of c b on a variety of substrates including activation antibody/antigen complexes cl q -activated cl bacterial lipopolysaccharide and cell membranes. the formation of c b is necessary for the amplification and progression of the complement cascade through both pathways. it is the primary opsonin for many pathogenic microorganisms and, in addition, promotes the clearance as well as solubilization of immune complexes. both the classical and alternative pathways converge at cs, which is cleaved to form products with multiple proinflammatory effects (see fig. ). csa is the most potent anaphylatoxin, inducing alterations in smooth muscle and vascular tone, as well as vascular permeability '. it is also a powerful 'chemotaxin' and activator of both neutrophils and monocytes. csa-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of multiple additional inflammatory mediators, including hydrolytic enzymes, cytokines, arachidonic acid metabolites and reactive oxygen species. cs cleavage also leads to the formation of csb- , or the membrane attack complex (mac). there is now strong evidence that the mac may play an important role in inflammation in addition to its role as a lytic pore-forming complex, as it also stimulates the release of many of the same proinflammatory molecules as csa and promotes thrombosis following deposition on platelets and endothelium with improved methodology for the detection of activated components in inflamed tissue and biological fluids, the complement system has been increasingly implicated as contributing to the pathogenesis of numerous disease states (see tabler•. these include immunological diseases characterized by antibodymediated classical pathway activation, as well as vascular inflammatory conditions in which the alternative pathway is induced after reperfusion of ischaemic tissue. distinct mechanisms of complement activation are associated with some diseases, such as alzheimer's disease, since the [ -amyloid protein has been shown to bind to c q directly and thereby to activate the classical pathway in vitro • in many conditions there is evidence for simultaneous activation of both classical and alternative pathways. because activation of cs represents a critical step in the inflammatory cascade and as there are no known naturally existing molecules that uniquely block cs activation, the cs molecule represents an attractive target for development of a monoclonal antibody-based complement inhibitor. therapeutic inhibition of the complement cascade at cs would block the formation of the potent inflammatory mediators csa and csb- via both the classical and alternative pathways, while preserving the patient's • ······················ new technology ability to generate the critical immunoprotective and immunoregulatory functions of c b-mediated opsonization and immune clearance. therefore, we have developed recombinant cs-specific monoclonal antibodies and their engineered derivatives as soluble antiinflammatory biopharmaceuticals. the principle of anti-cs monoclonal antibody therapy of inflammatory disease has been examined in several preclinical models. using a monoclonal antibody specific for mouse cs, we have shown that systemic anti-cs monoclonal antibody administration efficiently inhibited complement in vivo (inhibiting serum haemolytic activity for as long as six to seven days after a single intravenous injection), and that treatment with anti-cs monoclonal antibody was therapeutically effective in two distinct models of immune complex nephritis and autoimmune disease (y. wang et al., manuscript in preparation). in these models, continuous treatment with anti-cs monoclonal antibody for up to six months was not associated with any negative side effects. in murine collageninduced arthritis, anti-cs monoclonal antibody therapy not only prevented the onset of disease, but, most importantly, was also highly effective in ameliorating the course of established arthritis'". in an ex vivo model of cardiopulmonary bypass (cpb)-induced inflammation, administration of a prototype anti-human cs monoclonal antibody completely blocked csa and csb- generation in whole human blood, as well as both the platelet and leukocyte activation that normally occur during cpb". thus, anti-cs monoclonal antibody therapy effectively modulated inflammatory responses in both in vivo and ex vivo models. to generate a human cs-specific monoclonal antibody for clinical development, mice were immunized with purified human cs and candidate monoclonals were screened in high throughput in vitro assays for their ability to block both csa and csb- generation via the classical and alternative pathways. from this effort a highly potent anti-cs monoclonal antibody was derived with very high affinity (k. < pm), capable of blocking complement activation at monoclonal antibody/cs molar ratios as low as o.s:l. of this monoclonal antibody have been cloned and several recombinant forms have been engineered. both recombinant fab and scfv variants have been derived and shown to bind cs with similar affinity and to block cs activation at the same molar ratio as the intact antibody. in addition, humanized recombinant anti-cs monoclonal antibody and scfv that retain the binding affinity and complement inhibitory activity of their murine counterparts have been produced by cdr grafting (m. evans, manuscript in preparation). the ability of an scfv derivative of a cs-specific monoclonal antibody to inhibit complement in vivo was tested by generating and administering intravenously a cs-specific scfv cross-reactive with primate complement'". this scfvinhibited serum complement haemolytic activity for up to hours following administration of a single intravenous bolus, consistent with the more rapid clearance of scfvs relative to intact monoclonal antibodies. cs inhibition by an intravenously administered scfv also demonstrated that the functional domains of the immunoglobulin constant region were not required for complement inhibition in vivo. the efficacy and pharmacokinetic profile of the anti-cs scfv suggest that it may be a useful antiinflammatory agent in acute settings such as cpb or ischaemia/reperfusion injury associated with myocardial infarction. in summary, we have shown that inhibition of cs activation with highaffinity monoclonal antibodies represents a novel and potentially safe and effective approach to ameliorate inflammation in a variety of clinical settings. the potent anti-cs activity of the scfv molecule further suggests that it may be possible to define minimal peptide sequences that retain cs inhibitory activity, and thereby ultimately design orally available small molecule peptidomimetics. with recent advances in antibody engineering, monoclonal antibody-based therapeutic approaches appear to be well poised to achieve their anticipated clinical potential. immunohistochemistry detection kit for researchers using rodent models in research. the histomouse sp kit from zymed laboratories is designed to detect mouse primary antibodies on mouse tissue and cells immunologically, without causing background staining (resulting from the anti-mouse secondary antibody binding to endogenous mouse igg in the sample). zymed's approach is said to block the endogenous mouse igg in the tissue. the new kit contains blocker, biotinylated secondary antibody, streptavidin-horseradish peroxidase, the aec substrate system, counterstain and mounting solution. it can be used to detect mouse, rabbit, rat and guinea pig primary antibodies on mouse or rat tissue and cells. building antibodies from their genes immunosuppressive therapy as a determinant of transplantation outcomes pharmacodynamics of chimeric glycoprotein lib/lila integrin antiplatelet anti· body fab e in high-risk coronary angioplasty correlative imaging with monoclonal antibodies in colorectal, ovarian, and prostate cancer making antibodies by phage display technology antibody-targeted modifications antigen-specific human monoclonal antibodies from mice engineered with human ig heavy and light chain y acs molecular organization and function of the complement system membrane signaling by complement c b- , the membrane attack complex clinical complementology: recent progress and future trends beta amyloid activates com rat, hamster, cat and guinea pig origin. it can be used in immunohistochemistry, immunoprecipitation, immunoassays, radioimmunoimaging, antibody-<lrug targeting to the lung, and in the estimation of lung vessel injury in animal models. neurotrophic factors, receptor and antibodies.both human and recombinant rat cil-iary neurotrophic factor (cntf) can be obtained from r&d systems. the activities of these cntf proteins have been measured in a human erythroleukaemic cell line (tf- ) bioassay as well as by their ability to support the survival and neurite outgrowth of cultured embryonic chick dorsal root ganglia. in addition, r&d systems offers two anti-cntf antibodies -a polyclonal and a monoclonal anti-human cntf. soluble gp protein and both monoclonal and polyclonal anti-human gp antibodies are available. the anti-gp antibodies block the cell-surface human gp -mediated bioactivities induced by il- , il- , lif and osm, in addition to cntf. rb (ab- ) staining of formalin-fixed, paraffinembedded normal human bladder after thermal pretreatment, left, and mib- (ab- ) staining of highly proliferative lung tumour. oncogene science recently announced the availability of two new antibodies directed against the rb tumour suppressor gene protein (pl ). rb (ab- ), clone lm . , is said to stain formalinfixed, paraffin-embedded human tissues and will immunoblot the rb protein. rb (ab- ), clone af , on the other hand, will react with the rb protein from a key: cord- -d iwkatn authors: henry, kevin a.; arbabi-ghahroudi, mehdi; scott, jamie k. title: beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: d iwkatn for the past years, phage display technology has been an invaluable tool for studies of protein–protein interactions. however, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. this review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. the filamentous bacteriophage (genera inovirus and plectrovirus) are non-enveloped, rod-shaped viruses of escherichia coli whose long helical capsids encapsulate a single-stranded circular dna genome. subsequent to the independent discovery of bacteriophage by twort ( ) and d 'hérelle ( ) , the first filamentous phage, f , was isolated in loeb ( ) and later characterized as a member of a larger group of phage (ff, including f , m , and fd phage) specific for the e. coli conjugative f pilus (hofschneider and mueller-jensen, ; marvin and hoffmann-berling, ; zinder et al., ; salivar et al., ) . soon thereafter, filamentous phage were discovered that do not use f-pili for entry (if and ike; meynell and lawn, ; khatoon et al., ) , and over time the list of known filamentous phage has expanded to over members (fauquet et al., ) , including temperate and gram-positivetropic species. work by multiple groups over the past years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in marvin, ; rakonjac et al., ; rakonjac, ) . in the mid- s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by smith and colleagues (smith, ; parmley and smith, ) . based on the ideas described in parmley and smith ( ) , groups in california, germany, and the uk developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (bass et al., ; devlin et al., ; mccafferty et al., ; scott and smith, ; breitling et al., ; kang et al., ) . this technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. many excellent reviews are available on phage-display libraries and their applications (kehoe and kay, ; bratkovic, ; pande et al., ) . however, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. these tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. a final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( table ) . the display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. display may be achieved by fusing dna encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type display on pviii, type display on piii, etc.), resulting in fully recombinant phage. much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type display if both recombinant and wild-type pviii genes are on the phage genome, type + display if the parmley and smith ( ), mcconnell et al. ( ) , rondot et al. ( ) hybrid (type and + systems) type + system < smith and scott ( ) , smith and petrenko ( ) pvi hybrid (type + system) yes < > kda hufton et al. ( ) pvii fully recombinant (type system) no ∼ > kda kwasnikowski et al. ( ) hybrid (type + system) yes < gao et al. ( ) pviii fully recombinant (landscape phage; type system) no ∼ - residues kishchenko et al. ( ) , petrenko et al. ( ) hybrid (type and + systems) type + system ∼ - > kda scott and smith ( ) , greenwood et al. ( ) , smith and fernandez ( ) pix fully recombinant (type + * system) yes ∼ > kda gao et al. ( ) hybrid (type + system) no < gao et al. ( ) , shi et al. ( ) , tornetta et al. ( ) asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. the copy number depends on polypeptide size; typically < copy per phage particle but for pviii peptide display can be up to ∼ % of pviii molecules in hybrid virions. the total number of pviii molecules depends on the phage genome size; one pviii molecule is added for every . nucleotides in the viral genome. recombinant gene is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type * + display). by far the most commonly used coat proteins for display are the major coat protein, pviii, and the minor coat protein, piii, with the major advantage of the former being higher copy number display (up to ∼ % of recombinant pviii molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number ( - per phage particle). while pviii display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than per virion (sidhu et al., ) . for the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in e. coli cells. early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (meynell and lawn, ) and that only the major coat protein, pviii, and the minor coat protein, piii, are targeted by antibodies (pratt et al., ; woolford et al., ) . thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la cruz et al. ( ) . the phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pviii display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pviii or piii (de la cruz et al., ) . as library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; geysen et al., ; knittelfelder et al., ) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; table ) , including malaria and human immunodeficiency virus type (hiv- ). when displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein were immunogenic in mice and rabbits (de la cruz et al., ; greenwood et al., ; willis et al., ; demangel et al., ) , and antibodies raised against the latter cross-reacted with the full-length protein. various peptide determinants (or mimics thereof) of hiv- gp , gp , gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (minenkova et al., ; di marzo veronese et al., ; de berardinis et al., ; scala et al., ; chen et al., ; van houten et al., van houten et al., , , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di marzo veronese et al., ; scala et al., ) . the list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( table ) . while in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (irving et al., ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (van regenmortel, ); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. more recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. immunization with phage displaying alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (frenkel et al., (frenkel et al., , esposito et al., ; tanaka et al., ) , possibly reduced amyloid plaque formation in mice (frenkel et al., ; solomon, ; esposito et al., ) , and may have helped maintain cognitive abilities in a transgenic mouse model of alzheimer's disease (lavie et al., ) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. yip et al. ( ) found that antibodies raised in mice against an erbb /her peptide could inhibit breast-cancer cell proliferation. phage displaying peptide ligands of an anti-ige antibody elicited antibodies that bound purified ige molecules (rudolf et al., ) , which may be useful in allergy immunotherapy. several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. for example, immunization with phage displaying follicle-stimulating hormone peptides on pviii elicited antibodies that impaired the fertility of mice and ewes (abdennebi et al., ) . phage displaying or chemically rubinchik and chow ( ) conjugated to sperm antigen peptides or peptide mimics (samoylova et al., a,b) and gonadotropin-releasing hormone (samoylov et al., ) are also in development. for the most part, peptides displayed on phage elicit antibodies in experimental animals ( table ) , although this depends on characteristics of the peptide and the method of its display: piii fusions tend toward lower immunogenicity than pviii fusions (greenwood et al., ) possibly due to copy number differences (piii: - copies vs. pviii: estimated at several hundred copies; malik et al., ) . in fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (bsa) and keyhole limpet hemocyanin (klh; melzer et al., ; su et al., ) , and has comparatively few endogenous b-cell epitopes to divert the antibody response from its intended target (henry et al., ) . excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. the overall picture is considerably bleaker than that painted by table , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. irving et al. ( ) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. while the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (henry et al., ) . this may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see immunological mechanisms of vaccination with filamentous phage below). the filamentous phage has been used to a lesser extent as a carrier for t-cell peptide epitopes, primarily as fusion proteins with pviii ( table ) . early work, showing that immunization with phage elicited t-cell help (kölsch et al., ; willis et al., ) , was confirmed by several subsequent studies (de berardinis et al., ; ulivieri et al., ) . from the perspective of vaccination against infectious disease, de berardinis et al. ( ) showed that a cytotoxic t-cell (ctl) epitope from hiv- reverse transcriptase could elicit antigen-specific ctls in vitro and in vivo without addition of exogenous helper t-cell epitopes, presumably since these are already present in the phage coat proteins (mascolo et al., ) . similarly, efficient priming of ctls was observed against phage-displayed t-cell epitopes from hepatitis b virus (wan et al., ) and candida albicans (yang et al., a; wang et al., wang et al., , d , which, together with other types of immune responses, protected mice against systemic candidiasis. vaccination with a combination of phagedisplayed peptides elicited antigen-specific ctls that proved effective in reducing porcine cysticercosis in a randomized controlled trial (manoutcharian et al., ; morales et al., ) . while the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (plotkin, ) , in certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (gram et al., ) . e. coli f a-g adhesin (van gerven et al., ) , hepatitis b core antigen (bahadir et al., ) , and hepatitis b surface antigen (balcioglu et al., ) all elicited antibody responses when displayed on piii, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. phage displaying schistosoma mansoni glutathione s-transferase on piii elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (rao et al., ) . two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. immunization with phage displaying the e idiotype scfv (mimicking a vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from vibrio anguillarum challenge (xia et al., ) . a chemically linked phage-bcl tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a b-cell lymphoma model (roehnisch et al., ) , and was welltolerated and immunogenic in patients with multiple myeloma (roehnisch et al., ) . one study of dna vaccination with an anti-laminarin scfv found that dna encoding a piii-scfv fusion protein elicited stronger humoral and cell-mediated immune responses than dna encoding the scfv alone (cuesta et al., ) , suggesting that under some circumstances, endogenous phage t-cell epitopes can enhance the immunogenicity of associated proteins. taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional t-cell help to displayed or conjugated proteins. however, the low copy number of piii-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (figure ) . the phage particle is immunogenic without adjuvant in all species tested to date, including mice (willis et al., ) , rats (dente et al., ) , rabbits (de la cruz et al., ) , guinea pigs (frenkel et al., ; kim et al., ) , fish (coull et al., ; xia et al., ) , non-human primates (chen et al., ) , and humans (roehnisch et al., ) . various routes of immunization have been employed, including oral administration (delmastro et al., ) as well as subcutaneous (grabowska et al., ) , intraperitoneal (van houten et al., ) , intramuscular (samoylova et al., a) , intravenous (vaks and benhar, ) , and intradermal injection (roehnisch et al., ) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. antibodies are generated against only three major sites on the virion: (i) the surface-exposed n-terminal ∼ residues of the pviii monomer lattice (terry et al., ; kneissel et al., ) ; (ii) the n-terminal n and n domains of piii (van houten et al., ) ; and (iii) bacterial lipopolysaccharide (lps) embedded in the phage coat (henry et al., ) . in mice, serum antibody titers against the phage typically reach : - : after - immunizations, and are maintained for at least year postimmunization (frenkel et al., ) . primary antibody responses against the phage appear to be composed of a mixture of igm and igg b isotypes in c bl/ mice, while secondary antibody responses are composed primarily of igg and igg b isotypes, with a lesser contribution of igg c and igg isotypes (hashiguchi et al., ) . deletion of the surface-exposed n and n domains of piii produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van houten et al., ) . figure | types of immune responses elicited in response to immunization with filamentous bacteriophage. as a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. the phage likely activates t-cell independent antibody responses, either via phage-associated tlr ligands or cross-linking by the pviii lattice. after processing by antigen-presenting cells, phage-derived peptides are presented on mhc class ii and cross-presented on mhc class i, resulting in activation of short-lived ctls and an array of helper t-cell types, which help prime memory ctl and high-affinity b-cell responses. frontiers in microbiology | www.frontiersin.org although serum anti-phage antibody titers appear to be at least partially t-cell dependent (kölsch et al., ; willis et al., ; de berardinis et al., ; van houten et al., ) , many circulating pviii-specific b cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates t-cell-independent b-cell responses in addition to highaffinity t-cell-dependent responses (murira, ) . filamentous phage particles can be processed by antigen-presenting cells and presented on mhc class ii molecules (gaubin et al., ; ulivieri et al., ) and can activate t h , t h , and t h helper t cells (yang et al., a; wang et al., d) . anti-phage t h responses were enhanced through display of ctla- peptides fused to piii (kajihara et al., ) . phage proteins can also be cross-presented on mhc class i molecules (wan et al., ) and can prime two waves of ctl responses, consisting first of short-lived ctls and later of long-lived memory ctls that require cd + t-cell help (del pozzo et al., ) . the latter ctls mediate a delayed-type hypersensitivity reaction (fang et al., ; del pozzo et al., ) . the phage particle is self-adjuvanting through multiple mechanisms. host cell wall-derived lps enhances the virion's immunogenicity, and its removal by polymyxin b chromatography reduces antibody titers against phage coat proteins (grabowska et al., ) . the phage's singlestranded dna genome contains cpg motifs and may also have an adjuvant effect. the antibody response against the phage is entirely dependent on myd signaling and is modulated by stimulation of several toll-like receptors (hashiguchi et al., ) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (molenaar et al., ) , particularly of the liver and spleen, where it is retained for days (zou et al., ) , potentially activating marginal-zone b-cell responses. thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former soviet union and eastern europe (reviewed in sulakvelidze et al., ) . the filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of inovirus and plectrovirus includes many pathogens of medical importance, including salmonella, e. coli, shigella, pseudomonas, clostridium, and mycoplasma species. in an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "trojan horse" to introduce various antibacterial agents into cells. m and pf phage engineered to express either bglii restriction endonuclease (hagens and blasi, ; hagens et al., ) , lambda phage s holin (hagens and blasi, ) or a lethal catabolite gene activator protein (moradpour et al., ) effectively killed e. coli and pseudomonas aeruginosa cells, respectively, with no concomitant release of lps (hagens and blasi, ; hagens et al., ) . unfortunately, the rapid emergence of resistant bacteria with modified f pili represents a major and possibly insurmountable obstacle to this approach. however, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (hagens et al., ) or phage engineered to repress the cellular sos response (lu and collins, ) . filamentous phage f infection inhibited early stage, but not mature, biofilm formation in e. coli (may et al., ) . thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. more advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (figure ) . the first work in this area showed as proof-of-concept that phage encoding a gfp expression cassette and displaying a her specific scfv on all copies of piii were internalized into breast tumor cells, resulting in gfp expression (poul and marks, ) . m or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of staphylococcus aureus growth than high-concentration free chloramphenicol (yacoby et al., ; vaks and benhar, ) . m phage loaded with doxorubicin and displaying a targeting peptide on piii specifically killed prostate cancer cells in vitro (ghosh et al., a) . tumorspecific peptide:pviii fusion proteins selected from "landscape" phage (romanov et al., ; abbineni et al., ; fagbohun et al., fagbohun et al., , lang et al., ; wang et al., a) were able to target and deliver sirna-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (jayanna et al., a; wang et al., a wang et al., ,b,c, b bedi et al., bedi et al., , bedi et al., , ; they were non-toxic and increased tumor remission rates in mouse models (jayanna et al., b; wang et al., b,c) . using the b -ova tumor model, eriksson et al. ( ) showed that phage displaying peptides and/or fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (eriksson et al., ) . phage displaying an scfv against β-amyloid fibrils showed promise as a diagnostic (frenkel and solomon, ) and therapeutic (solomon, ) reagent for alzheimer's disease and parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (ksendzovsky et al., ) . similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (rakover et al., ) . the advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. first and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating lps, in the range of ∼ - endotoxin units (eu)/ml (boratynski et al., ; branston et al., ) , which have the potential to cause severe adverse reactions. lps is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (smith and gingrich, ; branston et al., ) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (boratynski et al., ; zakharova et al., ) , polymyxin b chromatography (grabowska et al., ) , and treatment with detergents such as triton x- or triton x- (roehnisch et al., ; branston et al., ) . these strategies routinely achieve endotoxin levels of < eu/ml as measured by the limulus amebocyte lysate (lal) assay, well below the fda limit for parenteral administration of eu/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated lps which may be undetectable. a second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in dalmasso et al., ) or for detection of foodborne pathogens post-production (reviewed in schmelcher and loessner, ) . filamentous phage displaying a tetracysteine tag on piii were used to detect e. coli cells through staining with biarsenical dye . m phage functionalized with metallic silver were highly bactericidal against e. coli and staphylococcus epidermidis . biosensors based on surface plasmon resonance (nanduri et al., ) , piezoelectric transducers (olsen et al., ) , linear dichroism (pacheco-gomez et al., ) , and magnetoelastic sensor technology (lakshmanan et al., ; huang et al., ) were devised using filamentous phage displaying scfv or conjugated to whole igg against e. coli, listeria monocytogenes, salmonella typhimurium, and bacillus anthracis with limits of detection on the order of - bacterial cells/ml. proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (li et al., b) and eggs (chai et al., ) . the filamentous phage particle is enclosed by a rod-like protein capsid, ∼ nm long and nm wide, made up almost entirely of overlapping pviii monomers, each of which lies ∼ angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (henry et al., ) . the regularity of the phage pviii lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (figure ) . the most commonly used approach is functionalization of amine groups with nhs esters (van houten et al., (van houten et al., , yacoby et al., ) , although this can result in unwanted acylation of piii and any displayed biomolecules. carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (li et al., a) . carrico et al. ( ) developed methods to specifically label pviii n-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (ng et al., ) or enzymes (chen et al., ; hess et al., ) , but this can be cumbersome and is less general in application. for more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. it has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (welsh et al., ) . lee et al. ( ) engineered m phage to display a zns-binding peptide on piii and showed that, in the presence of zns nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament hess et al., ) , this pioneering figure | chemically addressable groups of the filamentous bacteriophage major coat protein lattice. the filamentous phage virion is made up of ∼ , - , overlapping copies of the -residue major coat protein, pviii, arranged in a shingle-type lattice. each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). the n-terminal residues generally exposed to the immune system for antibody binding are in bold underline. figure adapted from structural data of marvin, , freely available in pdb and scope databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (mao et al., (mao et al., , , nanoparticles , and nanocomposites (oh et al., ; chen et al., ) . using hybrid m phage displaying co o -and gold-binding peptides on pviii as a scaffold to assemble nanowires on polyelectrolyte multilayers, nam et al. ( ) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (nam et al., ) . the electrochemical properties of such batteries were further improved through piii-display of single-walled carbon nanotube-binding peptides (lee et al., ) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. phagebased nanomaterials have found applications in cancer imaging (ghosh et al., b; yi et al., ) , photocatalytic water splitting (nam et al., a; neltner et al., ) , light harvesting (nam et al., b; chen et al., ) , photoresponsive technologies (murugesan et al., ) , neural electrodes (kim et al., ) , and piezoelectric energy generation (murugesan et al., ) . thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. it is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. magnetic alignment of high-concentration filamentous phage in solution can partially order dna, rna, proteins, and other biomolecules for measurement of dipolar coupling interactions (hansen et al., (hansen et al., , dahlke ojennus et al., ) in nmr spectroscopy. because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in husimi, ; wichman and brown, ; kawecki et al., ; hall et al., ) . the filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. first and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on piii. libraries of variant fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (marks et al., ; bradbury et al., ) . however, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; koide and koide, ) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. iterative methods have been developed to combine computationally designed mutations (lippow et al., ) and circumvent the screening of combinatorial libraries, but these have had limited success to date. recently, esvelt et al. ( ) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (pace), which allows multiple rounds of evolution per day with little experimental intervention. the authors engineered m phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene iii expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone dna polymerases. by supplying limiting amounts of receptive e. coli cells to the engineered phage variants, esvelt et al. ( ) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. carlson et al. ( ) later showed that pace selection stringency could be modulated by providing small amounts of piii independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated piii variant that impairs infectivity in a dominant negative fashion. pace is currently limited to protein functions that can be linked in some way to the expression of a gene iii reporter, such as protein-protein interaction, recombination, dna or rna binding, and enzymatic catalysis (meyer and ellington, ) . this approach represents a promising avenue for both basic research in molecular evolution (dickinson et al., ) and synthetic biology, including antibody engineering. filamentous bacteriophage have been recovered from diverse environmental sources, including soil (murugaiyan et al., ) , coastal fresh water (xue et al., ) , alpine lakes (hofer and sommaruga, ) and deep sea bacteria (jian et al., ) , but not, perhaps surprisingly, the human gut (kim et al., ) . the environmental "phageome" in soil and water represent the largest source of replicating dna on the planet, and is estimated to contain upward of viral particles (ashelford et al., ; chibani-chennoufi et al., ; suttle, ) . the few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from to % of all viral particles (demuth et al., ; pina et al., ; hofer and sommaruga, ) . there was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (hofer and sommaruga, ) . environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. the existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (roux et al., ) and reclaimed and potable water (rosario et al., ) but have much higher frequencies in wastewater and sewage (cantalupo et al., ; alhamlan et al., ) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. there are no data describing the population dynamics of filamentous phage and their host species in the natural environment. at the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. this can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. temperate filamentous phage may also play a role in genome evolution (reviewed in canchaya et al., ) . perhaps the best-studied example of virulence modulation by filamentous phage is that of vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding ctxφ phage (waldor and mekalanos, ) . integration of ctxφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs φ, and a satellite filamentous phage, tlc-knφ (hassan et al., ) . thus, filamentous phage species interact and coevolve with each other in addition to their hosts. infection by filamentous phage has been implicated in the virulence of yersinia pestis (derbise et al., ) , neisseria meningitidis (bille et al., (bille et al., , , vibrio parahaemolyticus (iida et al., ) , e. coli :k :h (gonzalez et al., ) , xanthomonas campestris (kamiunten and wakimoto, ) , and p. aeruginosa (webb et al., ) , although in most of these cases, the specific mechanisms modulating virulence are unclear. phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen ralstonia solanacearum (yamada, ) . since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (lin et al., ) . finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (petrenko and makowski, ) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (curtis et al., (curtis et al., , . engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (curtis et al., ). the filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. while novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. kh and js conceived and wrote the manuscript. ma-g read the manuscript and commented on the text. evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization generating fsh antagonists and agonists through immunization against fsh receptor n-terminal decapeptides metagenomics-based analysis of viral communities in 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new millennium a new filamentous bacteriophage with sex-factor specificity diversity and abundance of single-stranded dna viruses in human feces genetically engineered bacteriophage delivers a tumor necrosis factor alpha antagonist coating on neural electrodes expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector structure of a foreign peptide displayed on the surface of bacteriophage m epitope structures recognised by antibodies against the major coat protein (g p) of filamentous bacteriophage fd (inoviridae) mimotope vaccination-from allergy to cancer affinity maturation of single-domain antibodies by yeast surface display genetics of the immune response. i. the immune response to the phage fd in high and low responding inbred strains of mice convection-enhanced delivery of m bacteriophage to the brain multivalent display system on filamentous bacteriophage pvii minor coat protein phage immobilized magnetoelastic sensor for the detection of salmonella typhimurium specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen efrh-phage immunization of alzheimer's disease animal model improves behavioral performance in morris water maze trials ordering of quantum dots using genetically engineered viruses fabricating genetically engineered high-power lithium-ion batteries using multiple virus genes chemical modification of m bacteriophage and its application in cancer cell imaging direct detection of salmonella typhimurium on fresh produce using phagebased magnetoelastic biosensors mimotopes selected with a neutralizing antibody against urease b from helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination phage display for site-specific immunization and characterization of high-risk human papillomavirus specific e monoclonal antibodies inhibition of bacterial conjugation by phage m and its protein 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photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes stamped microbattery electrodes based on self-assembled m viruses biologically templated photocatalytic nanostructures for sustained light-driven water oxidation virus-templated assembly of porphyrins into light-harvesting nanoantennae spr biosensor for the detection of l. monocytogenes using phage-displayed antibody production of hydrogen using nanocrystalline protein-templated catalysts on m phage quantitative synthesis of genetically encoded glycopeptide libraries displayed on m phage graphene sheets stabilized on genetically engineered m viral templates as conducting frameworks for hybrid energy-storage materials affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of salmonella typhimurium detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism phage display: concept, innovations, applications and future antibody-selectable filamentous fd phage vectors: affinity purification of target genes potential applications of phage display to bioremediation a library of organic landscapes on filamentous phage abundance, morphology and distribution of planktonic viruslike particles in two high-mountain lakes correlates of protection induced by vaccination targeted gene delivery to mammalian cells by filamentous bacteriophage conditional lethal mutants of the small filamentous coliphage m . ii. two genes for coat proteins selection of antigenic and immunogenic mimics of hepatitis c virus using sera from patients towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region can induce antibodies crossreacting with a large number of viral variants filamentous bacteriophages: biology and applications, " in els (the encyclopaedia of life sciences) filamentous bacteriophage: biology, phage display and nanotechnology applications antigen-specific therapy of eae via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope expression of a -kilodalton glutathione s-transferase antigen of schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential structural requirements for the activity of the mirb ferrisiderophore transporter of aspergillus fumigatus chemically linked phage idiotype vaccination in the murine b cell lymphoma model phage idiotype vaccination: first phase i/ii clinical trial in patients with multiple myeloma phage display selection of peptides that affect prostate carcinoma cells attachment and invasion a helper phage to improve single-chain antibody presentation in phage display metagenomic analysis of viruses in reclaimed water assessing the diversity and specificity of two freshwater viral communities through metagenomics recombinant expression and neutralizing activity of an mhc class ii binding epitope of toxic shock syndrome toxin- epitope-specific antibody response to ige by mimotope immunization some physical-chemical and biological properties of the rod-shaped coliphage m generation and characterization of phage-gnrh chemical conjugates for potential use in cat and dog immunocontraception phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife infective and inactivated filamentous phage as carriers for immunogenic peptides vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants the use of filamentous bacteriophage fd to deliver hla-a -restricted peptides and to induce strong antitumor ctl responses selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera application of bacteriophages for detection of foodborne pathogens searching for peptide ligands with an epitope library de novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pix fusion proteins high copy display of large proteins on phage for functional selections filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface effect of dna copy number on genetic stability of phage-displayed peptides hydroxyapatite chromatography of phage-display virions phage display libraries of peptides and proteins displayed on filamentous phage generation of anti-β-amyloid antibodies via phage display technology towards alzheimer's disease vaccination filamentous bacteriophage as a novel therapeutic tool for alzheimer's disease treatment comparison of phage pviii and klh as vector in inducing the production of cytokines in c bl/ j mice bacteriophage therapy viruses in the sea a mimotope peptide of aβ fibril-specific antibodies with aβ fibrillation inhibitory activity induces anti-aβ conformer antibody response by a displayed form on an m phage in mice accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases antibody fab display and selection through fusion to the pix coat protein of filamentous phage an investigation on the nature of ultra-microscopic viruses antigenic properties of hcmv peptides displayed by filamentous bacteriophages vs. synthetic peptides in vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines presentation of the functional receptor-binding domain of the bacterial adhesin f a-g on bacteriophage m engineering filamentous phage carriers to improve focusing of antibody responses against peptides filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide basic research in hiv vaccinology is hampered by reductionist thinking lysogenic conversion by a filamentous phage encoding cholera toxin induction of hepatitis b virus-specific cytotoxic t lymphocytes response in vivo by filamentous phage display vaccine crosspresentation of phage particle antigen in mhc class ii and endoplasmic reticulum marker-positive compartments bio-mimetic nanostructure self-assembled from au@ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with mcf- -specific phage fusion protein paclitaxel-loaded peg-pe-based micellar nanopreparations targeted with tumor specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors hybrid phage displaying slaqvkytsassi induces protection against candida albicans challenge in balb/c mice protective immune responses against systemic candidiasis mediated by phage-displayed specific epitope of candida albicans heat shock protein in c bl/ j mice enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein paclitaxel-loaded polymeric micelles modified with mcf- cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity cytoplasmic delivery of liposomes into mcf- breast cancer cells mediated by cell-specific phage fusion coat protein bacteriophage and phenotypic variation in pseudomonas aeruginosa biofilm development evidence for tilted smectic liquid crystalline packing of fd inovirus from x-ray fiber diffraction experimental evolution of viruses: microviridae as a model system immunological properties of foreign peptides in multiple display on a filamentous bacteriophage adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion sensitive and selective bacterial detection using tetracysteine-tagged phages in conjunction with biarsenical dye phage display particles expressing tumor-specific antigens induce preventive and therapeutic anti-tumor immunity in murine p model development of a phage displayed disulfide-stabilized fv fragment vaccine against vibrio anguillarum high frequency of a novel filamentous phage, vcyφ, within an environmental vibrio cholerae population targeting antibacterial agents by using drug-carrying filamentous bacteriophages filamentous phages of ralstonia solanacearum: doubleedged swords for pathogenic bacteria prophylactic vaccination with phage-displayed epitope of c. albicans elicits protective immune responses against systemic candidiasis in c bl/ mice epitope mapping of mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes m phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors comparison of phage piii, pviii and gst as carrier proteins for peptide immunisation in balb/c mice spontaneous assembly of viruses on multilayered polymer surfaces characterization of murine coronavirus neutralization epitopes with phage-displayed peptides purification of filamentous bacteriophage for phage display using size-exclusion chromatography conformational mimicry of a chlamydial neutralization epitope on filamentous phage f , a rodshaped male-specific bacteriophage that contains dna biodistribution of filamentous phage peptide libraries in mice this work was supported by funding from the national research council of canada (kh, ma-g) and the canada research chair program (js). we thank jyothi kumaran and roger mackenzie for critical appraisal of the manuscript, and jasna rakonjac for inviting us to contribute it. this is national research council canada publication number . key: cord- - keu uay authors: kreer, christoph; gruell, henning; mora, thierry; walczak, aleksandra m.; klein, florian title: exploiting b cell receptor analyses to inform on hiv- vaccination strategies date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: keu uay the human antibody repertoire is generated by the recombination of different gene segments as well as by processes of somatic mutation. together these mechanisms result in a tremendous diversity of antibodies that are able to combat various pathogens including viruses and bacteria, or malignant cells. in this review, we summarize the opportunities and challenges that are associated with the analyses of the b cell receptor repertoire and the antigen-specific b cell response. we will discuss how recent advances have increased our understanding of the antibody response and how repertoire analyses can be exploited to inform on vaccine strategies, particularly against hiv- . in order to protect from a vast number of different pathogens, human b cells are able to generate a remarkable diversity of different b cell receptors (bcrs). during b cell development and maturation, these receptors are built by recombination and mutation processes resulting in a virtually unlimited number of different antibodies (i.e., soluble bcrs). however, certain pathogens, such as hiv- , challenge the immune system by the ability to rapidly escape from immune pressure [ , ] , resulting in an ongoing adaptation of the immune response against these pathogens. this evolutionary arms race between pathogens and the immune system leaves footprints in our immunological memory that can describe developmental pathways towards an adapted immune response. deciphering these pathways holds the potential to greatly improve our understanding of critical steps in lymphocyte receptor development and may inform on novel vaccination strategies. for a long time, experimental setups and bioinformatics pipelines were limited in assessing the diversity of lymphocyte receptor repertoires or in identifying subtle immunological imprints after infections or vaccinations. the advent of advanced single cell cloning and next generation sequencing (ngs) methods has revolutionized the field and opened the door to investigate adaptive immune receptor repertoires (airr) at an unprecedented depth. in this review, we summarize recent developments that have fostered our understanding of b cell biology and antibody responses. focusing on the development of highly potent broadly neutralizing antibodies against hiv- , we discuss how a detailed knowledge of the human b cell repertoire may support the development of novel vaccination strategies. the initial diversity of the b cell repertoire results from the assembly of the b cell receptor during early b cell development in the bone marrow. the recombination-activating gene (rag) / enzymes recombine variable (v), diversity (d), and joining (j) gene segments of the immunoglobulin heavy (igh) chain locus to first assemble the heavy chain variable region, followed by v and j gene segment recombination within the ig kappa (igk) and ig lambda (igl) loci [ ] . junctional diversity is further increased by rag / and other enzymes through the generation of palindromic (p) nucleotides, as well as by the terminal deoxynuclotidyl transferase (tdt) through the addition of non-template (n) nucleotides [ ] (figure b ). heavy and light chain v genes exclusively encode for two complementarity determining regions (cdr and cdr ) that are usually structurally exposed at the tip of the antibody and contribute to antigen recognition. a third cdr (cdr ) is generated by the v(d)j recombination process and is the most variable part within b cell receptors and antibodies. the cdrs are interspersed and flanked with framework regions (fwr) that mainly function as a scaffold for the overall immunoglobulin (ig) fold. however, mutations in fwrs have been shown to also influence binding affinity as well as neutralizing activity [ , ] . there are currently at least functional v, d, and j genes described for the igh locus [ ] , which results in a theoretical combinatorial number of different heavy chain variable regions. the igk locus can recombine to different kappa light chain variable regions from v and j genes, whereas the igl locus might recombine up to different lambda light chains from v and j genes [ ] . combinatorial pairing of heavy and light chains yields a theoretical diversity of about . × different antibodies. including the p/n nucleotides, the theoretical number of different antibody sequences is vastly higher than the total number of estimated b cells in the human body ( ) [ ] . however, all those antibody sequences are not equally likely to be generated. their generation probability spans orders of magnitude for igh alone [ , ] , with additional diversity being generated by insertions and deletions [ ] . of note, due to sampling issues, the number of b cell clones whose size falls below the detection threshold is unknown, rendering estimates of total b cell counts unreliable [ , ] . naïve b cells circulate between secondary lymphoid tissues (e.g., lymph nodes and spleen) until they recognize their cognate antigen [ ] . upon antigen contact, b cells can be recruited to lymphatic structures called germinal centers present in secondary lymphoid tissues. there, the recognition event is able to trigger a second step of diversification called affinity maturation. affinity maturation is mediated by the enzyme activation-induced deaminase (aid) as well as b cell expansion and selection [ ] . aid introduces shm including substitutions, insertions, and deletions into the variable regions, generating possible progenies that express b cell receptors that get selected for higher antigen affinity ( figure b) [ , ] . shm is favored but not limited to hot spot motifs and multiple shm hotspots have been identified [ , ] . in addition to their context preference, shms tend to occur close to each other along the sequence [ ] . they are typically more pronounced in the cdrs than in the framework regions due to positive selection as well as higher frequency of aid motifs [ ] . including shm into the calculation of potential bcrs results in an almost infinite number of different receptors. finally, aid activity is able to mediate heavy chain class switch from igm/igd to igg, iga, or ige. different b cell subtypes and antibody classes are of critical importance and their functions have been reviewed elsewhere [ , , ] . dissecting the humoral immune response is a challenging task. analyses of antibodies on the serum level, for example by elisa, affinity chromatography, or mass spectrometry [ ] , are usually limited to characterizing the polyclonal antibody response. genetic b cell analyses, on the other side, facilitate single cell (i.e., single antibody) resolution. to this end, antibody-coding nucleic acids (dna or rna) are extracted from b cells, amplified, and sequenced ( figure ). importantly, complete sequences of matched heavy and light chains allow for recombinant production of antibodies and thus allow studying antibody functions on a monoclonal level ( figure ). in this section, we describe the challenges arising at the different steps of genetic bcr analyses and discuss advantages and disadvantages of individual strategies. section, we describe the challenges arising at the different steps of genetic bcr analyses and discuss b cells can be subdivided into different subsets. these comprise (i) b cells at different developmental stages (e.g., pro-b cells, immature b cells, mature b cells), (ii) antigen-naive and antigen-experienced b cells, (iii) functional subsets, such as regulatory, effector, or memory b cells, or iv.) b cells with defined specificity (e.g., hiv- env -reactive). depending on the scientific question, it is often required to analyze an individual b cell subset and identification of such subsets can take place at distinct steps of an experimental pipeline ( figure , first row). different b cell subsets can be enriched or isolated by sorting techniques such as magnetic-activated cell sorting (macs) or fluorescence-activated cell sorting (facs) [ , ] . importantly, facs allows collecting and further processing target cells either in bulk approaches or as single cells in multi-well plates. recently, novel microencapsulation systems have been used to encapsulate single b cells into picoliter droplets [ , ] . the combination of single cell encapsulation with fluorescence-activated sorting (fluorescence-activated droplet sorting (fads), reviewed in [ ] ) allows processing of single antigen-specific b cells in compartments that are a million times smaller than the wells of multi-well plates, which significantly increases the throughput capacity. in order to isolate antigen-specific b cells, one of the following approaches (reviewed in [ , ] ) can be applied ( figure , first row): (i) antigen-derived baits that are fluorescently labeled can be used to identify and sort antigen-reactive b cells. this can be achieved by fluorescently-labeled proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] , antigens presented on virus-like particles [ , ] or cells [ ] , or by pathogens themselves [ ] . (ii) antibody libraries that are expressed on phages or yeast cells can be selected for binding to antigen-derived proteins or whole pathogens [ , ] . of note, however, the random pairing of heavy and light chains in combinatorial libraries does not allow to infer a representative picture of the underlying antibody response. (iii) single b or plasma cells or immortalized b cells can be expanded and stimulated to secrete antibodies that can be tested for antigen-binding or neutralizing activity [ ] [ ] [ ] [ ] . importantly, recombinant proteins and other baits that are used for selecting antigen-specific antibodies can critically differ in their structure and glycosylation pattern from their native counterpart. indeed, the generation of optimized bait proteins [ ] or native-like envelope trimers [ ] were critical steps to improve the isolation of potent hiv- broadly neutralizing antibodies (bnabs) by antigen-specific sorting strategies [ , [ ] [ ] [ ] . very recently, a combination of single cell co-encapsulation and dna-tagged recombinant proteins has even been used to directly map antibody sequences to their antigen specificity [ ] . direct heavy or light chain rt-pcr and sequence analyses from bulk-sorted b cells allow to infer b cell repertoire characteristics such as clonal distributions, v(d)j recombination, and somatic hypermutation [ ] [ ] [ ] [ ] [ ] [ ] . however, the native pairing information of heavy and light chains is essential to fully describe an individual antibody, e.g., for recombinant expression. although pairing information can be restored to some extent from bulk analyses (i.e., by bioinformatic approaches) [ ] , the most robust way to achieve these information is either by single cell sorting into multi-well plates [ ] [ ] [ ] [ ] or by co-encapsulation of single cells and rna-capture or barcode beads (e.g., with the × genomics chromium system) in picoliter droplets [ , , [ ] [ ] [ ] [ ] [ ] (figure , second row). single cell sorting into multiwell plates is typically limited in throughput to tens of thousands of cells, whereas encapsulation systems allow throughputs of hundreds of thousands of cells. however, droplet occupancy follows a poisson distribution and requires limiting dilutions of the sample and beads [ ] . as a consequence, the majority of all droplets remains empty or contains only unpaired cells or beads, which can lead to a high loss of input material. alternatives for heavy and light chain paring comprise combinatorial yeast or phage display libraries that can be screened for reactivity (reviewed in [ ] ). however, due to their stochastic nature, such display approaches also contain artificial heavy and light chain pairs. all current approaches require an initial pcr-based amplification of the bcr-encoding dna or mrna/cdna. the diversity of the b cell repertoire poses distinct challenges to this amplification step: (i) all possible v gene segments need to be covered by the pcr and (ii) priming sites may have been somatically hypermutated and are therefore prone to decreased amplification efficiencies (figure , third row). pcr amplification of antibody heavy and light chains has thus been performed with v gene-specific multiplex primer mixes [ , , [ ] [ ] [ ] [ ] [ ] . the majority of these primers were designed against the end of the coding region of the v gene, which is sufficient for amplifying most antibody sequences. however, hiv- bnabs, for instance, have been shown to accumulate high levels of shm as well as insertions and deletions [ , , , ] . in order to increase priming efficiency, primer mixes have been designed against the end of the less-mutated leader region that encodes the antibody secretion peptide. these mixes have been demonstrated to be superior for the isolation of highly mutated hiv- -reactive antibodies [ , ] . whereas primer sets perform well in single cell cloning approaches, they may introduce primer biases in bulk pcr amplification approaches [ ] . this can pose a critical disadvantage. a method that is able to overcome this limitation is the rapid amplification of cdna ends ( race) [ ] , which has been adapted to bulk [ ] and single cell approaches [ , ] . commonly applied protocols include template-switching (ts) reverse transcription, which introduces a ts-oligo during cdna synthesis. the ts-oligo bears a universal priming site that can be used together with a constant region reverse primer to amplify any antibody variable region independent of the incorporated v gene segment. sequence analysis of amplified heavy and light chains can be demanding in several aspects ( figure , fourth row). first, depending on the b cell subset of interest, the required throughput can vary from a few hundred to millions of antibody sequences. second, the sequencing method needs to reliably cover the whole region of interest (variable region~ - bp). third, shm needs to be distinguishable from sequencing errors, thus requiring low sequencing error rates or error-correction techniques. classical sanger sequencing is frequently employed in the analyses of b cell receptor subsets such as antigen-specific memory b cells in the blood [ , , ] . due to the inability to sequence bulk-amplified heavy and light chains, next generation sequencing (ngs) techniques such as pyro-, ion-semiconductor-, or illumina dye sequencing are typically preferred over sanger sequencing for high-throughput analyses [ ] [ ] [ ] [ ] [ ] [ ] . however, they often suffer from shorter read lengths and higher error rates. molecular barcoding techniques and bioinformatics pipelines have been developed to account for both pcr-and sequencing-induced errors [ ] . to this end, unique molecular identifiers (umi) are introduced during cdna generation by template-switching reverse transcription. moreover, protocols for long read parallel sequencing (e.g., smrt and nanopore sequencing) have been recently applied to analyze bcr sequences [ , ] . finally, high-throughput analyses of millions of different antibody sequences require advanced bioinformatics pipelines. several bioinformatics tools have been reported [ ] and standardized protocols on reporting antibody sequences have been developed by the adaptive immune receptor repertoire (airr) community [ , ] . a detailed description of the methods applied is beyond the scope of this review and they are covered in previous reviews [ , ] . isolated highly potent bnabs can serve as templates for the development of such strategies. over the last decade, numerous bnabs have been identified by bait-specific single cell sorts or b cell microcultures. most of these antibodies were obtained from the memory b cell pool as well as, occasionally, from plasma cells [ , , ] . this suggests that both, memory and antigen-secreting b only a small fraction of hiv- -infected individuals develop highly potent bnabs and detailed analyses of b cell receptors and antibodies at a single cell level have been limited to a few dozen subjects. nevertheless, these critical investigations have revealed sequence and structural characteristics of potent hiv- neutralizing antibodies that were repeatedly observed across different individuals. these features can include high levels of somatic hypermutation, the presence of unusual insertions or deletions, and/or long heavy chain cdr (cdrh ) regions. thus, vaccine-mediated hiv- bnab induction may require specifically tailored strategies for b cell activation and maturation. isolated highly potent bnabs can serve as templates for the development of such strategies. over the last decade, numerous bnabs have been identified by bait-specific single cell sorts or b cell microcultures. most of these antibodies were obtained from the memory b cell pool as well as, occasionally, from plasma cells [ , , ] . this suggests that both, memory and antigen-secreting b cells, can in principle serve as a valuable source for bnab isolation and characterization. all hiv- bnabs target epitopes on the hiv- envelope protein (env) that include the cd binding site (cd bs), glycan-dependent targets on the variable env loops (v /v , v ), the fusion peptide and the membrane-proximal external region (mper) of gp , and sites spanning the gp and gp subunits [ , ] . in clinical trials, bnabs have been shown to suppress viremia and delay the time to viral rebound after interruption of antiretroviral therapy (art) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of most relevance for hiv- vaccine efforts, however, bnabs are highly effective in preventing infection in animal models [ ] [ ] [ ] [ ] . as proof-of-concept trials for passive immunization using the cd bs bnab vrc in humans are ongoing (clinicaltrials.gov: nct , nct ), it is widely believed that induction of potent bnabs by vaccination will confer protection from hiv- . among the highly potent hiv- bnabs, antibodies of the vrc -and anc -classes target the cd binding site (cd bs) on the hiv- env. they are particularly noteworthy for their restricted v gene usage [ , , ] facilitating the vh - or vh - gene segments. importantly, members of the potent vrc -class of bnabs have now been identified in at least individuals, demonstrating their capacity to be reproducibly induced [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . such a reproducible development of very similar antibodies in different individuals is often referred to as convergent or stereotypical antibody responses or described as "public antibodies". identifying convergent immune responses is informative for vaccine design because strategies that induce such a repeatedly observed type of immune reaction may be broadly applicable on a population-level. indeed, b cell analyses have revealed convergent v gene responses not only against hiv- but several pathogens after infection and/or vaccination (table ) . vrc -class cd bs bnabs demonstrate the same mode of cd bs recognition that is dominated by the cdr of the heavy chain (cdrh ) [ ] . to avoid steric clashes, they share an additional restriction for use of an unusually short (five amino acids) light chain cdr (cdrl ), which is found in only~ % of antibodies [ , ] . finally, they show extensive levels of somatic hypermutation of up to > % on the nucleotide level (i.e., > mutations) from their inferred antibody germline sequences [ , , , ] . members of hiv- bnab classes targeting other epitopes are generally less restricted in terms of their v gene usage but often share other sequence and structural characteristics. for example, bnabs binding to the v /v apex region typically carry cdrh s of extraordinary length that are required to penetrate the extensive env glycan shield [ , ] . compared to the average cdrh lengths of approximately amino acids in the naïve and memory b cell receptor repertoires [ ] , v /v -targeting bnabs have been identified that have > -fold longer cdrh s (e.g., vrc . and pgdm with cdrh lengths of ≥ aa [ , ] ). similarly, relatively long cdrh s are also found in hiv- bnabs targeting other glycan-related epitopes (e.g., v loop, gp /gp interface) or the gp mper [ ] . in addition, some bnabs display poly-and/or autoreactivity [ , ] , features that are often associated with long cdrh s and are generally counterselected during b cell maturation [ ] . overall, the consistent observation of one or multiple rare features in potent hiv- bnabs highlights some of the difficulties for their induction through vaccination. however, several antibodies with considerable breadth and potency but lower levels of somatic hypermutation and more regular cdrh lengths have now been identified [ , , , ] . these antibodies may be more readily inducible and serve as blueprints for facilitating vaccine strategies. due to the unusual sequence and structural characteristics of most highly potent hiv- bnabs, unconventional approaches to vaccination are likely to be required. strategies that have been proposed include epitope-based and antibody lineage-based vaccine designs [ ] . epitope-based vaccination strategies use immunogens that mimic the general structure of vulnerable env sites, in principle allowing for the development of multiple bnab classes against the same target region. however, when reverted to their inferred germline sequence, many hiv- bnabs show considerably reduced or fully abrogated binding to hiv- env [ , [ ] [ ] [ ] . to this end, antibody-lineage based vaccination strategies employ designed immunogens that interact with inferred unmutated bnab precursors to initiate the development of a particular bnab lineage [ ] . although bnab precursor cell frequency in the repertoire is only one of a number of factors that will determine the potential success of lineage-based vaccine design, a comprehensive understanding of the composition of the b cell receptor repertoire in healthy individuals can provide critical information to guide the development of vaccination pathways [ ] . as seen for the majority of antibodies, most potent hiv- bnabs are strongly dependent on interactions mediated by the cdr of the heavy chain. compared to the distribution in the overall memory b cell repertoire, many hiv- bnabs have relatively long cdrh s which have been suggested to be largely generated during vdj recombination [ ] . particularly long cdrh s are required for many bnabs targeting the v /v apex region of env. notably, among näive b cell repertoires of healthy individuals, cdrh lengths of amino acids and more have been identified in less than . % of sequences [ ] , and a cdrh length of amino acids as seen for bnab pg was found to be exceedingly rare ( . %) [ ] . although this suggests difficulties for cdrh -based hiv- vaccination, the potential contribution of bcr repertoire analyses to vaccine design was recently demonstrated when precursor frequencies of the cdrh -dominated bnab bg [ , ] were determined to inform on the selection of an immunogen targeting bg -like precursors [ ] . among × cdrh sequences from a total of healthy donors, bg -like sequences were identified in all individuals [ , ] . importantly, rare immunogen-reactive b cells could subsequently be isolated from additional healthy donors [ ] . moreover, antibody lineage-based vaccination strategies that aim to engage precursors of the vrc -class of cd bs bnabs have entered the clinical stage with the germline-targeting immunogen eod-gt (clinicaltrials.gov: nct ) [ ] . as mentioned before, antibodies of this class are particularly restricted for usage of the vh - * allele and a amino acid cdrl [ ] . bcr repertoire analyses revealed that potential vrc -class precursor b cells are exceptionally rare [ ] [ ] [ ] . in addition, allelic variation can result in the lack of naïve b cells derived from the key vh - * allele [ ] . nevertheless, eod-gt -reactive naïve b cells could be identified in a majority of hiv- -negative donors ( / ) [ , ] , providing repertoire analysis-based support for advancing the eod-gt immunogen to be evaluated in a clinical setting. while germline-targeting immunogens are designed to initiate a particular b cell lineage, subsequent immunizations with additional antigens will likely be required to induce the development of broad and potent mature antibodies through additional rounds of affinity maturation [ ] . to this end, interrogations of the natural development of bnabs in hiv- elite neutralizers may be highly informative for immunogen design. high-throughput parallel sequencing methods of the b cell receptor repertoire combined with bioinformatical processing and phylogenetic analyses have facilitated to reconstruct the inferred development of antibody lineages [ , [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of note, when informed by template sequences of bnabs obtained through single cell approaches, high-throughput bcr sequencing methods can identify antibodies with higher breadth and potency [ ] . of particular relevance for vaccine design, longitudinal studies that investigate the co-evolution of hiv- and the neutralizing antibody response in single individuals may provide guidance for the design of antigens driving bnab potency and breadth. for example, several studies revealed that development of broad neutralization was preceded by viral diversification and/or supported by antibody helper lineages that selected for viral variants that drove bnab development [ , , , [ ] [ ] [ ] , ] . while these observations support a stepwise immunization approach, "dead-end" limbs of antibody lineages appear during the affinity maturation process. therefore, sequential immunogens will need to be carefully selected [ , , , ] . besides the lineage specific analyses, high-throughput ngs approaches have also been used to investigate the whole b cell receptor repertoire of hiv- -infected individuals either from combinatorial libraries [ ] [ ] [ ] or from pbmcs or purified b cells [ , , [ ] [ ] [ ] [ ] . a recent study by waltari et al. detected slight shifts in v gene family usage, higher degrees of somatic hypermutation, and longer cdrh s for hiv- -infected individuals [ ] . however, other studies could not find any differences but report variation within healthy or hiv- -infected individuals to be as large as between the different cohorts [ , ] . the current sampling and sequencing depths might therefore still hamper the identification of hiv- infection-induced changes on the b cell receptor repertoire. only a small fraction of hiv- -infected individuals is able to mount a broadly neutralizing serum activity against hiv- . over the last decade, advances in screening methods and single cell cloning techniques enabled the isolation of numerous broadly neutralizing antibodies. these antibodies have been shown to be promising candidates for hiv- treatment and prevention. however, molecular analyses also revealed special characteristics such as v gene restriction, long cdrh s, and/or high loads of shm, which may restrict the development of highly potent bnabs in natural infection and hamper their induction by current vaccination strategies. to overcome potential roadblocks for the induction of bnabs through vaccination, a number of strategies have been proposed. all of these, however, will require the interaction of one or multiple immunogens with b cell receptors to effectively drive bnab development. thus, a detailed understanding of the naïve b cell receptor repertoire and the constantly adapting antibody response in the context of hiv- infection can be highly informative for vaccine design. novel experimental and bioinformatics pipelines have the capacity to integrate neutralization, antibody sequence, and structural data. these methods hold great promise to identify common pathways of potent immune responses that will be critical for developing effective vaccination strategies. measurement of the mutation rates of animal viruses: influenza a virus and poliovirus type identification and characterization of conserved and variable regions in the envelope gene of htlv-iii/lav, the retrovirus of aids igg subclasses and allotypes: from structure to effector functions mechanisms of programmed dna lesions and genomic instability in the immune system lack of n regions in antigen receptor variable region genes of tdt-deficient lymphocytes hierarchy and extremes in 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human antibody genes of different isotypes antibody repertoires in humanized nod-scid-il rgamma(null) mice and human b cells reveals human-like diversification and tolerance checkpoints in the mouse sequence and structural convergence of broad and potent hiv antibodies that mimic cd binding antibodies in hiv- vaccine development and therapy openprimer for multiplex amplification of highly diverse templates toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding rapid production of full-length cdnas from rare transcripts: amplification using a single gene-specific oligonucleotide primer construction of representative immunoglobulin variable region cdna libraries from human peripheral blood lymphocytes without in vitro stimulation amplification and analysis of cdna generated from a single cell by -race: application to isolation of antibody heavy and light chain variable gene sequences from single b cells cross-specificity of protective human antibodies against klebsiella pneumoniae lps o-antigen repertoire and neutralizing activity of antibodies against hepatitis c virus e peptide in patients with spontaneous resolution of hepatitis bioinformatic and statistical analysis of adaptive immune repertoires in-depth determination and analysis of the human paired heavy-and light-chain antibody repertoire ireceptor: a platform for querying and analyzing antibody/b-cell and t-cell receptor repertoire data across federated repositories computational strategies for dissecting the high-dimensional complexity of adaptive immune repertoires potent and broad hiv-neutralizing antibodies in memory b cells and plasma identification of near-pan-neutralizing antibodies against hiv- by deconvolution of plasma humoral responses recent progress in broadly neutralizing antibodies to hiv virologic effects of broadly neutralizing antibody vrc administration during chronic hiv- infection viraemia 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targeting the cd supersite in donors multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for hiv- neutralization by vrc -class antibodies focused evolution of hiv- neutralizing antibodies revealed by structures and deep sequencing identification of a cd -binding-site antibody to hiv that evolved near-pan neutralization breadth rapid and focused maturation of a vrc -class hiv broadly neutralizing antibody lineage involves both binding and accommodation of the n -glycan delineating antibody recognition in polyclonal sera from patterns of hiv- isolate neutralization lower igg somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent b cell response b cell gene signature with massive intrahepatic production of antibodies to hepatitis b core antigen in hepatitis b virus-associated acute liver failure infant and adult human b cell responses to rotavirus share common immunodominant variable gene repertoires vh - is the dominant immunoglobulin heavy chain gene segment in rotavirus-specific memory b cells expressing the intestinal homing receptor alpha beta immunodominance of the vh - antibody gene segment in the primary repertoire of human rotavirus-specific b cells is reduced in the memory compartment through somatic mutation of nondominant clones ability to develop broadly neutralizing hiv- antibodies is not restricted by the germline ig gene repertoire structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine rapid development of broadly influenza neutralizing antibodies through redundant mutations electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization b-cell repertoire dynamics after sequential hepatitis b vaccination and evidence for cross-reactive b-cell activation human 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targeting cryptic epitopes on envelope domain iii preferential use of the vh - gene segment by the human immune response to code for antibodies against the v domain of hiv- structural basis for germ-line gene usage of a potent class of antibodies targeting the cd -binding site of hiv- gp structure of hiv- gp v /v domain with broadly neutralizing antibody pg a broadly neutralizing antibody targets the dynamic hiv envelope trimer apex via a long, rigidified, and anionic beta-hairpin structure large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv- antibodies hiv- neutralizing antibodies: understanding nature's pathways hiv- neutralizing antibodies with limited hypermutation from an infant early antibody lineage diversification and independent limb maturation lead to broad hiv- neutralization targeting the env high-mannose patch hiv- vaccines based on antibody identification, b cell ontogeny, and epitope structure recombinant hiv envelope proteins fail to engage germline versions of anti-cd bs bnabs structural basis for broad and potent neutralization of hiv- by antibody vrc . science the effects of somatic hypermutation on neutralization and binding in the pgt family of broadly neutralizing hiv antibodies b-cell-lineage immunogen design in vaccine development with hiv- as a case study when designing vaccines, consider the starting material: the human b cell repertoire human peripheral blood antibodies with long hcdr s are established primarily at original recombination using a limited subset of germline genes long antibody hcdr s from hiv-naive donors presented on a pg neutralizing antibody background mediate hiv neutralization coexistence of potent hiv- broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller structural characterization of a highly-potent v -glycan broadly neutralizing antibody bound to natively-glycosylated hiv- envelope a generalized hiv vaccine design strategy for priming of broadly neutralizing antibody responses hiv- broadly neutralizing antibody precursor b cells revealed by germline-targeting immunogen the human naive b cell repertoire contains distinct subclasses for a germline-targeting hiv- vaccine immunogen differences in allelic frequency and cdrh region limit the engagement of hiv env immunogens by putative vrc neutralizing antibody precursors somatic populations of pgt - hiv- -neutralizing antibodies identified by de novo identification of vrc class hiv- -neutralizing antibodies by next-generation sequencing of b-cell transcripts developmental pathway for potent v v -directed hiv-neutralizing antibodies maturation and diversity of the vrc -antibody lineage over years of chronic hiv- infection maturation pathway from germline to broad hiv- neutralizer of a cd -mimic antibody of the mper-directed hiv- -neutralizing antibody e viral variants that initiate and drive maturation of v v -directed hiv- broadly neutralizing antibodies hiv envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a v apex broadly neutralizing antibody lineage staged induction of hiv- glycan-dependent broadly neutralizing antibodies enhanced potency of a broadly neutralizing hiv- antibody in vitro improves protection against lentiviral infection in vivo co-evolution of a broadly neutralizing hiv- antibody and founder virus cooperation of b cell lineages in induction of hiv- -broadly neutralizing antibodies characterization of human igg repertoires in an acute hiv- infection deep sequencing and circos analyses of antibody libraries reveal antigen-driven selection of ig vh genes during hiv- infection the potential of the human immune system to develop broadly neutralizing hiv- antibodies: implications for vaccine development dynamics of immunoglobulin sequence diversity in hiv- infected individuals rapid amplification of cdna ends and illumina miseq reveals b cell receptor features in healthy adults, adults with chronic hiv- infection, cord blood, and humanized mice multi-donor longitudinal antibody repertoire sequencing reveals the existence of public antibody clonotypes in hiv- infection we thank all members of the klein lab for helpful discussions. the authors declare no conflict of interest. key: cord- -la svzml authors: strohl, william r. title: current progress in innovative engineered antibodies date: - - journal: protein cell doi: . /s - - - sha: doc_id: cord_uid: la svzml as of may , , antibody-based molecules have been approved by a regulatory authority in a major market. additionally, there are and antibody-based molecules in phase iii and phase i/ii clinical trials, respectively. these total antibody-based clinical stage molecules include naked iggs, antibody-drug conjugates, bispecific antibodies, total fc fusion proteins, radioimmunoglobulins, antibody fragments, and immunocytokines. new uses for these antibodies are being discovered each year. for oncology, many of the exciting new approaches involve antibody modulation of t-cells. there are over antibodies in clinical trials targeting t cell checkpoints, t-cell-redirected bispecific antibodies, and chimeric antigen receptor (car) cell-based candidates (all currently in phase i or ii clinical trials), totaling more than t cell interacting clinical stage antibody-based candidates. finally, significant progress has been made recently on routes of delivery, including delivery of proteins across the blood-brain barrier, oral delivery to the gut, delivery to the cellular cytosol, and gene- and viral-based delivery of antibodies. thus, there are currently at least antibody-based clinical stage molecules or cells, with incredible diversity in how they are constructed and what activities they impart. these are followed by a next wave of novel molecules, approaches, and new methods and routes of delivery, demonstrating that the field of antibody-based biologics is very innovative and diverse in its approaches to fulfill their promise to treat unmet medical needs. this year, , marks the th anniversary of the approval by the united states food and drug administration (us fda) of rituxan® (rituximab) and zenapax® (daclizumab), for treatment of b cell malignancies and for use to suppress organ rejection in renal transplants, respectively (table ) . while two antibodies had previously been approved by the fda (table ) , the approval of rituxan® and zenapax® in was a watershed moment in the history of monoclonal antibody (mab) therapeutics. the reasons are very different for each molecule. rituxan® has become both a huge medical and commercial success, with indications in b cell malignancies as well as in the treatment of rheumatoid arthritis (ra) (storz, ) . rituxan® is currently the fourth best-selling innovative drug of any kind with worldwide sales of $ , mm (table ) , about % of those sales coming in cancer indications and the other % from sales for treatment of ra (la merie publishing, ) . including rituxan®, seven of the top ten selling innovative drugs in the world in were proteins, six of which were antibodyrelated molecules (table ) . zenapax®, on the other hand, was the first humanized antibody to be fda approved but it never achieved significant commercial success and was eventually withdrawn from the market in . daclizumab, however, has been approved recently under the tradename zinbryta® for treatment of relapsing forms of multiple sclerosis (ms). to date, unique, innovative antibodies and fc fusion proteins have been approved for treatment of diseases in at least one major market (i.e., us, eu, japan) (table ) . of these, seven have been withdrawn from marketing either due to lack of efficacy, poor toxicity to efficacy profiles, or lack of market interest (table ) . of the approved antibody-based molecules, five contain completely murine (table ) . thus, as of may , , there are at least known antibody and fc fusion protein clinical-stage candidates (table ) . of these, are "naked" iggs, are "naked" antibody fragments (in both cases, "naked" refers to antibodies that are not antibody-drug conjugates [adcs], bispecific antibodies, radioimmunotherapeutics, or immunocytokines), are adcs, are bispecific antibodies, are fc fusion proteins, are conjugated with radioisotopes either as therapeutics or imaging agents, and are immunocytokines (table and fig. ). it is notable that, with the exception of fc fusion proteins, most of the non-"naked" antibodies are skewed towards the phase i/ii clinical stages, likely due to the more recent development of the various innovative technologies incorporated into those molecules (table ). in addition to these protein antibody-derived clinical stage molecules, there are documented phase i or ii clinical stage chimeric antigen receptor (car)-t cell or natural killer (nk) cell candidates that incorporate antibodies as their cars (table and fig. ) . thus, there are at least protein and cell based antibody-derived constructs either approved for medical use or being evaluated for their safety and efficacy in clinical trials. the unique antibody-based molecules/cells in development or approved for therapeutic use (table ) target unique antigens (table ). because several targets are important for multiple disease areas (e.g., vascular endothelial growth factor [vegf] as a significant target in both oncology and ophthalmology indications), there are more uses listed than antibodies. thus, unique molecules are used in different major therapeutic area indications (table ) , and the unique targets are distributed amongst major uses (table ) . about % of these protein and recombinant cell-based candidates are directed against targets in oncology (table ) . not surprisingly, all of the current car-t and car-nk candidates are in clinical evaluation for cancer indications. there are, however, preclinical efforts to generate car-t cells against viruses and virus-infected cell targets (sahu et al., ; liu et al., ; hale et al., ) , so this may change in the near future. another % of the clinical candidates are directed against targets in the immunology therapeutic area (including autoimmune and asthma, but excluding ms) ( antigen related cell adhesion molecule ], muc [mucin , cell surface associated]), are essentially "postal addresses" to which killing mechanisms can be targeted directly. these killing mechanisms can include, either individually or in combinations, antibody-dependent cellular cytotoxicity (adcc) (ochoa et al., ) , antibody-dependent cellular phagocytosis (adcp) (shi et al., ) , complement-dependent cytotoxicity (cdc) , antibody-drug conjugates (adc) (tsuchikama and an, ; beck et al., ) , antibody-induced apoptosis (sun et al., ; wang et al., ) , antibody-induced, non-apoptotic programmed cell death (alduaij et al., ) , bispecific antibody-redirected killer t or nk cells (lum and thakur, ; satta et al., ; suzuki et al., ) , or car-t/car-nk cells (ruella and gill, ; ruella and june, ; smith et al., ) . the second group, which overlaps with the first group, are receptors which may be targeted to block ligand binding and signal transduction (esparis-ogando et al., ; zhang and zhang, ) . the final category are checkpoint modulators, either to block t cell inhibitory pathways or to directly stimulate t or nk cells or macrophages. there are about t-cell related oncology targets in this category. of the unique targets for antibody-based drug candidates, the most widely targeted antigen is cd , which is recognized by clinical candidates, of which are cars ( table ). the second most targeted protein is cd e, found in clinical stage or approved molecules, of which are t cell-redirecting bispecific antibody candidates (table ) . thus, the two top targets, cd and cd e, are responsible for the engineered retargeting of t cells, either as car-t cells (ruella and gill, ; ruella and june, ; smith et al., ) or t-cell redirecting bispecific antibodies (lum and thakur, ; satta et al., ; suzuki et al., ) , to kill cancer cells. of the non-t-cell related targets, the proteins currently most widely targeted are erbb (her ), egfr, ms a (cd ), cd , pdcd (pd- ), msln (mesothelin), and erbb (her ), all for cancer indications. the th cytokine, il a, to which antibody-related biologics are directed, is currently the top non-oncology target (table ). there are unique molecules or recombinant cars directed against the top targets shown in table , representing about % of all of the clinical stage or approved antibody-based molecules/cells; the remaining (∼ %) candidates target the remaining unique targets. the approved mabs and fc fusion proteins are directed against unique targets, with tnf (tnf-α) and ms a (cd ) being the most widely targeted, with five antibody-based molecules each (table ). the five most valuable targets for approved mabs and fc fusion proteins are tnf (tnf-α), vegf, erbb (her ), ms a (cd ), and pdcd (pd- ) ( table ) . antibodies against the first four of these targets were approved more than ten years ago, so the market value has built up over time. remarkably, however, the anti-pd- antibodies, keytruda® and opdivo®, were approved , making pdcd (pd- ) a very fast rising target of value (table ) . the top ten antibody-based therapeutic targets (table ) comprise % of the value of the total targets, with the anti-tnf molecules leading the way with a market share of % (table ) . based on sales figures, recombinant proteins comprised seven of the top best selling drugs worldwide (table ) . of these seven proteins, five (humira®, rituxan®, remicade®, avastin®, herceptin®) are mabs and one (enbrel®) is an fc fusion protein (table ) . finally, since january (the past . years), antibodies and fc fusion proteins have comprised % ( / ) of innovative united states food and drug administration (us fda) drug approvals (fig. ) . this represents the greatest percentage ever since the beginning of the antibody era. thus, it is clear that mabs and fc fusion proteins are making an enormous impact on the pharmaceutical industry, both as novel approaches to treat difficult diseases and meet unmet medical needs, as well as providing an exciting new growth area for the industry. human iggs have been engineered in a multitude of ways to generate different effects (strohl and strohl, ) , as shown in table . in the earlier days of antibody engineering, the focus was on manipulation of the variable regions to humanize and affinity-mature antibodies, or to generate different types of antibody fragments such as scfvs (bird et al., ; huston et al., ) , diabodies (holliger et al., ) , pegylated fabs (choy et al., ) , tandabs (kipriyanov et al., ) , and domain antibodies (ward et al., ) . the next wave of antibody engineering focused more on the generation and application of "fit for purpose" antibodies (strohl, ) with tuned fc functions such as increased adcc, adcp, and cdc (strohl and strohl, ; brezski and georgiou, ; sondermann and szymkowski, ; barnhart and quigley, ) , or muted or silenced fc functions (labrijn et al., ; vafa et al., ; schlothauer et al., ; lo et al., ; borrok et al., ) . these functions have been approached using both glyco-engineering strategies such as low or no fucosylation for higher fcγriiia binding and increased adcc (shields et al., ; ferrara et al., ; malphettes et al., ; golay et al., ) , higher levels of sialylation for dampened immune responses (anthony and ravetch, ) , or non- abbreviations: fc, fragment crystallizable; ms, multiple sclerosis; gpcr, g-protein coupled receptor. * these numbers add up to more than the unique targets noted in table because several targets have major indications in multiple therapeutic areas (e.g., anti-vascular endothelial growth factor [vegf] antibodies with major indications in both oncology and ophthalmology). ** mostly single-pass membrane targets, either as monomeric cell-bound proteins, homodimeric receptors, or heterodimeric receptors. glycosylated antibodies for partly subdued fc effector function (walker et al., ; nesspor et al., ) . these and more fc effector modulations can also be generated with amino acid changes in the lower hinge and fc (shields et al., ; lazar et al., ; strohl, ; strohl and strohl, ; vafa et al., ; brezski and georgiou, ; sondermann and szymkowski, ; barnhart and quigley, ) . there are currently three approved antibody-based molecules with modified fc functionality. these include the ctla -fc fusion proteins, abatacept (orencia®) and belatacept (nujolix®), both of which have modified hinges to reduce fc functionality (davis et al., ) , and the humanized anti-c mab, eculizumab (soliris®), which has an igg /igg hybrid fc to reduce fc functionality (rother et al., ) . all three of these molecules bind to immune (gray et al., ) followed by commonly used names in parentheses. system components and the muted fc design was intended to increase the safety margin. additionally, two glyco-engineered antibodies with improved adcc activities have been approved in at least one major market. the first, mogamulizumab (poteligeo®), is an afucosylated anti-ccr mab approved in japan for adult t-cell leukemia/lymphoma that is produced by a cell line with a mutation in the fut (α- , -fucosyltransferase) gene (potelligent® technology from biowa; yamane-ohnuki et al., ; kanda et al., ; malphettes et al., ) . the second, obinutuzumab (gazyva®), an anti-ms a (cd ) mab with low fucose content, has been approved for treatment of chronic lymphocytic leukemia (cll) (golay et al, ) . the low fucose of obinutuzumab is due to the addition to the producing cell line of a β- , -n-acetylglucosaminyltransferase iii (gntiii) gene which adds the bisecting n-acetylglucosamine (glcnac) that interferes with fucosylation (glycart glycomab® technology acquired by roche in ) (ferrara et al., ) . moreover, there are four glycoengineered (low or no fucose) and one aglycosyl-igg phase iii clinical candidates in the late stage clinical pipeline. currently there are no approved iggs with amino acidmodified increased fc effector function, although there are two such fc-modified, increased fc effector function iggs in late stage clinical trials, the anti-cd mab, mor (morphosys, xencor), in phase ii/iii clinical trials for treatment of b cell malignancies (nct ), and the anti-erbb (her ) mab, margetuximab (merck, macrogenics), in phase iii clinical trials for breast cancer (nct ). moreover, there have been many efforts to modulate aspects of igg biology beyond just increasing or decreasing fc effector function (table ). the first of these is modulation of half-life via modified interaction of the fc with the recycling receptor fcrn (roopenian and akilesh, ; abbreviations: cd, cluster of differentiation; egfr, epidermal growth factor receptor; her , human epidermal growth factor receptor- ; ig, immunoglobulin; il, interleukin; pd- , programmed cell death protein- ; rank, receptor activator of nuclear factor kappa-b; tnf, tissue necrosis factor; vegf, vascular endothelial growth factor. * names given as hugo gene nomenclature committee (hgnc) names (gray et al., ) followed by commonly used names in parentheses. ** rounded to one decimal point. data abstracted from la merie, . *** $ . b of $ . b is % of total mab and fc fusion protein value in ( of total actively marketed antibody-based products). baker et al., ) . the most important of these modifications has been the "yte" mutation (dall' aqua et al., ; dall'aqua et al., ) from medimmune (astrazeneca), which has been incorporated into a few early stage candidates at this point (robbie et al., ) . other half-life extension mutations of the fc also have been made, including xencor's xtend technology (zalevsky et al., ) , which has been incorporated into at least two early stage clinical candidates, alexion's anti-c mab, alxn , and the national institutes of health's (nih's) anti-cd mab, vrc ls. several other igg engineering technologies have been reported that have the potential to modulate the capabilities of existing and future clinical candidates (table ). these include protease activated "probody" iggs for tumor-localized activity (erster et al., ; desnoyers et al., ; polu and lowman, ) , protease-resistant iggs that are stable in the tumor micro-environment (kinder et al., ) , hexameric antibodies with high c q binding and concomitant cdc effector function (cook et al., ; de jong et al., ) , ph-dependent binding (igawa et al., a; chaparro-rogers et al., ; devanaboyina et al., ) and sweeping antibodies ) that improve upon the elimination profile for soluble antigens (igawa et al., ) , engineering variable regions for improved solubility and developability (clark et al., ; seeliger et al., ) , modulation of the pi or charge of the antibody variable sequences for improved half-life (igawa et al., b; li et al., ; datta-mannan et al., ) and/or separation and purification (sampei et al., ) , and mutation of protein a binding site for improved purification of a heterobispecific igg (tustian et al., ) . another area of antibody engineering that is starting to see significant activity is the engineering of igms as therapeutics, especially where high avidity effects are desired (chromikova et al., ; wang et al., b) . in a recent example, an anti-tnfrsf b (dr ) igm demonstrated -fold greater avidity and -fold greater killing effect than a similar igg (wang et al., b) . adcs target a cytotoxic drug to a tumor to kill cancer cells while lowering the systemic exposure of the active moiety, with the goal of increasing the size of the efficacy/toxicity window of highly toxic anti-tumor drugs (strohl and strohl, ; tsuchikama and an, ; beck et al., ) . adcs morrison et al., humanization mouse cdrs in human frameworks jones et al., ; queen et al., generation of scfvs fv domains fused with linker bird et al., ; huston et al., fc fusion proteins igg fc fused with peptides or proteins capon et al., affinity maturation improvement in binding to target hawkins et al., isotype switching for modified fc functionality fc functionality - change in fc activity greenwood et al., aglycosyl igg n x mutation to generate aglycosylated igg to reduce fcγr activity bolt et al., ; nesspor et al., heterodimeric consist of three components, the targeting antibody, the cytotoxic payload, and the linker that couples those two components together (fig. ) . with those three components come five considerations for the design and construction of an adc: first, the targeting antibody must bind to a protein that is found either exclusively on cancer cells or significantly overexpressed on cancer cells as compared with expression on normal tissues. the best targets for adcs may be oncofetal antigens or targets that may be overexpressed in cancer cells but present in normal tissues at low copy number or in tissues in which the toxicity is tolerable. the cell surface proteins most widely targeted with clinical stage (or approved) adcs currently are her (five adcs targeting), cd (four adcs targeting), cd (three adcs targeting), and mesothelin ( adcs targeting). cea-cam , egfr (wild-type), egfr (variant iii), cd , and cd each have two clinical stage adcs targeting them. the properties of good adc targets, as well as descriptions of candidate adc targets, have been reviewed (teicher, ; strohl and strohl, ). an interesting strategy being employed by cytomx to increase the tumor specificity of their adcs is the use of pro-antibodies that possess a peptide sequence covering the paratope, preventing binding to their target until it reaches the tumor microenvironment (tme). once in the tme, the paratope-shielding peptide is cleaved by matrix metalloproteinases (mmps), which are in high concentrations in most tmes, allowing the antibody to bind to targets in that local environment (desnoyers et al., ; polu and lowman, ) . second, the adc-directing antibody must be rapidly internalized upon ligation to its targeted receptor. antibodies that bind cell surface receptors may or may not internalize rapidly, so when isolating the antibody, incorporation of internalization screens into the discovery process is critical (poul et al., ; zhou et al., ) . third, the identity, number, and type of linker attachment sites is a critical issue. in first generation adcs, the linkers were typically attached to the ɛ-amine of lysine residues (tsuchikama and an, ; beck et al., ) . given that there are about lysine residues in a typical igg, ten of which can be accessed for chemical coupling (tsuchikama and an, ) , the results of such conjugations are highly heterogeneous. even with optimization, conjugation to lysines results in a drug to antibody ratio (dar) of about - , with a range of - (lazar et al., ; tsuchikama and an, ; beck et al., ) . there are multiple challenges with heterogeneous adcs including analytical challenges, batch-to-batch consistencies, the stability of the adc, and the potential for variable pharmacokinetics if conjugation sites in some antibodies interfere with normal fcrn-mediated recycling (beck et al., ) . site specific conjugation, which has been achieved through a variety of methods and can result in very tight dars and increased homogeneity (junutula et al., ; panowski et al., ; perez et al., ; beerli et al., ; ihospice et al., ; siegmund et al., ; thompson et al., ; tsuchikama and an, ; beck et al., ) , appears to be a significant advancement. new approaches using extension sequences, such as developed by mersana, can achieve a drug/antibody ratio of (yurkovetskiy et al., ) . fourth, the stability of the linker can have a huge influence on the efficacy and toxicity of the adc. in theory, a more stable linker which is only degraded within the lysosome should have the best safety profile. unfortunately, it is not that simple, as there are cases in which highly stable linkers resulted in safety issues. some of these may be due to mannose receptor, or potentially also fcγr-mediated binding and internalization of adcs, which could result in "off-target" toxicity issues (gorovits and krinos-kiorotti, ; beck et al., ) . finally, not all cancer cells within a tumor are target antigen-positive (singh et al., ) , thus allowing potential abbreviations: bbb, blood brain barrier; cdc, complement-dependent cytotoxicity; cdrs, complementarity determining regions; cns, central nervous system; ert, enzyme replacement therapy. innovative antibodies review escape of the antigen-negative cells from targeted therapies. it has been demonstrated that membrane permeability of the cytotoxin is a critical factor for potential bystander activity . thus, design of future adcs will need to take the chemistry of the resultant adc into account to optimize bystander effect and efficacy. there currently are clinical stage adcs, including three approved adcs, nine in phase iii development, and another in phase i/ii clinical development. the three approved adcs include mylotarg® ( , withdrawn in ), the cd -targeting adcetris®, and the erbb (her )-targeting kadcyla®. these clinical stage adc molecules are directed against at least different known targets, although a few have not been disclosed, so the actual number may be higher. the most targeted cell surface receptors currently are erbb and cd ( adcs against each), and cd , cd , and msln (mesothelin) ( adcs against each). there are known different classes of drugs incorporated into clinical stage adcs, of which are small molecule classes and five of which are protein-based. the most widely used drug class incorporated into clinical stage adcs are the auristatins (employed times), followed by the maytansanoids (in adcs), and benzodiazepines (used in adcs) (table ) . of the biologics, pseudomonas exotoxin pe is incorporated into four adcs (table ) . even though three adcs have been approved for therapeutic use, this technology is still relatively early in the developmental cycle and many of the "rules" for optimized adcs are still being sorted out (drake and rabuka, ; beck et al., ) . more details on the design and construction of adcs can be found in tsuchikama and an ( ) and in beck et al. ( ) . fc fusions are fusions of the igg fc domain with either a protein or peptide. in theory, the fusion can be to either the c-or n-terminus of the fc, but most fc fusions on the market and in clinical development today are n-terminal fusions. the primary reason for generating fc fusions is to extend the half-life of pharmacologically relevant protein or peptide by using the fcrn-mediated recycling of the fc (strohl and strohl, ; strohl, ) . currently, fc fusion proteins (strohl, ) , with the pharmacologically active "head groups" being blood factors, such as f (factor ix) and f (factor viii), peptides such as gcg (glp- ) and a thpo (thrombopoietin) analogue, and an enzyme, such as the tissue non-specific alkaline phosphatase (tnsalp; millan et al., ) in asfotase alfa (strensiq®) (hofman et al., ) . certain human cytokines such as il have been approved (marketed name, proleukin®) for systemic delivery and use in severe diseases such as metastatic melanoma and metastatic renal cell carcinoma (dutcher, ) . systemic delivery of the t cell-activating cytokine, il , however, brings with it the potential for adverse events. the concept of using antibodies to target cytokines to either tumors or to specific tissues came into fruition around the turn of the century (penichet and morrison, ; halin et al., ) . since that time, there has been an effort to target il , or other cytokines such as il and tnf, to the tumor microenvironment, where the desired activity can take place with reduced adverse systemic effects (neri and sondel, ) . this approach has been actualized by the fusion of cytokines to antibodies to make immunocytokines that may target vasculature associated with tumors (pasche et al., ; hemmerli and neri, ) , tumor cell surface antigens (klein et al., ) , or targets that would assist in accumulation in inflamed joints (hughes et al., ) . immunocytokines come in two major formats, cytokine-scfv (or other fragment) fusions which have a short circulating half-life and cytokine-igg fusions, which retain a long half-life (neri and sondel ) . antibody-directed modulation of immune cell checkpoint receptors has become one of the most exciting and important new areas in antibody therapeutics over the past few years. most efforts have been focused on t cell checkpoint modulation, but there is increasing interest in b cell, nk cell, and myeloid cell checkpoint modulation as well. t cell activation is regulated by a series of three signals. the first signal is provided by the interaction of the t cell receptor (tcr) with major histocompatibility complex (mhc, hla) class i (for cd t cells) or mhc (hla) class ii (for cd t cells) on antigen presenting cells (apcs). the secondary signal is provided through one of several checkpoint receptors (table ) , which can either provide a costimulatory signal to activate the t cells, or a blocking signal to dampen t cell response (topalian et al., ) . the third signal comes from the production of either pro-inflammatory, t cellactivating cytokines or anti-inflammatory cytokines that would act to reduce t cell response (chikuma et al., ; schirdewahn et al., ) . cancer cells can express ligands for t cell inhibitory receptors such as pdcd (pd- ) (ligand is cd [pd-l ]), ctla- (ligands are cd and cd ), and havcr (aka tim ) (ligand reported to be gal ) to inhibit t cell activation and cytolytic t cell responses. ligation of these receptors can lead to t cell anergy or exhaustion, resulting in the inability of the immune system to kill cancer cells. inhibition of the blocking responses to t cell activation using anti-pdcd , anti-ctla , or anti-cd antibodies has proven clinically to result in improved responses for a subset of patients with metastatic melanoma, nsclc, and potentially other forms of cancer (achkar and tarhini, ; kim et al., ) . additionally, efforts are ongoing to use combinations of anti-pd and anti-ctla antibodies to increase the percentage of patients experiencing durable responses, i.e., "raising the tail of the survival curve" (harris et al., ) . alternatively, several clinical candidates are agonists of t-cell activating receptors such as tnfrsf (ox ), cd , tnfrsf (cd , - bb), tnfrsf (gitr), icos (cd ), cd , or cd to stimulate t cell responses (antonia et al., ; table ) . additionally, t cell checkpoint pathways are potentially important in infectious diseases, in which t cell exhaustion halts t cells from eliminating viral and bacterial pathogens (dyck and mills, ) . finally, antibody intervention in t cell checkpoint pathways may play a role in autoimmune diseases, where blocking the activating signals or increasing the blocking signals may result in lowering the t cell activation response (van der vlist et al., ) . five mabs and two fc fusion proteins that target t cell/ apc checkpoints have been approved (table ) . two more t cell checkpoint inhibitor antibodies are currently in phase iii clinical trials and are in phase i/ii clinical trials, covering different t cell checkpoint targets. some of these checkpoint targets are being tested in both immune and oncology related diseases. for example, cd , cd , and tnfrsf (ox ) antagonists are in early stage clinical trials for treatment of various immune disorders, whereas cd , cd , and tnfrsf (ox ) agonists are in early stage clinical trials for various cancer indications (table ) . checkpoint ligands expressed on cancer cells also are potentially excellent targets, both because they can block the inhibitory checkpoint interaction as well as targeting the ligand-expressing cancer cells with fc-active antibodies. for this approach, there are now three approved anti-pd-l antibodies and another seven in clinical trials, as well as three clinical stage anti-cd (cd ligand) mabs and one cd -targeting car-t cell product in phase i clinical trials., as well as four anti-cd (b h ) antibodies are currently in phase i clinical trials. b cell transitional checkpoints are centered around b cell homeostasis and the choice of whether the b cell should mature or proceed to apoptosis. this process ensures that b cells expressing autoreactive immunoglobulins are purged (cancro et al., ) . key regulators of b cell maturation that function in b cell checkpoints are tnfsf b (soluble blys, ligands b lymphocyte stimulator; also known as b cell activating factor [baff] ) and tnfsf (april, a proliferation- * abbreviations: imm, immunology; onc, oncology. ** known preclinical programs that should progress to clinical trials by end of . *** names given as hugo gene nomenclature committee (hgnc) names (gray et al., ) followed by commonly used names in parentheses. inducing ligand). tnfsf b can bind the tnfsf b receptor (br ; also known as baff-r) to promote b cell survival, and both tnfsf b and tnfsf can bind tnfrsf b (transmembrane activator- and calcium modulator and cyclophilin ligand-interactor, taci) and tnfrsf (b cell maturation antigen, bcma), both of which result in ig class switching and t cell-dependent responses (cancro et al., ). overexpression of tnfsf b can lead to autoimmune consequences, such as system lupus erythematosus (sle) or sjögren's syndrome (cancro et al., ). one b cell checkpoint inhibitor (anti-tnfsf b mab, benlysta®) is approved, two more are currently in phase iii clinical trials, and three are in phase i/ii clinical trials, all targeting the b cell activating factor regulatory pathway. another approach that has gained interest in very recent years is the immunomodulation of nk cells. nk cells, as well as cd t cells, express a series of inhibitory receptors including klrc -form a (nkg a), tigit, cd , and kir family members (carotta, ) . as an immune defense mechanism, tumor cells express ligands to bind to these receptors to inhibit unwanted activation of nk cells. currently there are six antibodies in phase i/ii clinical trials binding these targets to remove the brake on nk cell activation. finally, another checkpoint that regulates the activity of macrophages and their phagocytosis of target cells is the cd /sirpa (signal regulatory protein alpha) and calr (calreticulin)/lrp pathway. the cd /sirpa ligation is often referred to as the "don't eat me" signal, whereas calr/ lrp ligation is known as the "eat me" signal (mccracken et al., ) . blocking of cd by antibodies or fc fusion proteins can lead to an imbalance and a pro-"eat me" response (mccracken et al., ) . currently, four anti-cd antibodies or fc fusion proteins are being evaluated in clinical trials for treatment of cancer (table ) . one approach that has gained interest in recent years is the combination or mixture of antibodies, usually against a single target, included into a single dosage (raju and strohl, ; carvalho et al., ) . thus far, antibody mixtures are being used mostly for oncology and infectious disease indications. the danish biotechnology company, symphogen, has led this space, with four antibody mixtures currently being tested in clinical trials. these include sym , a mixture of two anti-egfr mabs, sym , a mixture of six antibodies against the erbb (erb-b receptor tyrosine kinase) family of receptors (ellebaek et al., ) , sym , a mixture of two antibodies targeting met (cmet), and sym , an undisclosed mixture of antibodies partnered with genentech for an infectious disease target. at least nine other antibody mixtures are being evaluated in clinical trials, all of which are against infectious diseases targets such as ebola virus, botulinum toxin, and other viruses. one very interesting new approach in this area that could see significantly greater upside in the coming years is the generation of fully human antibody mixtures, or polyclonal mixtures, in transgenic (tg) cattle (matsushita et al., (matsushita et al., , . these may, if found safe and efficacious, at least partially replace "specific" intravenous immunoglobulin (ivig), which is igg purified from individuals who have been vaccinated or from convalescing patients who have produced iggs against a specific target (llewelyn et al., ; mire et al., ) . the upside of tg cattle-produced human iggs is supply, consistency across lots, and the ability to vaccinate the cows with antigens not available for human vaccination due to regulatory and safety considerations. one such polyclonal mixture from tg cattle already being evaluated in clinical trials is sab- (sab therapeutics), a polyclonal mixture of human iggs targeting middle east respiratory (mers) virus (nct ; luke et al., ) . bispecific antibodies, first conceptualized in (milstein and cuello, ) , are antibodies that can bind two different antigens simultaneously. there are five fundamental groups of bispecific antibody formats: (i) asymmetric bivalent, bispecific igg-like antibodies with heterodimeric heavy chains (hcs) (ridgeway et al., ; merchant et al., ; gunasekaran et al., ; strop et al., ; klein et al., ; labrijn et al., von kreudenstein et al., brinkmann and kontermann, ) ; (ii) tetravalent multispecific antibodies that are comprised of iggs, with additional binding domains, e.g., scfvs, fvs, vhh domains, or non-antibody binding scaffolds such as fynomers (brack et al., ; silacci et al., ) , fused to either the n-or c-termini of either the heavy or light chains (lcs) (coloma and morrison, ) ; (iii) engineered binding domains within the normal igg structure, such as the "two-in-one" bispecific approach from genentech (bostrom et al., ; eigenbrot and fuh, ) and the f-star approach of designing novel second binding sites within the c h domain , (iv) engineered antibody fragments linked by short peptide linkers which can be made into bivalent, trivalent, or tetravalent formats addressing two to three targets (mack et al., ; holliger and winter, ; kipriyanov et al., ; reusch et al., ; egan et al., ) . these may be fused to an fc domain or other half-life extending molecule ; and (v) iggs that are chemically coupled to generate igg-igg conjugates (e.g., brennan et al., ; garrido et al., ) . examples of these five basic formats are shown in fig. . many variations on these central themes have been reviewed multiple times (kontermann, ; spiess et al., ; kontermann and brinkmann, ; ha et al., ; brinkmann and kontermann, ) . over the past decade there has been a literal explosion of novel bispecific antibody technologies, approaches, and clinical candidates. today there are at least bispecific or bifunctional antibodies in clinical trials that are made from at least different bispecific platform technologies (table ) . these include ten asymmetrical igg-based platforms ( bispecific antibodies), five appended igg platforms ( bispecific antibodies), a single platform for chemically coupled iggs (four bispecific antibodies), eight fragment-based platforms ( bispecific antibodies), and one igg-based bispecific generated with an unknown platform (table ) . two bispecific antibodies have thus far been approved for medical use, both in the field of oncology. the first bispecific antibody of any kind to be approved was catumaxomab (removab®), a bivalent, trifunctional, hybrid mouse igg arat igg b antibody targeting cd e with one arm and epcam with the other. catumaxomab, approved in (only in the european union) for treatment of malignant ascites, was generated by the three-way fusion of a mouse b-cell, a rat b-cell, and a myeloma cell to form a quadroma cell line (triomab® technology) (zeidler et al., ) . the second bispecific antibody to be approved was the anti-cd e x anti-cd "bispecific t cell engager" (bite) mt- , constructed by linking two scfvs with a five residue (g s) linker (mack et al., ) . this bite®, now known as blinatumomab (trade name, blincyto®), was approved in for treatment of b-cell acute lymphoblastic leukemia (all). the current clinical stage bispecific antibodies are used for a variety of different purposes. for example, of them bind two soluble antigens such as il and il (e.g., sar ; nct ), nine bind two receptors on the , igg-scfv fusion, mabtyrin (igg with non-antibody binding scaffold "centyrin" fused to c-terminal end of heavy chains); (c) iggs to which additional antigen combining sites have been added within the structure (e.g., two-in-one antibodies, mat "modular antibody technology" platform from f-star); (d) engineered antibody fragments linked by short peptide linkers which can be made into bivalent, trivalent, or tetravalent formats addressing two to three targets (e.g., bispecific t-cell engager (bite), nanobody platform, dual-affinity re-targeting (dart) antibodies, "tandem antibody" structures (tandabs)); (e) chemically coupled iggs. brennan et al., ; garrido et al., innovative antibodies mack et al., ; schlereth et al., ; baeuerle et al., tandab antibody fragment- william r. strohl same cell surface such as egfr and met (e.g., jnj- ; nct ), and four bind a cell surface target such as delta like canonical notch ligand (dll ) with one combining site and a soluble ligand such as vegf with the other (e.g., navicixizumab; nct ). two current clinical stage bispecific antibodies are biparatopic, i.e., both arms bind to the same receptor, albeit at two different nonoverlapping epitopes (e.g., zymeworks zw , which binds two non-overlapping epitopes of erbb ; nct ). the most significant use of bispecific antibodies, however, is for t cell redirection, in which one combining site is directed toward a cell surface target on a cancer cell and the other combining site binds cd e on t cells to redirect those cells to the targeted cancer cell (see below). twenty-seven clinical stage bispecific antibodies are immune cell redirection bispecific antibodies. one of these targets fcgr a (cd a) for nk cell redirection, while the other bispecific antibodies target cd e on t cells to redirect the cytotoxic t cells (ctls) to kill and lyse cancer cells. of these, are constructed from antibody fragments, seven are asymmetric bispecific iggs, four are conjugated bispecific iggs that are used to activate t cells ex corporally (brennan et al., ; garrido et al., ) , and two are bispecific iggs with appended domains (table ). the two appended iggs also utilize an asymmetric fc format so that only one cd e-binding arm is present. it is generally accepted that the most potent t cell redirecting antibodies are fragments, with unmodified bites and darts (dualaffinity re-targeting antibodies) demonstrating sub-picomolar ic values for in vitro killing activities (moore et al., ) . of the two approved antibodies, blincyto® is a mouse bite, while removab® is an asymmetric rat/mouse igg. given that both are "first generation" t cell redirecting, fully mouse antibodies for very different indications, it is difficult to say today which type of platform (fragment vs. igg-based) will ultimately be the most efficacious for treatment of diseases. the larger igg-based forms appear to be significantly less potent based on in vitro activities and in vivo preclinical dosing than are fragments (unpublished data). thus, there is a balance between sheer potency, which can be achieved with small size, and long half-life, which typically brings with it greater size and less potency. additionally, both the size of the cell surface receptor of the target cells and the epitope to which the antibody binds appear to be critical factors in potency as well (bluemel et al., ) . moreover, the potency of t cell redirected bispecific antibodies depends on the affinity of the arms for each antigen. typically in the case of bispecific t cell redirection antibodies, the affinity for the cancer cell surface target is much higher (i.e., -fold or more) than the affinity for the cd e chain on t cells (zhukovsky et al., ) . in summary, factors that may influence potency in t cell redirected antibodies are size of the antibody, size of the target cell surface protein, epitope on that protein to which the antibody binds, and affinity. another area that has not yet been fully investigated with respect to t cell redirection is the role of fc functionality. the triomab® platform, on which removab® is designed, has a highly active fc domain that interacts with human fcγrs to increase the immune response (chelius et al., ; hess et al., ) . on the other hand, most of the current fragment-fc, asymmetric igg, or appended igg platforms have used muted or silenced fcs so as not to over stimulate the immune system via interactions with myeloid effector cells. even with the absence of fc activity, many treatments with t cell redirecting bispecific antibodies are accompanied by cytokine storms that need to be addressed as part of the therapeutic paradigm . thus, it seems likely that most t cell redirecting antibodies made in the future will continue to avoid fc activity in an effort to limit the release of pro-inflammatory cytokines by t cells and other effector cells in the tumor microenvironment. cars are anti-tumor targeted antibodies that have been fused genetically to a stalk or linker, a transmembrane domain, and intracellular t cell activation domains that have been borrowed from activation checkpoint receptors such as cd , tnfrsf (cd ), and/or tnfrsf (ox ) ( fig. m ; figueroa et al., ; van der stegen et al., ; ruella and gill, ; smith et al., ; ruella and june, ; lim and june, ) . while the concept of car-t cells has been around since the early s (eshhar et al., ) , the advancement of technologies required to turn this into a viable "manufacturable" process was only realized in recent years. thus, similar to bispecific antibody technology, while conceptually old, truly developable car-t technology is still relatively young and still developing (lim and june, ) . there are fundamentally two types of cars. the first is autologous, in which a patient's t cells are collected by a process known as apheresis, and then either as a whole pool, or a fractioned pool of cd t cells, cd t cells or possibly both cd and cd t cells, are transduced with the cars using either viral vectors such as lentivirus or transposons such as sleeping beauty or piggyback (figueroa et al., ; lim and june, ) . the recombinant t cells, now armed with cars targeting a tumor expressed on their surface, are activated and infused back into the patients from which they were derived to kill cancer cells bearing the antigen (figueroa et al., ) . the second major type of car is allogeneic, or universal. an "off-the-shelf" cell line is constructed, typically devoid of mhc class i molecules (ren et al., a ) and endogenous t cell receptors (macleod et al., ; ren et al., a) to decrease the risk of host vs. graft (rejection) and graft vs. host disease (gvhd), respectively. this universal t cell line also would express cars for treatment of cancer or possibly viral infections. thus far, the barriers to generate truly off the-shelf allogeneic cell lines are still quite high, with control of proliferation, continued activation of the cells once they are engrafted, and incorporation of kill switches for safety purposes as critical issues still to be worked out. nevertheless, significant progress has been made in just the past year suggesting that fully modified allogeneic car-t cell therapy is quickly becoming a reality (ren et al., a, b) . to date, there are four generations of autologous car-t cell constructs. the first generation typically consisted of the extracellular, cancer cell-targeting scfv fused to the cd stalk and transmembrane domain followed by cd (aka cd ζ), which provided the activation signal (park and brentjens, ; figueroa et al., ; lim and june, ) . the first generation cars possessed ample cytotoxicity but lacked proliferative and survival signals. the second-generation cars typically linked the exodomain scfv to the transmembrane domain of cd , tnfrsf (cd , - bb), or tnfrsf (ox ) to provide a proliferation signal, followed by cd (cd ζ) to provide the cytolytic activation signal. the third generation cars have typically linked the targeting scfv to the cd transmembrane domain, followed by either the tnfrsf (cd , - bb), or tnfrsf (ox ) activation domains, and then cd (cd ζ) (park and brentjens, ; figueroa et al., ; smith et al., ; lim and june, ) . these cars combined cytolytic activity with both proliferation and survival signals to enhance both their activity and their persistence in the patient's serum. fourth generation cars add new activities such as a suicide mechanism to kill off the cars in case they become overproliferative, or utilize t cells that have been conditioned to recognize viral antigens which can be used as "vaccines" to increase the persistence of the car-t construct (chmielewski et al., ; smith et al., ; lim and june, ) . there are currently different car constructs in clinical trials. as stated earlier, all of the car candidates are in phase i or ii clinical trials. almost half ( / ) of the current cars originated in china, with originating in the us, and originating in europe. cars have been generated against different targets, of which are cell-surface proteins on cancer cells and one, wt , an mhc-displayed peptide target derived from an intracellular antigen (rafiq et al., ) . fifty-three (∼ %) clinical car candidates are directed against cd . the next most targeted antigens are gd and msln (mesothelin) ( cars each), erbb (her ) and cd ( cars each), and gpc (glypican- ) and tnfrsf (cd ) ( cars each). most of the current clinical stage car constructs are autologous car-t constructs generated from αβ t cells (table ) , but there are a few examples of other formats, including early formats of allogeneic car-t cells, autologous car γδ t cells, both autologous and allogeneic car-nk cells, car-nkt cells, and cars made from tcrs (table ) . it is too early to judge the success of the car field, although it is clear that this area has generated an enormous amount of interest, as well as funding well exceeding $ b. it is noteworthy that novartis recently ( / / ) filed a biologics license application (bla) to the us fda for treatment of relapsed and refractory b-cell acute lymphoblastic anemia (b-all) with ctl (tisagenlecleucel-t), making it the first car construct to be submitted for regulatory approval (kingwell, ) . moreover, kite pharma announced shortly thereafter ( / / ) that they had completed their rolling bla submission for treatment of non-hodgkin lymphoma (nhl) using kte-c (axicabtagene ciloleucel). if either ctl or kte-c is, or both are, approved within the next year, it will mark a huge milestone in this exciting new field. an area that has been of interest for many years, but has proven challenging, is the targeting of antibodies to compartments into which they do not normally go. these include, for examples, targeting antibodies to the gut via an oral route, to the brain by crossing the blood-brain barrier, or to the cytosolic intracellular compartment. all of these compartments present significant challenges, but in the past few years, significant strides have been made for all of them. the most advanced tissue-targeted antibody-based product the bone-targeted enzyme replacement-fc fusion, asfotase alpha (strensiq®), which was approved by the us fda for treatment of hypophosphatasia (hofman et al., ) . asfotase alpha (tnsalp-fc-deca-aspartate fusion protein) is targeted to bone with a deca-aspartate peptide fused to the c-terminus of the fc (millan et al., ) . the second area of antibody targeting that is represented by clinical candidates is based on the route of delivery to get the antibodies to the desired compartment. at least three orally-delivered, antibody-related proteins targeted to the intestinal tract are currently being evaluated in clinical trials. these include prx- (protalix®), an anti-tnf plant cellexpressed and delivered fc fusion protein in phase ii clinical trials (nct ) for the treatment of ulcerative colitis biologics for certain types of cancer where the tumor is more accessible (zeltsman et al., ) . a novel approach for delivering mabs and/or fc fusion proteins is via delivery of the gene or genes that produce them, either as naked dna, rna, or by a viral-based vector. this is not an entirely new approach, since studies were done around the turn of the century showing that rna (giraud et al., ) and viral (lewis et al., ) delivery of igg genes could result in demonstration of in vivo igg activity. nevertheless, there was not much interest until the past few years, when it has become evident that vectored or nucleic acid delivery of igg could potentially be a significant new approach to deliver antibodies for therapeutic use. one of the more exciting forms of delivery is the intramuscular injection of adeno-associated viruses (aavs) encoding antibodies, followed by years of consistently high expression of those antibodies in non-human primates greig et al., ) . it is important to note that aavs exist in the muscle cells as extrachromosomal elements and do not integrate, which increases the safety of their use for long term expression of antibodies or other proteins (greig et al., ) . this suggests that such an approach might be appropriate for delivery of anti-hiv antibodies to help patients either to become cured or, minimally, less reliant on highly active anti-retroviral therapy (haart) (schnepp and johnson, a; . there are several very promising, potent anti-hiv antibodies in clinical trials currently, some of which have been expressed in vivo using gene-based delivery of antibodies for potential therapeutic use (schnepp and johnson, b; yang and wang, ; . similarly, but with a different twist, aav-delivered antibodies to the nasal passages of mice have demonstrated excellent prophylaxis against flu virus (limberis et al., ; balazs et al., ; adam et al., ) . since these aavs enter epithelial cells that are sloughed off over several months, this provides a potentially safe route for delivery of prophylactic anti-flu antibodies that would cover the entire flu season. the potential significance of this approach is that there are several ha-binding and neutralizing antibodies available now that are nearly universal influenza virus inhibitors. these could potentially be used in clinical trials to determine whether or not this prophylactic, pan-influenza nasal delivery approach might be feasible. finally, the concept of using oncolytic viruses to deliver anti-tumor or checkpoint modulating antibodies to a tumor is very exciting. oncolytic viruses have been engineered for years to deliver immune-modulating molecules such as csf (gm-csf) to the tme (bommareddy et al., ) , so it makes sense that they could be engineered to deliver tme modulating antibodies (du et al., ) . (adelfinger et al., ; liikanen et al., ; fajardo et al., ) . over the past decade there has been a significant shift from discovery and development of basic antibodies, e.g., naked igg isotype antibodies with no additional engineering other than perhaps humanization and affinity maturation, to more sophisticated forms of antibodies in all kinds of shapes and sizes. these newer forms include fc-modified, glyco-engineered, bispecific, drug-conjugated, and cell surface expressed antibodies (i.e., cars) as new weapons to fight difficult to treat diseases. we now see this dramatic shift in the types and numbers of modified antibodies now reaching clinical trial studies. this new phase of antibody drug discovery and development represents an exciting and bold new era that should see antibody-based therapeutics expanding their influence in many types of diseases. in the next few years we will likely see the first regulatory approvals of car-t based antibodies and immunocytokines, as well as approvals of additional new bispecific antibodies, new adcs, fc engineered antibodies, and glyco-engineered antibodies. additionally, we should see new advances in targeting antibodies to the cns and intracellular compartments, as well as nucleic acid or viral-vectored delivery. what an exciting time to be an antibody engineer! abbreviations aavs, adeno-associated viruses; adc, antibody-drug conjugates; adcc, antibody-dependent cellular cytotoxicity; adcp, antibodydependent cellular phagocytosis; adcs, antibody-drug conjugates; car, chimeric antigen receptor; ccr , c-c motif chemokine receptor; cdc, complement-dependent cytotoxicity; cxcr , c-x-c motif chemokine receptor ; egfr, epithelial growth factor receptor; epcam, epithelial cell adhesion molecule; erbb , erb-b receptor tyrosine kinase ; gpcrs, g-protein coupled receptors; hiv, human immunodeficiency virus; mab, monoclonal antibody; ms, multiple sclerosis; nk, natural killer; ra, rheumatoid arthritis; rsv, respiratory syncytial virus; tnf-α, tumor necrosis factor-alpha; vegf, vascular endothelial growth factor william r strohl declares that he has financial interest in johnson & johnson, for whom he was recently an employee, but no other potential conflicts of interest. this article does not contain any studies with human or animal subjects performed by the author. structure and function of the blood-brain barrier the blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells the use of immunotherapy in the treatment of melanoma adeno-associated virus -mediated airway expression of antibody protects old and immunodeficient mice against influenza virus preclinical testing oncolytic vaccinia virus strain glv- b expressing an anti-vegf singlechain antibody for canine cancer therapy novel type ii anti-cd monoclonal antibody (ga ) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in b-cell malignancies a novel 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immunoglobulin fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins. front immunol engineering hiv-resistant, anti-hiv chimeric antigen receptor t cells enhancement of the antitumor activity of interleukin- by targeted delivery to neovasculature immuno-oncology combinations: raising the tail of the survival curve selection of phage antibodies by binding affinity. mimicking affinity maturation the antibody-based targeted delivery of interleukin- and to the tumor neovasculature eradicates tumors in three mouse models of cancer mor /es , a novel bispecific antibody targeting psma for the treatment of metastatic castration-resistant prostate cancer cancer therapy with trifunctional antibodies: linking innate and adaptive immunity asfotase alfa: enzyme replacement for the treatment of bone disease in hypophosphatasia diabodies: small bispecific antibody fragments diabodies": small bivalent and bispecific antibody fragments targeting of viral interleukin- with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli antibody recycling by engineered ph-dependent antigen binding improves the duration of antigen neutralization reduced elimination of igg antibodies by engineering the variable region engineered monoclonal antibody with novel antigen-sweeping activity in vivo sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens from circulation sitespecific conjugation of monomethyl auristatin e to anti-cd antibodies improves their pharmacokinetics and therapeutic index in rodent models a plant cell-expressed recombinant anti-tnf fusion protein is biologically activity in the gut and alleviates immune-mediated hepatitis and colitis effector cell recruitment with novel fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo b-cell depletion replacing the complementarity-determining regions in a human antibody with those from a mouse site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index comparison of cell lines for stable production of fucose-negative antibodies with enhanced adcc endosomal acidic ph-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape survival benefit of immune checkpoint inhibitors according to the histology in non-small-cell lung cancer: a meta-analysis and review engineered proteaseresistant antibodies with selectable cell-killing functions car t therapies drives into new terrain bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics progress in overcoming the chain association issue in bispecific heterodimeric igg antibodies cergutuzumab amunaleukin (cea-il v), a cea-targeted il- variant-based immunocytokine for combination cancer immunotherapy: overcoming limitations of aldesleukin and conventional il- -based immunocytokines vectorization in an oncolytic vaccinia virus of an antibody, a fab and a scfv against programmed cell death- (pd- ) allows their intratumoral delivery and an improved tumor-growth inhibition medi : a pcsk ab-glp- fusion molecule that elicits robust antidiabetic and antihyperlipidaemic effects in rodents and non-human primates easd dual targeting strategies with bispecific antibodies bispecific antibodies sales of recombinant therapeutic antibodies & proteins. weikersheim efficient generation of stable bispecific igg by controlled fab-arm exchange oral administration of okt mab to patients with nash, promotes regulatory t-cell induction, and alleviates insulin resistance: results of a phase iia blinded placebo-controlled trial analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry engineered antibody fc variants with enhanced effector function clinical use of blinatumomab for b-cell acute lymphoblastic leukemia in adults a her -specific modified fc fragment (fcab) induces antitumor effects through degradation of her and apoptosis generation of neutralizing activity against human immunodeficiency virus type in serum by antibody gene transfer fusion protein from rgd peptide and fc fragment of mouse immunoglobulin g inhibits angiogenesis in tumor framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge intracellular released payload influences potency and bystander-killing effects of antibody-drug conjugates in preclinical models oncolytic adenovirus expressing monoclonal antibody trastuzumab for treatment of her -positive cancer the principles of engineering immune cells to treat cancer self-assembled protein nanocarrier for intracellular delivery of antibody intranasal antibody gene transfer in mice and ferrets elicits broad protection against pandemic influenza novel cd -based bispecific chimeric antigen receptor designed for enhanced anti-hiv potency and absence of hiv entry receptor activity mgd , a cd x cd dualaffinity retargeting bi-specific molecule incorporating extended circulating half-life for the treatment of b-cell malignancies discovery of antibodies effector attenuating substitutions that maintain antibody stability and reduce toxicity in mice enhancing endosomal escape for intracellular delivery of macromolecular biologic therapeutics human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo targeting t cells with bispecific antibodies for cancer therapy a small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity integration of a cd car into the tcr alpha chain locus streamlines production of allogeneic gene-edited car t cells conversion of ctla- from inhibitor to activator of t cells with a bispecific tandem single-chain fc ligand highly efficient deletion of fut in cho cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies delivery of antibodies to the cytosol triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production species-specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle molecular pathways: activating t cells after cancer cell phagocytosis from blockade of cd "don't eat me" signals an efficient route to human bispecific igg enzyme replacement therapy for murine hypophosphatasia hybrid hybridomas and their use in immunohistochemistry passive immunotherapy: assessment of convalescent serum against ebola virus makona infection in nonhuman primates application of dual affinity retargeting molecules to achieve optimal redirected t-cell killing of b-cell lymphoma beat® the bispecific challenge: a novel and efficient platform for the expression of bispecific iggs chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains humanized porcine vcam-specific monoclonal antibodies with chimeric igg /g constant regions block human leukocyte binding to porcine endothelial cells immunocytokines for cancer treatment: past, present and future avidity confers fcγr binding and immune effector fuction to aglycosylated igg increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle antibody-dependent cell cytotoxicity: immunotherapy strategies enhancing effector nk cells sitespecific antibody drug conjugates for cancer therapy adoptive immunotherapy for b-cell malignancies with autologous chimeric antigen receptor modified tumor targeted t cells the antibody-based delivery of interleukin- to the tumor neovasculature eradicates murine models of cancer in combination with paclitaxel antibody-cytokine fusion proteins for the therapy of cancer antibody-drug conjugates: current status and future directions probody therapeutics for targeting antibodies to diseased tissue selection of tumor-specific internalizing antibodies form phage libraries a humanized antibody that binds to the interleukin receptor optimized t-cell receptormimic chimeric antigen receptor t cells directed toward the intracellular wilms tumor antigen potential therapeutic roles for antibody mixtures multiplex genome editing to generate universal car t cells resistant to pd- inhibition a versatile system for rapid multiplex genome-edited car t cell generation a tetravalent bispecific tandab (cd /cd ), afm , efficiently recruits t cells for the potent lysis of cd (+) tumor cells. mabs knobs-into-holes' engineering of antibody ch domains for heavy chain heterodimerization a novel investigational fc-modified humanized antibody, motavizumab-yte, has an extended halflife in healthy adults fcrn: the neonatal fc receptor comes of age development of pf- , a highly potent anti-p-cadherin/anti-cd bispecific dart molecule with extended half-life for the treatment of cancer discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria how to train your t cell: genetically engineered chimeric antigen receptor t cells versus bispecific t-cell engagers to target cd in b acute lymphoblastic leukemia chimeric antigen receptor t cells for b cell neoplasms: choose the right car for you anti-hiv designer t cells progressively eradicate a latently infected cell line by sequentially inducing hiv reactivation then killing the newly gp -positive cells identification and multidimensional optimization of an asymmetric bispecific igg antibody mimicking the function of factor viii cofactor activity redirection of t-cell effector functions for cancer therapy: bispecific antibodies and chimeric antigen receptors the third signal cytokine il- rather than immune checkpoint inhibitors contribute to the functional restoration of hepatitis d virus-specific t-cells eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain ep-cam-/cd -bispecific antibody construct novel human igg and igg fc-engineered antibodies with completely abolished immune effector functions adeno-associated virus delivery of broadly neutralizing antibodies vector-mediated in vivo antibody expression boosting antibody developability through rational sequence optimization improved cancer therapy and molecular imaging with multivalent, multispecific antibodies trastuzumab triggers phagocytic killing of high her cancer cells by interaction with fcγriv on immune cells high resolution mapping of the binding site on human igg for fc gamma ri, fc gamma rii, fc gamma riii, and fcrn and design of igg variants with improved binding to the fc gamma r lack of fructose on human igg n-linked oligosaccharide improves binding to human fcgamma rlll and antibody-dependent cellular toxicity spontaneous isopeptide bond formation as a powerful tool for engineering site-specific antibody-drug conjugates discovery and characterization of cova , a clinical stage bispecific tnf/il- a inhibitor for the treatment of inflammatory diseases quantitative characterization of in vitro bystander effect of antibody-drug conjugates a novel, native-format bispecific antibody triggering t-cell killing of b-cells is robustly active in mouse tumor models and cynomolgus monkeys chimeric antigen receptor (car) t cell therapy for malignant cancers: summary and perspective harnessing fc receptor biology in the design of therapeutic antibodies alternative molecular formats and therapeutic applications for bispecific antibodies in vitro and ex vivo strategies for intracellular delivery rituximab. how approval history is reflected by a corresponding patent filing strategy optimization of fc-mediated effector functions of monoclonal antibodies isotype selection and fc engineering: design and construction of fit-for-purpose therapeutic antibodies fusion proteins for half-life extension of biologics as a strategy to make biobetters therapeutic antibody engineering: current and future advances driving the strongest growth area in the pharma industry generating bispecific human igg and igg antibodies from any antibody pair nivolumab effectively inhibit platinum-resistant ovarian cancer cells via induction of cell apoptosis and inhibition of adam expression chimeric antigen receptos and bispecific antibodies to retarget t cells in pediatric oncology armed oncolytic adenovirus expressing pd-l mini-body enhances anti-tumor effects of chimeric antigen receptor t-cells in solid tumors cytotoxic mechanisms of immunotherapy: harnessing complement in the action of antitumor monoclonal antibodies antibody-drug conjugate targets straightforward glycoengineering approach to site-specific antibodypyrrolobenzodiazepine conjugates generation of binding molecules immune checkpoint blockade: a common denominator approach to cancer therapy antibody-drug conjugates: recent advances in conjugation and linker chemistries development of purification processes for fully human bispecific antibodies based upon modification of protein a binding avidity engineered glycoforms of an anti-neuroblastoma igg with optimized antibody-dependent cellular cytotoxic activity an engineered silent fc variant of an igg eliminates all immune effector functions via structural perturbations improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design aglycosylation of human igg and igg monoclonal antibodies can eliminate recognition by human cells expressing fcγri and/or fcγrii receptors study of natural antibodies against vascular endothelial growth factor receptor in hepatocarcinoma multimeric anti-dr igm antibody displays potent cytotoxicity in vitro and promotes tumor regression in vivo binding activities of a repertoire of single immunoglobulin variable domains secreted from escherichia coli brain penetration, target engagement, and disposition of the bloodbrain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin establishment of fut knockout chinese hamster ovary cells; an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity passive immunization against hiv/aids by antibody gene transfer boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates a polymer-based antibody-vinca drug conjugate platform: characterization and preclinical efficacy enhanced antibody half-life improves in vivo activity simultaneous activation of t cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing surgical immune interventions for solid malignancies receptor tyrosine kinases in carcinogenesis (review) internalizing cancer antibodies from phage libraries selected on tumor cells and yeast displayed tumor antigens bispecific antibodies and cars: generalized immunotherapeutics harnessing t cell redirection (ilan et al., ) . the plant cells are thought to protect the fc fusion while traversing through the stomach. over the last decade, the mouse anti-cd e mab, okt , has been evaluated in clinical trials for oral delivery to the gut for treatment of nonalcoholic steatohepatitis (nash; nct ), with results suggestive of clinical activity (lalazar et al., ) . a second anti-cd e mab formulated for oral delivery is foralumab (ni- ; novimmune, tiziana life sciences), a fully human mab currently being prepared for phase ii clinical trials for oral delivery for the treatment of nash. getting antibodies to cross the blood-brain barrier has been a goal for well over two decades. igg levels in the human brain are approximately . % of the serum concentration of - mg/ml (abbott et al., ) . this differential is due to the blood-brain barrier (bbb) which effectively keeps antibodies out of the brain. considering the wealth of potential targets for biologics in the central nervous system (cns), there has been a great effort to find mechanisms to improve the ability to translocate biologics into the cns. in recent years, significant progress has been made in getting antibodies to traverse the bbb. yu et al. ( ) used a bivalent, bispecific antibody binding tfrc (transferrin receptor, cd ) with one arm and bace (β-secretase- ) with the other arm, to demonstrate that low affinity antibodies to tfrc were more efficient at transcytosis than high affinity antibodies. while they only achieved about -fold higher accumulations of antibody in the brain over controls, they clearly demonstrated anti-bace pharmacological activity of the antibody, proving that the antibody had accumulated within the brain (yu et al. ) . they also generated a bivalent, bispecific antibody targeting human and non-human primate (nhp) tfrc with one arm and human bace with the other arm . the best variants, which were low-to-moderate affinity antibodies to tfrc, were accumulated -fold higher in the brain than control antibodies and they demonstrated in vivo pharmacological activity in nhps .neiwoehner et al. ( ) compared the efficiency of transcytosis using a tetravalent, bispecific antibody with two arms each binding to tfrc and app (amyloid-beta, aβ) to a trivalent, bispecific antibody with only one arm binding tfrc. they found that monovalent binding to tfrc promoted efficient transcytosis whereas bivalent binding to tfrc resulted in shuttling the complex towards lysosomal degradation. they demonstrated a -fold improvement in target engagement over the control (neiwoehner et al. ) . in contrast to these studies in which monovalent targeting of the transcytotic receptor was optimal, the anti-tmem a (α( , )-sialoglycoprotein), llama single-domain antibody, fc (abulrob et al., ) , appeared to be transcytosed more efficiently as a dimer rather than a monomer (farrington et al., ) . recently, fc was fused in an scfv format to the n-terminus of the hc of an anti-grm (glutamate metabotropic receptor , mglur ) antagonist igg to shuttle it across the bbb (webster et al., ) , achieving pharmacological activity with a -fold enrichment of the antibody in the brain parenchyma (webster et al., ) . thus, it still appears that there is much to be learned about optimizing antibodies for transcytotic delivery of proteins to the cns.william pardridge and his colleagues have isolated an anti-human insr (insulin receptor) antibody that can be transcytosed by insr on endothelial cells lining the vasculature in the brain (boado et al., ) . they have used the anti-insr antibody as a transcytotic carrier to move enzymes across the bbb for cns enzyme replacement therapy (ert) (boado et al., (boado et al., , . these candidates are constructed by fusion of the enzymes to the c-terminus of the bbb-traversing anti-insr igg "hirmab" (boado et al., (boado et al., , . agt- , which is a tetravalent (two antibody arms and two enzymes) fusion of an anti-insr antibody and α-l-iduronidase (ali) (boado et al., ) , is being evaluated in phase i clinical trials (nct ) for the treatment of mucopolysaccharidosis i (mps i; hurler syndrome). agt- was recently demonstrated to be taken up by nonhuman primate brain at . % of injected dose as compared to % injected dose of α-l-iduronidase alone (boado and pardridge, ) , demonstrating the pharmacological relevance of the bbb-traversing bispecific antibody. agt- , comprised of a fusion of iduronate -sulfatase (ids) to the c-termini of the anti-insr hcs (boado et al., ) , is under phase i clinical testing (nct ) for the treatment of mucopolysaccharidosis ii (mps ii; hunter syndrome).the final delivery-related technology that has gotten very interesting in recent years is the delivery of mabs to the cytosol of cells via pinocytosis and endosomal escape (marschall et al., ; lönn et al., ; stewart et al., ; lim et al., ) . multiple approaches have been taken to get biologically active antibodies into the cytosol of cells, including the use of cell penetrating peptides (marschall et al., ; lönn et al., ; lim et al., ) . just recently, a unique antibody has been generated for the delivery of an igg to the cytosol of cells via endosomal escape (choi et al., ) . this antibody, which has a unique sequence in its light chain variable region, has been matured to increase the proportion of igg that enters the cytoplasm . this, and other cell penetration technologies (marschall et al., ; lönn et al., ; lim et al., ) bring hope that one day, antibodies will be used to target cytosolic antigens. traditional forms of delivery for mabs and fc fusion proteins has been via either intravenous (iv) or subcutaneous (sc) administration of formulated proteins. generally, high dose mabs for oncology indications are limited to iv dosing, whereas low dose antibodies such as adalimumab, golimumab, and ustekinumab can easily be delivered in sc doses. additionally, in recent years there has been increased interest in intratumoral dosing of antibodies and other key: cord- -b o roz authors: verhoef, jan; snippe, harm title: immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites date: journal: principles of immunopharmacology doi: . / - - -x_ sha: doc_id: cord_uid: b o roz despite the introduction of effective health measurements, vaccination and antimicrobial therapy infectious diseases continue to threaten human life. the reasons are numerous and diverse: antibiotic resistance, hospital-invading pathogens, new emerging infectious diseases, bioterrorism, biological warfare. this chapter is an introduction to several aspects of infectious diseases viewed from the host as well as from the pathogen (bacterium, virus and parasite). furthermore the basic principles of innate and adaptive immune responses, especially in debilitated patients, are described. detailed information is given on the pathogenesis of septic shock, aids and vaccination strategies. in the middle of the th century,it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means.at the beginning of the th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms in order to prevent the diseases in question (rabies, diphtheria, tetanus, etc.) and due to the discovery of antimicrobial chemicals (ehrlich) and antibiotics (fleming), the threat of infectious diseases seemed to be minimized. largescale vaccination programs against childhood diseases (diphtheria, whooping cough and polio) started in the early fifties, giving hope to finally eradicate these diseases from the planet. this approach was successful for smallpox ( ) ; however, new infectious diseases emerged (e.g., legionella, hiv, helicobacter, sars, etc.). new vaccines and antibiotics are needed. furthermore, due to the intensive medical treatment with antibiotics and immunosuppressive drugs,hospital infections are a growing problem. bacteria hitherto deemed harmless are causing opportunistic infections in immunocompromised patients.the pathogens develop multiple resistances to antibiotics and sometimes no effective antibiotics are available to treat those patients. to make the story evermore serious, man is surrounded and populated by a large number of different non-pathogenic micro-organisms. in the normal -healthy -situation, there is a balance between the offensive capabilities of micro-organisms and the defences of the human body. the body's defences are based on vital non-specific and specific immunological defence mechanisms. an infection means that the microorganism has succeeded in penetrating those lines of defence, signaling a partial or complete breakdown of the body's defence system. the body's first line of defence comprises the intact cell layers of skin and mucous membrane, which form a physical barrier. the skin's low ph level and bactericidal fatty acids enhance the protection provided by this physical barrier.the defence in the respiratory tract and the gastrointestinal tract is mucous, the 'ciliary elevator' of the epithelium, and the motility of the small intestine. the presence of normal microbial flora (colonization resistance) in the intestine also plays a role in protection against colonization. the most important humoral natural resistance factors are complement, lysozyme, interferon, and a number of cytokines. lysozyme, which is found in almost all body fluids, degrades sections of the cell wall of gram-positive and -in combination with complement -gram-negative bacteria. this causes the otherwise sturdy cell wall to leak and the bacterium to burst. interferons are glycoproteins which may inhibit the replication of viruses. within several hours after the onset of a virus infection, interferons are produced in the infected cell and help protect the neighbouring unaffected cells against infection.this protection is brief, but high concentrations of inter-ferons are produced at a time when the primary immunological response is relatively ineffective. cytokines, such as il- (interleukin- ), gm-csf (granulocyte-macrophage colony-stimulating factor), and tnf-α (tumor necrosis factor α), stimulate non-specifically the proliferation, maturation, and immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites jan verhoef and harm snippe a function of the cells involved in defence (see chapter a. ). innate immune cells recognize microbes by toll-like receptors (see section pathogenesis of shock), giving rise to the above production of cytokines in the early phase of the response. micro-organisms that succeed in penetrating the first line of defence are ingested, killed, and degraded by phagocytic cells (leukocytes, monocytes, macrophages), which are attracted to a microbial infection through chemotaxis. the ingestion by phagocytic cells of the micro-organism is enhanced by serum proteins (opsonins),such as antibodies and the c b component of complement, for which these phagocytes have a receptor.after ingestion, the particle is surrounded by the membrane of the phagocyte, forming a vacuole known as a phagosome .the phagosome then fuses with some of the countless lysosomes in the phagocyte,thus allowing the lysosomal microbicidal agents and enzymes to do their work.in the case of leukocytes,the formation of toxic oxygen radicals greatly contributes to the killing and elimination of the ingested micro-organism ( fig. ) (see chapter a. ). a special role in cellular natural resistance is reserved for the nk (natural killer) cells, which display considerable cytotoxic activity against virusinfected cells. this nk activity is stimulated by inter-ferons and,at a very early stage in the infection,serves to reinforce the non-specific defence mechanism. in the specific immune response, elements of the natural defence mechanism are directed against a specific enemy. depending on the micro-organism, either the cellular defence mechanism (tuberculosis) or the humoral antibody-dependent defence mechanism (influenza) is of primary importance. in many cases, a joint cellular and humoral response is needed to provide an effective defence (typhus). both t lymphocytes and macrophages play a role in cellular defence. during the first contact with an antigen, macrophages process the antigen and present its protein fragments (t-cell epitopes) to t cells, which then proliferate and remain present for years in the body as memory cells. when a second encounter occurs, t cells produce lymphokines, which activate the macrophages. these activated macrophages grow larger, produce more and better degrading enzymes, and are now able to eliminate micro-organisms which otherwise would have survived intracellularly (tuberculosis, typhoid fever). macrophages from non-immune animals are not able to eliminate these micro-organisms. five different classes of antibodies can be distinguished in man, namely, igg, iga, igm, igd, and ige. they differ from one another in size, charge, amino acid composition, and glycosylation (see chapter a. , c. ). in principle, the structure of the antibodies is the same, i.e., heavy and light chains: it is the variable part of these chains which recognizes the micro-organism.the biological function (see below) is determined by the constant part (fc) of the heavy chain.with the exception of igd, all these antibodies are important in antimicrobial activity. -iga,which is found in all external secretions,reacts with the surface of micro-organisms, preventing them from adhering to sensitive cells and mucous membranes. -igg neutralizes microbial toxins. -igg, igm, and c b serve as opsonins, which promote phagocytosis. -igg, igm, and to a lesser extent iga activate the complement system after binding to the microorganism.activation products c a and c a ensure that the phagocytes are attracted to the inflammatory response. schematic representation of the progressive steps of phagocytic endocytosis. -ige is of importance in parasite infections. at the site of the infection,mast cells,bearing specific ige, release large quantities of vasoactive amines, which cause the contraction of smooth muscle tissue and increase the permeability of the blood vessels. in the intestine, this results in worms being detached and eliminated. several non-invasive bacteria, i.e., those that do not invade the body, cause disease through the production of exotoxins (tetanus, diphtheria, cholera). the immune system neutralizes the toxin with the aid of antibodies (igg, igm). if the individual has not been inoculated, the toxin will act on certain cells in the body directly through a receptor. this bond is very strong (i.e., has a high affinity), and is difficult to break by the administration of antibodies. in practice, if there are clinical symptoms of the disease, then large doses of antitoxins must be administered.if one is trying to prevent the development of the disease, then the presence of small quantities of specific antibodies (igg) is sufficient. the adherence of bacteria to cells is effectively blocked by iga. oral vaccination against cholera, for example,is aimed at obtaining sufficient specific iga in the intestine, so that no colonization of this bacterium can take place, and the cholera toxin can no longer adhere to its receptor. in general, defence against invasive bacteria is provided by antibodies (igg, igm) that are directed against bacterial surface antigens. in many cases, these bacteria have a capsule which interferes with effective phagocytosis. antibodies against these capsule antigens neutralize the interference, with subsequent elimination of the bacteria by phagocytes. antibodies (igm, igg, iga) in combined action with complement kill bacteria by producing holes in the cell wall of the bacterium. although intracellular bacteria (tuberculosis, leprosy, listeriosis, brucellosis, legionellosis, and salmonellosis) are ingested by macrophages, they are able to survive and multiply. in these cases, cellular immu-nity alone provides the defence, since antibodies are not effective. only activated macrophages are capable of killing and degrading these bacteria. antibodies neutralize viruses (fig. ) directly and/or indirectly by destroying infected cells that carry the virus antigen on their surface. the mechanisms of this defence resemble those of humoral defence against bacterial surfaces. the antibodydependent cellular cytotoxicity reaction is specific for the defence against viruses. cells which carry on the surface an antigen encoded by the virus are attacked by cytotoxic k cells, bearing antibodies which fit the antigen on the target cell (k cells have fc receptors for igg). not only humoral, but also cellular immunity plays an important role in virus infections. people with a genetic t-cell deficiency are highly susceptible to virus infections. in cellular defence, it is primarily the virus-infected cells which are attacked and eliminated. cytotoxic t cells recognize mhc- -presented t-cell epitopes on the surface of virus-infected cells and kill them. the fungi responsible for human diseases can be divided into two major groups on the basis of their growth forms or on the type of infection they cause. pathogens exist as branched filamentous forms or as yeasts, although some show both growth forms. the filamentous types (trichophyton) form a 'mycelium'. in asexual reproduction, the fungus is dispersed by means of spores; the spores are a common cause of infection after inhalation. in yeast-like types (cryptococcus), the characteristic form is the single cell, which reproduces by division or budding.dimorphic types (histoplasma) form a mycelium outside, but occur as yeast cells inside the body. candida shows the reverse condition and forms a mycelium within the body. in superficial mycoses, the fungus grows on the body surface, for example skin, hair, and nails (epidermophyton, trichophyton), the disease is mild, and the pathogen is spread by direct contact. in deep mycoses (aspergillus, candida, cryptococcus, histoplasma), internal organs are involved and the disease can be life-threatening and is often the result of opportunistic growth in individuals with impaired immunocompetence. many of the fungi that cause disease are free-living organisms and are acquired by inhalation or by entry through wounds. some exist as part of the normal body flora (candida) and are innocuous unless the body's defences are compromised in some way. the filamentous forms grow extracellularly, while yeasts can survive and multiply within phagocytic cells. neutrophils kill yeasts by means of both intraand extracellular factors. some yeasts (cryptococcus neoformans) form a thick polysaccharide capsule in order to prevent phagocytic uptake. in addition, many cell-wall components of yeasts cause suppression of cell-mediated immune responses.the role of humoral and cellular immunity in controlling infections caused by fungi is not yet well defined, but cellular immunity is the cornerstone of host defence against (some) fungal infections.as a consequence, hiv infection, which affects the cellular arm of the immune system, results in previously uncommon infections such as those caused by c. neoformans. the immunological defence systems against parasites are considerably more complex than those against bacteria and viruses. this is due to various factors. in the first place, each parasite has its own life cycle, consisting of various stages with specific antigen compositions.moreover,parasites are able to avoid the host defence system (mimicry), to combat it (immunosuppression), or to mislead it (antigenic variation). both humoral and cellular immunity are important for the defence against parasites growing intercellularly, as we have seen in the case of bacteria and viruses. antibody concentrations (igm, igg, ige) are often elevated. ige also plays a special role in the removal of parasites (especially worm infections) from the intestine (see above). in gram-negative (fig. ) bacterial infections, the interaction between bacterial endotoxin and various host-cell systems has been implicated in the pathogenesis of septic shock. in particular, the release of tnf-α (also called cachectin) and interleukin- (il- ), after the activation of host cells by endotoxin, induces hemodynamic shock. several lines of evidence support the current hypothesis that the monocyte-macrophage is the principal cellular mediator of endotoxicity. first, c h/hej mice carrying a single gene defect are nonresponsive to lipopolysaccharide (lps). the transfer of macro-phages of a closely related lps-sensitive strain makes the mice responsive. second, when the host is challenged with endotoxin, soluble factors are produced by macrophages that mediate fever and an acutephase response. these factors include the proinflammatory cytokines, il- , il- , il- , and tnf-α. together, tnf-α and il- stimulate endothelial cells to produce and express proteins on their membrane that have adhesive properties for leukocytes, promoting the migration and passage of polymorphonuclear leuko-cytes (pmns) from blood vessels through the endothelial layer, leading to pmn influx into the tissue. adhesion molecules that mediate the binding of pmns appear on the endothelium after an inflammatory stimulus, followed by molecules that are specific for adhesion of monocytes or lymphocytes, which may be why neutrophils enter before mononuclear cells. molecules that are currently known to be involved in leukocyte-endothelium interactions belong to three structural groups: the immunoglobulin gene superfamily, the integrin family, and the selectin family. concomitant with cytokine release, lps induces the activation of pmns,macrophages,and many other cells,resulting in the release of toxic oxygen radicals, which lead to tissue damage.at the same time, membrane-associated phospholipases are activated and products of the arachidonic-acid cascade are released through the cyclooxygenase and/or lipooxygenase pathways (see chapter a. ). plateletactivating factor (paf) is also generated, partly in response to the same signals.all these products contribute to a generalized inflammatory state with influx of pmns, capillary-leak syndrome, disturbances in blood coagulation, and myocardial suppression. endotoxin and tnf-α also produce multiple abnormalities in coagulation and fibrinolysis, leading to microvascular clotting and diffuse intravascular coagulation.they also induce endothelial cells to produce plasminogen activator and il- , which is an important modulator of the production of acutephase proteins by the liver. interestingly, despite having important structural differences, tnf-α and il- have multiple overlapping and few distinct biological activities,act synergistically,and mimic the whole spectrum of toxicity caused by lps (see chapter a. ).il- is an important chemoattractant and activator of neutrophils and is crucial in the early stages of inflammation. infusion of endotoxin in healthy humans leads to an early and transient increase in plasma levels of tnf-α (detectable after min, peaking after - min, and undetectable after - h), which coincides with the development of clinical symptoms and pathophysiological responses encountered in gramnegative septicemia. tnf-α, il- , il- , and il- levels are also increased in patients with sepsis syndrome, with high levels of these cytokines being correlated with severity of disease. all these observations support the concept that endotoxin largely acts by initiating an inflammatory response through the activation of monocytes-macrophages and the subsequent release of cytokines. it also activates the complement system (leading to the generation of c a, which induces aggregation of pmns and pulmonary vasoconstriction) and factor xii of the intrinsic coagulation pathway (hageman factor). finally, it induces the release of endorphins, which are also involved in the complex interactions of the inflammatory response in endotoxic septic shock. gram-positive bacteria are frequently and increasingly cultured from blood obtained from patients in shock. unlike the pathophysiology of shock caused by gram-negative bacteria,not much is known about the sequence of events that controls the signaling of monocytes and macrophages that leads to the release of cytokines. cell-wall components, such as peptidoglycan and teichoic acid, are clearly important in the activation of these cells. exotoxins,however,may also play a role in the pathogenesis of gram-positive bacterial shock. cd is a cell surface glycoprotein that functions as a binding receptor for lps.however its membrane anchoring by a glycosylphosphatidyl inositol (gpi) linkage suggests little signaling and suggests the existence of additional coreceptors. recent studies indicate that innate immune cells recognize conserved pathogen-associated molecular patterns (pamps), including lps, through toll-like receptors (tlrs) (fig. ) . this family of proteins, that resemble the antimicrobial toll proteins of drosophila, has been identified in humans and mice. tlr was identified as the missing link in lps-induced cell signal transduction and responsiveness that is associated with md- and cd . the tlr family members are coupled to a signaling adapter protein (myd ) and form differential dimers that may explain the discrete responses to tlr ligands such as lipoprotens, heat shock proteins, unmethylated cpg dna, viral dsrna and bacterial flagellin. intracellular signaling involves several kinases depending on the tlrs involved and includes the map kinase and nf-κb pathways leading to a cellular response. recently other protein families have been identified via genetic screening that also participate in direct recognition of pathogens. a new protein was found to be involved in resistance to gram-positive bacterial infections and recognizes the cell wall component peptidoglycan. the human immunodeficiency virus (hiv) is a retrovirus that infects cells bearing the cd antigen, such as t-helper cells (th), macrophages, and dendritic cells.the cd molecule,together with other receptor molecules, like chemokine receptor , acts as a binding site for the gp envelope glycoprotein of the virus. in an attempt to respond to hiv antigens and concomitant secondary microbial infections, these cells are activated, thus inducing the replication of hiv in the infected cd t cells, which are finally destroyed. in contrast, hiv- infection of macro-phages is self-sustained and results in an inexorable growth of chronic active inflammatory processes in immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites schematic illustration of cell activation through toll-like receptors (tlrs). many tissue compartments including the central nervous system. infected cells bear the fusion protein gp and may therefore fuse with other infected cells.this helps the virus to spread and accounts for the multinucleated cells seen in lymph nodes and brain. as a result of the decreased numbers of cd positive t-helper cells and defects in antigen presentation, depressed immune responses in these patients are observed. during the progression of the disease, opportunistic infections by otherwise harmless micro-organisms can occur. these include candida albicans oesophagitis, mucocutaneous herpes simplex, toxoplasma in the central nervous system, and pneumonia caused by toxoplasma and pneumocystis carinii; kaposi's sarcoma also occurs frequently in these patients.this has been linked to the presence of a previously unknown type of herpes virus (hhv- ).this immune deficiency syndrome is called 'acquired immune deficiency syndrome' (aids). it has been suggested that infected monocytes/macro-phages carry the hiv virus into the brain where it replicates in microglia and infiltrating macrophages. as a consequence many aids patients develop cognitive and motor brain impairments. however, the picture is complicated by the various persistent infections already present in these patients, which give rise to their own pathology in the brain. these include toxoplasma gondii, cryptococcus neoformans and jc virus. so far a cure for hiv infection has not been achieved. the main effort in the prevention of hiv infection lies in mass public education programmes. treatment of infected individuals is possible but expensive. at this moment a triple therapy is being prescribed in the western countries (two reverse transcriptase inhibitors and one protease inhibitor, fig. ), each of which interfere with specific steps in the process of hiv replication. one major problem that has arisen is the increasing resistance to these drugs. blocking of the chemokine receptor , a recently described co-receptor on cd cells for hiv, may be an alternative treatment for infected persons. this notion is supported by a recent finding that a homozygous defect in this chemokine receptor accounts for resistance of multiple-exposed individuals to hiv- infection. preparing his vaccines, koch employed killed germs (cholera) as a vaccine. since diphtheria and tetanus cause disease by means of toxins, the next logical step in the development of vaccines was the use of detoxified toxins to induce protection against these diseases (diphtheria, von behring and tetanus, kitasato).von behring and kitasato were the first to demonstrate that the source of the protective activity induced by vaccines was present in blood serum. von behring was also the first to prove that protective immunity could be passed on via serum. the development of new vaccines had its ups (yellow fever) and downs (tuberculosis). with the arrival of antibiotics, all work on new bacterial vaccines was suspended or severely curtailed, although some researchers continued to work on viral vaccines, such as rubella, measles, polio, and mumps. since it has proved difficult to consistently develop new antibiotics to combat antibiotic-resistant bacteria, interest in vaccines has gradually increased over the last years (see chapter c. ). today, thanks to new insights into the immune system and modern molecular biological and chemical techniques of analysis and synthesis, it is possible to produce well-defined vaccines.these contain only those determinants of the pathogenic microorganism which induce protection (epitopes). these epitopes are usually short peptide or oligosaccharide chains, which can be produced synthetically or by means of recombinant dna techniques. the immunogenicity of these products can be enhanced by coupling them to a carrier (tetanus toxoid, liposomes) and/or by adding an adjuvant (a substance which strengthens the immune response non-specifically). the recombinant dna technique can also be used to obtain attenuated strains of micro-organisms, which are fully immunogenic and thus provide protection,but which are no longer virulent. one example of this is the development of a new cholera vaccine based on a bacterium which has all the characteristics of a virulent strain,except the toxin.the bacterium has retained all its adherence factors, which allow it to adhere to the intestinal mucosa; the length of time it spends in the intestine is sufficient to stimulate the local immune system.the newest trend in vaccinology is immunization by introducing plasmid dna into the host.success has been attained by this method for hepatitis b vaccination. not only are new vaccines being developed, but it is also possible to heighten natural resistance for longer or shorter periods. various interleukins (il- , gm-csf) and interferons are being studied in order to use them to combat infectious diseases. monoclonal antibodies (antibodies with one specificity) directed against the endotoxin of gram-negative bacteria are now being administered to patients with severe gram-negative sepsis (serum therapy). more work is still necessary, however, to refine this technique, as the therapeutic effect is still limited. as outlined above for a number of bacteria and viruses, effective vaccines have been developed and applied worldwide.the eradication of smallpox (variola major) virus in the seventies of the last century was a milestone for the world health organization. the next goal of the who is to eradicate the poliovirus in the coming years. major problems to be dealt with are the distribution of these vaccines, the costs involved, the registration and the compliance of the vaccinees and molecular techniques to trace the final bug. meanwhile new unexpected microbiological threats come into focus. hospital infections caused by multiple resistant micro-organisms due to the abundant use of antibiotics and exchange of genetic material between micro-organisms impose major problems on patients and healthcare workers. new antibiotics and/or vaccines should be developed and new strategies employed to contain these infections. due to crowding and high mobility of the world population, old and new pathogens, e.g., influenza and sars, threaten our society. on top of this terrorists might intentionally use micro-organisms (smallpox, anthrax, plague etc.), or bacterial toxins (botulism) to cause death and disease in humans or animals in a civilian setting.the recognition that an event was caused by a biological weapon presents a severe challenge to be prepared for such an attack, especially for medical care providers and public health officials. strategies to combat bioterrorism have to be worked out but with the experience of years of combating microorganisms with hygiene measures, vaccination, antibiotic and anti-viral treatment, there must be a way out. despite the introduction of effective health measurements, vaccination and antimicrobial therapy infectious diseases continue to threaten human life. the reasons are numerous and diverse: antibiotic resistance, hospital-invading pathogens, new emerging infectious diseases, bioterrorism, biological warfare. this chapter is an introduction to several aspects of infectious diseases viewed from the host as well as from the pathogen (bacterium, virus and parasite). furthermore the basic principles of innate and adap-tive immune responses, especially in debilitated patients, are described. detailed information is given on the pathogenesis of septic shock,aids and vaccination strategies. medical microbiology a history of immunology the beast in man: microbes and macrobes as intimate enemies. part ii.the battle of bugs the immune system in health and disease microbiology we thank dr. c.p.m. van kessel of the eijkman-winkler center for the design and layout of the artwork. key: cord- -kkaxha d authors: zhang, mao-yu; lu, jin-jian; wang, liang; gao, zi-chao; hu, hao; ung, carolina oi lam; wang, yi-tao title: development of monoclonal antibodies in china: overview and prospects date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: kkaxha d monoclonal antibodies (mabs) have become increasingly important as human therapeutic agents. yet, current research concentrates on technology itself and pays attention to developed countries. this paper aims to provide a comprehensive review of mabs development in china through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing r&d projects. the trends in therapeutic areas and industrialization process are also highlighted. development and research trends of mabs are analyzed to provide a future perspective of mabs as therapeutic agents in china. over the past three decades, monoclonal antibodies (mabs) have achieved a dramatic development from scientific tools to powerful human therapeutic agents [ ] (see figure ). sales of mabs therapies exceeded billion us dollars in and are expected to reach billion us dollars by [ ] . in , kohler and milstein firstly described the in vitro production of murine mabs from hybridomas [ ] , which was an innovative step towards the development of human mabs as therapeutic agents. in the late s, clinical development of murine mabs was initiated but then inhibited by numerous significant drawbacks [ ] . later, in attempt to overcome the inherent immunogenicity concerns and reduce effector function of murine mabs in human [ ] , chimeric mousehuman antibodies were developed [ , ] . then, humanization antibodies were developed [ ] , which significantly enlarged the clinical usage of mab. in the following research exploration, human antibodies developed by phage display [ ] [ ] [ ] and human ig mice advanced the development of mab greatly [ , ] . nowadays, human mabs are the fastest growing category of mab therapeutics entering clinical study. development of this class of therapeutic agents started as early as s but achieved no clinical or commercial success until when adalimumab became the first human mab approved by the us food and drug administration (fda) [ ] . clinical development of mabs in china, like many developed countries, started with murine mab [ ] . r&d of mabs in china began in the s [ ] and the first mab therapeutic agent (murine monoclonal antibody against human cd antigen of t lymphocyte for injection) was introduced in [ ] . although mabs development in china has made significant progress over the past decades [ ] , all the mabs currently approved by cfda are technologically outsourced from foreign firms, like avastin (roche) [ ] . these mabs mainly target cd [ ] [ ] [ ] , antitumor necrosis factor (tnf) [ , ] , vegfr [ ] [ ] [ ] , her [ , ] , and egfr [ , ] for the treatment of cancer or immunological disorders [ ] . recently, i-chtnf and humanized mab h-r have been developed and approved as the treatment for solid tumor after panorex [ ] . it is anticipated that continuous research on mabs will give rise to an expanding source of therapeutics. stepping into st century, the technology level of mabs in china has vastly improved, resulting in great research progress resulting in high expression and specificity. for example, mabs based on research of tnf were developed to treat rheumatoid arthritis (ra) for patients presenting with medium and severe symptoms [ ] . these mabs block the interaction between vegf and kdr molecule and exhibit high specificity and activity [ ] . this paper aims to provide a comprehensive review of mab development in china through systematic analysis of product registry, patent application, clinical trials, academic publication, and ongoing r&d projects. we used multiple sources for data collection. all mabs products approved for marketing in china were searched in the drug registry maintained by cfda. the united states patent and trademark office (uspto) was the data source for the analysis of patent applications submitted by chinese assignee. all clinical trials involving mabs approved and carried out in china were identified at the chinese clinical trial registry (chictr) database. academic publications about mabs published by researchers in mainland china were searched from thomson reuters' web of science (wos). information about the progress of the ongoing r&d mabs projects was collected from the main research institutes and firms in china. in this research, we focused primarily on the mabs development in mainland china and thus the corresponding data about hong kong, taiwan, or macao was not considered. based on the information from the abovementioned sources, this paper tries to present a comprehensive analysis of mabs development in china which will help provide some perspectives for continuous development of these therapeutic agents. murine monoclonal antibody against human cd antigen of t lymphocyte for injection was the only mab approved by cfda since until when cfda granted approval to the second mab. up to date, there are a total of eight mab products approved by cfda (see table ). most of the mabs approved as human therapeutic agents are approved for the treatment of cancer or immunological disorders [ , ] . of the eight mabs launched in china, three target antigens related to antineoplastic diseases, five target antigens related to immunological diseases, and two target antigens related to kidney transplant rejection. as shown in table , there are three products approved for oncological uses including stage iii/iv of nasopharyngeal carcinoma (npc), advanced liver cancer, and non-small cell lung cancer (nsclc). another three products are indicated for immunological diseases, namely, psoriasis vulgaris, subacute eczema, active rheumatoid arthritis, moderate and severe plaque psoriasis, and ankylosing spondylitis. there are also products which are used to prevent rejection after organ transplantation. targets. the target of a therapeutic antibody is a major determinant of its efficacy and safety profile [ ] . the eight mabs products which are approved by cfda target a total of seven unique antigens. these include cd , egfr, intracellular dna for tumor therapy, il- , tnf for immunological diseases, and cd and cd for prevention of organ rejection after transplantation. there is also one mab which target the nucleus of tumor cells. in terms of versions, among the eight mabs approved in china, three are murine, one is chimeric, two are humanized and two are recombinant fusion protein. at present, there is no fully human mab approved for marketing in china. the development of mab technology remains at an early stage in china. overall, the mabs being studied are "me-too" and "me-better" products acquired through internal and external cooperation. on one hand, drug companies and universities with sufficient capacity conducted joint r&d projects. on the other hand, mab producers in china directly purchased the production technology from foreign countries, as demonstrated by the technology purchase actions of shanghai mei' en (collaborate with university of southern california, usa) and shanghai celgen (transfer from condar co., ltd., usa). according to the uspto database, the first mab-related patent in china was granted to monoclonal antibody against hepatitis e virus or its fragment with binding activity and use thereof in . notably, there are a total of nine mab-related patents approved up to date. of these nine patents (see table ), three are related to cancer diagnosis and therapy along with four other mabs targeting tnf, efgr, and vegfr. two patents which are associated with infectious diseases focus on virus or its fragment with binding activity and use thereof. for patent assignee, academic institutions are the most common and powerful controller in china, such as the institute of basic medical sciences of chinese academy of medical sciences, shanghai cancer institute, and xiamen university. table . four candidates are in phase ii (bevacizumab, bimotuzumab and cetuximab), three in phase iii (rituximab, infliximab and bevacizumab), and five under postmarketing surveillance (ranibizumab, metuximab and rituximab). among these, seven were chimeric mabs while the other five were humanized mabs. among these twelve mab candidates, eight are studied as the treatment for cancer while the rest are tested for their use in ophthalmology and immunology. three of the eight mabs for cancer treatment are under review for market reassessment. the technology to develop mabs as antitumor drugs is considered mature. for example, as the first mab product used in non-hodgkin's lymphoma (nhl) therapy, rituxan has demonstrated great achievements in both clinical setting and market share once it has been approved. mabs for immunological diseases are mostly used to treat rheumatoid arthritis and lupus notes: "primary sponsor" is the experiment contract organization, "funding" represents the resource of fund, from which institutions or others. "a" is the independent innovation foundation to universities and colleges by jinan science and technology bureau. "nsclc" is "non-small-cell lung cancer. " "nhl" is short for non-hodgkin's lymphoma. "hcc" is hepatocellular carcinoma. "cpgj" is shanghai cp guojian pharmaceutical co., ltd. source: http://www.chictr.org/en/. myelopathy. rituximab which is indicated in lupus myelopathy has gained a great success in clinical application and it is now being explored further for any new indications. although mabs are not considered as the drug of choice in ophthalmology, they may possibly become the innovative treatment for neovascular glaucoma and diabetic macular edema as shown in table . the following examples are used to provide a holistic view of mabs research development in china. biotech pharmaceutical funded the cancer center in the radiology department of sun yat-sen university to conduct open and multicenter clinical research on the efficacy of cis-platinum combined with nimotuzumab as the first line treatment of npc. this clinical research will help shed light on the possibility of "taixinsheng" combination therapy. similarly, chengdu huasun bio-tech funded fudan university to conduct intervention study to improve clinical outcomes of the combination therapy of "licartin" and transcatheter arterial chemoembolization (tace). these involve eight clinical trials which evaluate the clinical effectiveness of mabs in combination therapies. this is an indication that mabs as part of a combination therapy has become the new and alternative trend in future research. academic publications related to mabs in china were extracted from thomson reuters' web of science (wos) database. this is a powerful database which contains bibliographic data for, and citation to, publications in over , of the world's most important academic journals dating back to . studies and research about mabs in china began in the st century [ ] . publications related to mabs between and were searched using the following strategies: topic = (monoclonal antibod * ) and title = (monoclonal antibod * ) and address = (china not hongkong not taiwan not macau). "mabs" was used as the keyword in the same way. in the query above, the asterisk ( * ) represents any group of characters or no character and the literature type is limited in "article. " when "monoclonal antibody" was used as the search word, articles were identified. of these, were retrieved after excluding articles that were irrelevant to our research questions. with "mab" as acronym words, articles were identified. of these, articles retrieved after excluding irrelevant records. finally a total of articles were collected after repetitions of literature search (see figure ). the data shows that the number of publications was the most abundant in the last five years ( - ) and peaked in , while the decrease in and implies the more challenges for chinese researchers to make breakthrough in more innovative mab research. this historical trend of research corresponded with the trends of mabs technology development in china. by using "result analysis, " research areas of mabs in china focused on biochemistry, molecular biology, immunology, biotechnology, applied microbiology, biochemical research methods, chemistry, chemical analytical, and chemistry applied. the data also showed that research in joint forces with international counterparts was a common practice in mainland china especially with the us. this paper described the development and achievement of research in four main areas, namely, approved products, patent, clinical trials, and publications. in addition, research results also showed that academic institutes and enterprises play table . [ ] , high expression, mabs purification [ ] , and platform construction of phage antibody library technology [ , ] . as fully human mabs are considered the most promising category of targeted therapeutic agents [ ] , china has also shown great interests. academy of military medical science, for instance, is dedicated to advancing the screening techniques of phage antibody libraries and optimization platform establishment necessary for the development of fully human mabs [ ] . drug companies in china also play an important role in the development of mabs, especially in biosimilars [ ] . being one of the representatives among the numerous creative and energetic mab enterprises in china, biotech pharmaceutical developed and produced the first humanized mab [ ] . biotech also formed collaboration with cuba center of molecular immunology, which is yet another example demonstrating the strong interests in international cooperation by chinese enterprises. the r&d institution network in shanghai focused mainly on mabs technology industrialization. one of the r&d companies in the zhangjiang hi-tech park is shanghai cp guojian pharmaceutical co., ltd. (cpgj) which was founded by china international trust and investment corporation (citic) and is now coinvested by shanghai lansheng guojian pharmaceutical co., ltd. contributions have been made to the r&d, pilot plant test, and industrialization of antibody based drugs like cpgj. there are other r&d companies in the park which focus on r&d, manufacturing and marketing of high quality recombinant protein for the treatment of immune disease and neoplastic disease such as shanghai celgen biopharma. to summarize, mabs play an important role as efficient agent for antitumor and immunology disease. eight products in total are launched by cfda currently, mainly are in chimeric and humanized types. the number of total publications has been increasing since st century. nevertheless higher quality articles are needed. concerned about the intellectual property, patents applied by chinese assignee grow rapidly. to monitor safety and effectiveness of mabs, enterprises give support to and fund clinical trials for antitumor and other new indications. the future for mabs in china is promising. it should be mentioned that antibody-drug conjugates are becoming an increasingly important subclass of antibodyrelated cancer therapeutics and glycoengineering is being developed as a method to enhance the pharmacological properties of mabs. china may also need to pay much more attention to these kinds of antibodies. this paper found that mabs have experienced the fastest growth among all human therapeutic agents in the past two decades and will continue to do so in coming years [ , ] and presented a lot of advancements which have been achieved in china [ , ] . chinese biosimilar antibodies may now be approved in europe [ ] ; at the same time, some of the most successful innovated biopharmaceuticals currently in the market (including recombinant insulin, human growth hormone, etc.) will see the expiration of their patents in the us over the next few years. this will provide an opportunistic market share which potentially worth billion dollars for biosimilars including mabs in china [ ] . however, there are still some challenges yet to be resolved and at the top of the list would be production capability for china [ ] . firstly, there is still a lack of capacity for large-scale cell and perfusion cultivation at present [ ] . to address this problem, improving the cells expression is necessary. china should attempt to work with developed countries because, comparing patent information related to us, the patents applied in both countries mainly involve three of these eight sections (section c, section a, and section g) and, as indicated by the patent search results, the mabs research in the us encompasses a lot more fields than in china [ ] , especially in protein expression. moreover, according to the data searched in wos, us has been the strategic partner to china in r&d, which as mentioned before indicates that china should be more active to track first-edge technological research to make up the lack of capacity. additionally, data from ims showed that in mab drugs accounted for . % of the worldwide total sale, but only . % in china [ ] , which was far below the global average [ ] . while limited by production capability, high price, and low recognition, mabs in chinese market perform not well as other countries. to change the status, actions should be taken by the government. the chinese government is particularly supportive to universities and enterprises by ensuring reliable sources of funding for biopharmaceutical research and development. some of the most important funding sources are national natural science foundation of china, national high technology research and developmental program of china, and national basic research program of china. as universities and institutions account for a significant proportion of patent applications, china is steering towards a research-oriented country in the area of mab development. accordingly, the government has adopted favorable policies to encourage university-industry cooperation [ ] . the government also provides financial support to create and maintain a healthy environment for the biopharmaceutical industry. the inclusion of cancer treatment into the national social security would provide prosperous future for mabs development in china once approved [ ] . inclusion to the medical insurance catalogues also provides another important incentive for thriving mabs development. for example, basiliximab is listed in the medical insurance catalogues in thirteen provinces while infliximab in nine provinces. last, measures should also be made to attract much more foreign talents to join in the r&d and benefit from technology around the world. with all of these government support and joint efforts of academic and industry, there the mab development in china may contribute to global mabs production and therapeutic innovation. indeed, we strongly believe that in the near future, there will be likely a rapid growth in the development and usage of mabs in china owing to the sustained support from government. monoclonal antibody therapeutics: history and future advances in the production and downstream processing of antibodies continuous cultures of fused cells secreting antibody of predefined specificity differences in promiscuity for antibody-fcrn interactions across species: implications for therapeutic antibodies immunogenicity of engineered antibodies monoclonal antibodies as innovative therapeutics chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains reshaping human antibodies: grafting an antilysozyme activity assembly of combinatorial antibody libraries on phage surfaces: the gene iii site a surface expression vector for antibody screening by-passing immunisation: human antibodies from synthetic repertoires of germline vh gene segments rearranged in vitro production of fully human antibodies by transgenic 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(avastin), a humanized anti-vegf monoclonal antibody for cancer therapy efficacy and concentration-response of murine anti-vegf monoclonal antibody in tumor-bearing mice and extrapolation to humans pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer antibodies to watch in : mid-year update molecular mechanisms of resistance to the egfr monoclonal antibody cetuximab tumor antigentargeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape monoclonal antibodies-focus point of anti-tumor drugs research therapeutic effect of infliximab on moderate and severe active rheumatoid arthriti construction of a single-chain fv antibody against epidermal growth factor receptor and its antitumor activity which are the antibodies to watch in opportunities and challenges brought by monoclonal antibodies rapid development to biopharmaceutical in china potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal 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biopharmaceutical industry advances in antibody manufacturing using mammalian cells the progress of therapeutic antibody drug and the industrial key-technology of antibody production contrastive analysis of technology patent situation between china and us the swot analysis of antibody therapeutics in china general situation and analysis of patent applications of monoclonal antibody in china the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- - mbqkzpu authors: cichon, g; boeckh-herwig, s; schmidt, hh; wehnes, e; müller, t; pring-akerblom, p; burger, r title: complement activation by recombinant adenoviruses date: - - journal: gene ther doi: . /sj.gt. sha: doc_id: cord_uid: mbqkzpu recombinant adenoviruses are currently the most important vector system in gene therapy. adenoviruses frequently cause upper respiratory tract infections in humans and anti-adenoviral antibodies are found in – % of the population. therefore in the majority of potential patients receiving adenoviral gene therapy, the contact of virus particles and blood will lead to the formation of antigen–antibody complexes. these complexes have the ability to induce inflammatory reactions via an activation of the complement system. we have determined the level of c a (the most reactive complement component) generated in isolated citrate plasma of healthy individuals after challenge with recombinant and wild-type adenoviruses in amounts corresponding to virus blood levels to be expected in patients during adenoviral gene therapy. all plasma samples containing anti-adenoviral antibodies showed a substantial, dose-dependent generation of c a. a virus plasma level of about . × ( ) particles/ml (which was calculated to be the highest blood level reached during clinical trials in the past) induced an average release of about ng/ml c a (baseline levels < ng/ml). analyzing the nature of anti-adenoviral antibodies showed, that not only antibodies with neutralizing properties (anti-ad ), but also non-neutralizing anti-adenoviral antibodies are capable of complement activation. this study suggests that complement activation can be ignored in local low-dose applications of recombinant adenoviruses, but warrants attention after systemic application of large viral quantities. in clinical protocols aiming at systemic virus application, measures for monitoring and controlling the complement system should be included on a regular basis. recombinant adenoviruses are highly efficient in vivo gene transfer vehicles and offer the basis for a broad spectrum of therapeutic strategies especially in cancer therapy. current concerns regarding their application in humans are derived from the appearance of severe lifethreatening inflammatory reactions which were seen in one patient during a clinical trial for the correction of an inborn liver defect. this patient received a dose of × ad particles/kg via hepatic artery infusion. , some hours after virus infusion he developed a complex of clinical symptoms including hyperpyrexia, clotting disorders, thrombocytopenia and jaundice. three days after virus application he died due to the onset of severe lung perfusion disturbances (adult respiratory distress syndrome, ards) and multiple organ failure (mof). four of five patients who received a comparable high virus dose also developed hyperthermia of about °c, but recovered without major problems. tory reaction, but the precise pathomechanism remains unclear. in this article, we would like to provide evidence that a strong complement activation might have been involved in the observed reactions. it has been known for a long time that adenoviruses are able to induce an antibody-dependent activation of the complement system in human plasma, but the level of activation which is reached during clinical adenoviral gene therapy (especially in intravascular infusions) might have been underestimated in the past. we will show that challenge of isolated human plasma with serotype adenoviruses in amounts corresponding to blood levels reached in the above-mentioned trial, generates a level of complement activation, which holds the potency to induce serious inflammatory reactions. complement activation is a physiological defense mechanism which induces rapid destruction and phagocytosis of inoculated microbes and foreign bodies. complement activation can occur by antigen-antibody complexes (classical pathway) and directly after plasma contact with a number of microbial components or foreign surface (alternative pathway). , the majority of adults carry antibodies against adenoviral hexon group antigens , due to previous adenoviral infections. the direct inoculation of viral particles into the circulation in such individuals will usually induce the formation of antigen-antibody complexes and subsequently complement activation. factors influencing the number of antigen-antibody complexes will depend on the route of administration (systemic or local), the virus dose, the amount and type of pre-existing antibodies and the activity of regulatory proteins. during the early phase of complement activation, the anaphylatoxins c a and c a are generated. these mediators induce a broad variety of inflammatory reactions, such as il- and tnf release, - activation of neutrophilic granulocytes and macrophages, contraction of pulmonary vasculature, changes in vascular permeability, and platelet aggregation. systemic activation of the complement system, as known from patients suffering from sepsis, severe burns or injuries, could induce autodestructive inflammatory reactions, such as adult respiratory distress syndrome (ards) or multiple organ failure (mof). [ ] [ ] [ ] [ ] [ ] both types of clinical symptom complexes have been observed in the patient who died during the otcd trial, which suggests an involvement of the complement system. in this article, we will discuss prophylactic measures which might help to control or avoid excess activation of the complement system after adenoviral gene therapy in humans. citrate blood was taken from healthy volunteers by brachial vein puncture and anti-adenoviral antibody titers were determined by elisa, complement fixation test (cbr) and neutralizing assay. sixteen of citrate plasma samples contained anti-adenoviral antibodies titers ranging between and u/ml (ibl-elisa). the presence of anti-adenoviral antibodies was also determined by cbr (titers / - / / ). in of samples, neutralizing antibodies were detected ( / - / ), while four samples did not contain antibodies with neutralising properties (Ͻ / ). two samples did not contain any detectable anti-adenovirus antibodies (negative in all three test systems). plasma samples were challenged with virus particles (ad ) in four different concentration ( × - × particles/ml plasma) at °c. reaction was stopped after min by addition of / vol . m edta and c a-desarg levels were determined by elisa ( figure ). to evaluate possible inhibitory effects of cellular components of the blood, citrate blood samples were challenged with equivalent amounts of virus particles ( % cellular volume was considered). after challenging plasma (blood) samples with × and × particles/ml, a moderate generation of c a-desarg ( - ng/ml and - ng/ml, baseline Ͻ ng/ml) was determined, while a substantial further increase in complement activation was measured after incubation with × and × particles ( - ng/ml and - ng/ml) ( figure ). the presence of cellular components of the blood reduced the level of complement activation by about % (data not shown). comparing c a-desarg levels of plasma samples containing neutralizing antibodies and those without neutralizing antibodies revealed gene therapy higher levels of activation in the first group (about ng/ml versus ng/ml). the data show that antibodydependent complement activation does also occur in the absence of neutralizing antibodies. to judge the complement-activating potency of ad particles, control experiments were performed with heat aggregated human igg (haag, - mg/ml) one of the most potent activators of the complement system ( figure ). a linear dose dependency of complement activation was seen after challenge with and mg/ml haag, while incubation in higher amounts ( and mg/ml) led to saturation of c a release. to investigate whether complement activation is a specific problem of serotype adenoviruses which might be solved by switching the adenoviral serotype, we challenged six plasma samples with ad serotype , , , and particles. complement activation was also found after challenge with serotype , and while only low values were seen after stimulation with ad (subgenus e) ( figure ). the level of c a-desarg release (after stimulation with the same viral serotype) varied between plasma samples from different volunteers on average by a factor of to . in samples with no detectable anti-adenovirus antibodies, no complement activation was noted after challenge with ad particles. this finding suggests a strict antibodydependence of complement activation in human plasma. to further verify the role of pre-existing antibodies for complement activation in this system, immunoglobulin was removed from citrate plasma samples by gel filtration on protein-g sepharose. the filtration procedure itself induced some complement activation, but after removal of the immunoglobulins, the addition of virus particles no longer induced c a release ( figure ). after reconstitution of ig-depleted plasma with polyvalent immunoglobulin (containing an average amount of neutralizing anti-ad antibodies at a titer of / ) to a concentration of . mg ig/ml (about % of physiological ig serum concentration), the previous responsiveness to adenovirus challenge was demonstrated. complement activation via the classical pathway by adenovirus was confirmed in an additional experimental system. plasma samples were incubated with adenoviruses in the presence of egta. the calcium-binding capacity of egta blocks the antibody-dependent classical pathway of complement activation, whereas the alternative pathway remains unaffected, thus allowing differentiation of both pathways. egta blocked the virus-induced complement activation completely (figure ). these findings reveal that the alternative, ie antibody-independent pathway of complement activation after challenge with adenoviruses, is not relevant in human plasma. complement activation after challenge with recombinant adenoviruses does not depend on the nature of the encoded transgene as no difference was observed after challenge with ad-p , ad-haat and ad-hldl (data not shown). to exclude a potential role of host cell proteins present as contaminants in the virus preparation, hela, a and cell lysates at different concentrations were used as additional control. the protein content in l virus suspension ( × particle/ml) was determined as . g. a comparable dose of cell lysate had no influence on complement activation (data not shown). the data presented suggest that complement activation could be expected in the majority of potential patients receiving adenoviral gene therapy. since complement activation is a physiological defense mechanism, controlled local activation does not carry any health risk for a patient. potential hazardous reactions could occur if complement-activating components enter the circulation and induce the formation of large numbers of circulating antigen-antibody complexes. therefore the level of induced complement activation will depend on the applied dose and the route of application (local, systemic). after local low-dose adenovirus applications, complement activation can be most likely ignored. however, direct intravascular high dose applications (> × particles/kg) might carry an increased risk for the development of adverse inflammatory reactions. in a previous study in rabbits, we have shown that local application via the portal vein cannot prevent a rapid spread of virus into the circulation. in clinical trials for the treatment of malignant tumors or the correction of monogenetic diseases, doses of up to × particles per kg body weight have been applied by intravascular application via the hepatic artery. immediately after virus infusion, the blood or blood plasma, respectively, will contain about about . × viral particles per ml. higher concentration could be expected at the site of inoculation. a particle concentration of . × ad particles per/ml induces an average level of complement activation (in anti-ad antibody carriers) of about ± ng c a-desarg/ml in isolated human plasma. an in vivo level of ng/ml c a would bear a high risk for serious inflammation of the vascular bed with subsequent organ damage. clinical experience regarding serious effects of complement activation is derived from patients suffering from burns or severe injuries. the mobilization of tissue factors from damaged tissue has the potency to induce systemic complement activation. clinical symptoms of complement-mediated inflammation normally arise in the first days after the initial trauma. characteristic manifestations are edema and gas exchange disturbances of the lungs (adult respiratory distress syndrome, ards) and failure of liver and kidney function (multiple organ failure, mof). patients often die not as a direct consequence of the initial accident, but from the subsequent inflammatory immune response. in clinical studies, the plasma values for the onset of these syndromes has been found in the range of ng c a-desarg/ml. , , the in vitro data presented here, only took into account blood-derived inhibitory factors. after in vivo application, additional regulatory mechanisms involving vascular endothelial cells and the reticulo-endothelial system (liver, spleen) will influence complement activation. however, the level of c a-desarg released after in vitro challenge strongly suggests complement activation to be relevant for in vivo applications, particularly in high-dose intravascular applications in humans. the studies show with serotype , , and , that there is a broad cross-reactivity between different serotypes with regard to complement activation and, therefore, serotype switching might be of only limited value. there is only a weak correlation between the level of complement activation and the number of pre-existing antibodies. it is interesting that it is the presence rather than the level of anti-adenovirus antibodies that appears to be important for triggering the complement system. apart from monitoring complement activation on a regular basis during the trial, the data indicate that a number of additional measures might be appropriate. before virus application, the level of all anti-adenoviral antibodies should be determined. measuring only the titer of neutralizing antibodies provides no information about the total amount of relevant, ie complement-activating antibodies. the individual differences in complement reactivity against adenoviruses (up to a factor of ) will make it difficult to define a dose threshold which would reliably exclude uncontrolled complement activation. it might be more appropriate to define an individual threshold performing in vitro complement studies with patients' plasma before gene therapy, in a kind of bedside gene therapy test to predict the amount of in vivo activation to be expected. patients showing a strong in vitro reactivity could benefit from appropriate pharmacological measures. prophylactic steroid treatment will have no influence on the extent of complement activation, but steroids are known to reduce subsequent inflammatory reactions, like cytokine release and macrophage activation. , c esterase inhibitors have been proven for many years to be beneficial for the treatment of hereditary angioedema. they are currently tested as a protective measure against complement-mediated inflammatory tissue damage [ ] [ ] [ ] and would help to avoid or reduce adenovirus-induced complement activation. recombinant adenoviruses hold an important potency especially for tumor therapy. current applications are restricted to patients in an advanced and otherwise untreatable state of malignant growth. measures which could help to control vector-related side-effects, might allow earlier intervention and improve the therapeutic benefits of adenoviral vector technology. adenovirus wild-type specimens of serotype , , , and were provided by the institute of virology of the medizinische hochschule hannover, germany. wild-type viruses were grown on hela and a cells. recombinant serotype -derived adenoviruses encoding the human p gene were kindly provided by wei-wei zhang (anderson cancer center, houston, tx, usa), and recombinant adenoviruses encoding the human alpha- antitrypsin (haat) gene and the gene for bacterial ␤galactosidase (lacz) were a kind gift of mark kay (stanford university, usa). a recombinant virus encoding the gene for the human ldl receptor was generated according to the procedure of mcgrory et al. recombinant viruses were propagated on cells and lysed by three freeze-thaw cycles. purification was performed by two rounds of cesium chloride density gradient centrifugation and cesium chloride was removed by gel filtration on sephadex g- columns (pharmacia, uppsala, sweden). equilibration was performed with a buffer containing mm kcl, mm mgcl , pbs. sterile filtration was carried out using a . m filter (schleicher and schuell fp / , dassel, germany). virus suspensions were supplemented with % glycerol and stored in aliquots at Ϫ °c. titration was carried out in endpoint dilution assays on cells in -well plates as described elsewhere. in addition, particles were counted spectrophotometrically as described by mittereder et al. biological titers were presented as infectious particles (i.p.)/ml and physical titers as particles/ml. particle concentration of adenoviral stock solutions ranged between - × particles/ml. biological titers were on average one order of magnitude below physical titers. citrate plasma was obtained from healthy volunteers (a-l) and anti-adenoviral antibodies were determined in a commercial elisa (anti-adenovirus; ibl, hamburg, germany). viral target antigens employed in the ibl-elisa are derived from serotype adenoviruses (subgenus c) only. in addition anti-adenoviral antibodies were also determined in a complement fixation test (cft) for adenovirus antibodies (virion-serion, wü rzburg, germany) and by neutralizing assay. the cft employs a mixture of adenovirus antigens derived from subtype , , , (subgenera b, c, e). the titers of neutralizing antibodies were determined by the ability of proband sera to prevent infection and subsequent replication of cells by recombinant viruses. l previously heat-inactivated ( min at °c) serum samples were serially diluted in three independent tests and incubated with l virus suspension, containing × i.p. ad-hldl (diluted in medium without fcs) for min at °c in -well plates. l of the serum-treated virus samples were transfered to cells, seeded in -well plates at a density of × cells/well (in l medium, % fcs) on the previous day. the neutralizing antibody titer was calculated from the reciprocal cytopathic effect observed days after infection. citrate blood was obtained from healthy volunteers by cubital vein puncture in . ml vials containing . m sodium citrate (vacutainer system ; becton dickinson, nj, usa). in one part of the sample citrate, plasma was immediately separated by centrifugation ( min, g) and aliquots of l plasma were incubated with l virus suspension/storage buffer containing × , × , × , and × particles/ml plasma for min at °c. for complement studies in full blood l citrate blood were incubated with the same amounts of virus particles as applied in the studies with plasma. the reactions were stopped by addition of / vol . m edta. plasma was separated from blood samples (after challenge with ads) by centrifugation and stored at Ϫ °c for further analysis. for discrimination of the classical activation pathway from the alternative pathway, citrate plasma was pretreated with egta ( / vol . m egta) before incubation with adenoviruses. in a parallel experiment, egta was supplemented with mm mgcl . comparable results were observed as with egta alone. the reaction was stopped after min at °c by addition of edta ( / vol. . m edta) and samples were immediately stored at Ϫ °c for further analysis. for studying complement activation in the absence of immunoglobulin, depletion was performed by gel filtration of the plasma samples twice using protein-g sepharose columns (high trap protein g columns; pharmacia, freiburg, germany). reconstitution experiments with purified ig showed in controls that the reactivity of the complement system was not impaired in the depleted plasma. human gamma-globulin from pooled plasma ( mg ig/ml; dianova, hamburg, germany) of a known anti-adenovirus antibody titer was used and incubations with four different concentrations of viral particles were performed as described above. c a-desarg levels were determined as previously described in an elisa system using a monoclonal antibody to a neoantigenic epitope of c a-desarg. , control experiments heat-aggregated igg (haag) was generated according to standard techniques. in brief, mg/ml human igg in pbs (dianova) were heated for min at °c. particle aggregates were removed by microcentrifugation ( min, g) and the remaining protein concentration was determined photometrically. heat-aggregated igg was diluted in pbs to a final concentration of , , and mg and l of these dilutions were incubated with l citrate plasma for min at °c. reactions were stopped by addition of / vol . m edta. a , hela and cell lysates were generated by harvesting and pelleting × cells of each line. cells were resuspended in ml complete medium ( % fcs), three freeze-thaw cycles were performed. protein content in the supernatant was measured spectrophotometrically using the bradford assay. citrate plasma was incubated with a mixture of complete medium and cell lysates in four different concentrations ( . , . , . and g/ml plasma). the reaction was stopped after min at °c by addition of edta as described above. the protein content at the highest virus dose employed ( × particles) was determined as . g. to exclude a direct interaction between the surface proteins of recombinant viruses and the c a-desarg elisa-system recombinant viruses ( × - × particles) were diluted in % bovine serum albumin or in heat-inactivated plasma, respectively. no activation was noticed after min incubation. in addition injection buffer (containing % glycerol) was 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permeability during cardiopulmonary bypass human platelet activation by c a and c a des-arg complement activation in injured patients occurs immediately and is dependent on the severity of the trauma inflammatory mediators in relation to the development of multiple organ failure in patients after severe blunt trauma complement activation and clearance in acute illness and injury: evidence for c a as a cell-directed mediator of the adult respiratory distress syndrome in man the inflammatory basis of trauma/shock-associated multiple organ failure complement activation and the prognostic value of c a in patients at risk of adult respiratory distress syndrome intravenous administration of recombinant adenoviruses causes thrombocytopenia anemia and erythroblastosis in rabbits a phase i/ii study of hepatic artery infusion with wtp -cmv-ad in metastatic malignant liver tumours white blood cell counts and plasma c a have synergistic predictive value in patients at risk for acute respiratory distress syndrome prostaglandin and complement interaction in clinical acute respiratory failure the effects of methylprednisolone on complement, immunoglobulins and pulmonary neutrophil sequestration during cardiopulmonary bypass modifying risk for extracorporeal circulation: trial of four anti-inflammatory strategies combined antithrombin iii and c -esterase inhibitor treatment decreases intravascular fibrin deposition and attenuates cardiorespiratory impairment in rabbits exposed to escherichia coli endotoxin effect of c inhibitor on inflammatory and physiologic response patterns in primates suffering from lethal septic shock c inhibitor in anti-inflammatory therapy: from animal experiment to clinical application a simple technique for the rescue of early region i mutations into infectious human adenovirus type evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy the c terminus of the anaphylatoxin c a generated upon complement activation represents a neoantigenic determinant with diagnostic potential quantitation of the anaphylatoxin c a in the presence of c by a novel sandwich elisa using monoclonal antibody to a c a neoepitope this work was supported by the bundesministerium fü r forschung und technologie ( kv / ). we are grateful for the technical assistance of beate goldbrich, christel uhse, gudrun kliem and hannelore scharfenorth. the authors would like to thank friedrich luft and charles coutelle for critical reading of the manuscript. key: cord- -ln g z authors: sissons, j.g. patrick; oldstone, michael b.a. title: antibody-mediated destruction of virus-infected cells date: - - journal: adv immunol doi: . /s - ( ) - sha: doc_id: cord_uid: ln g z this chapter describes the effect of antibody on virus-infected cells with special reference to the human system. the destruction by antibody of the infected cells through the mediation of complement is described in detail based in considerable part on the contributions of the authors. activation of the alternative pathway by the various infected cells is of special interest. the interesting effect of the antibody-dependent cell-mediated cytotoxicity (adcc) system involving viral antigens in cell killing is also presented. multiple additional topics are also covered, such as the effect of antibody on the expression of viral proteins both on the surface of the cell and intracellularly. serum antibody, produced in response to virus infections, is of major importance in preventing the spread of infection by virtue of neutralizing free virus in extracellular fluids. virus neutralization by antibody is enhanced by complement. antibody binding to the surface of virus-infected cells can affect virus production and release in the absence of an effector system. immunoglobulin (igg) antibody can mediate the destruction of virus-infected cells in conjunction with complement or cytotoxic lymphocytes. in addition, at a conceptual level there is evidence to suggest that antibody may enhance and confer specificity on basic nonspecific humoral and cell-mediated defense mechanisms. producing tissue injury, or encouraging virus persistence have attracted extensive interest in recent years. these interactions are inevitably intricate. added to the fundamental complexity of the immune response itself is the complicated nature of viruses as antigens, with their intracellular site, ability to replicate, multiple separate antigenic molecules, and, in some cases, ability to infect and replicate in lymphocytes, macrophages, and their precursor cells. a number of immunologic mechanisms have been defined by which virus-infected cells can be killed in vitro. antibody-independent cellmediated cytotoxicity against virus infected target cells has received great attention. this is especially so in the last years, since the demonstration that killing of virus-infected cells by specifically sensitized cytotoxic t cells occurs and is h- restricted in the mouse. this subject has recently been reviewed in this series by zinkernagel and doherty ( ) . even more recently the "spontaneous" cytotoxicity of murine and human lymphocytes for virus-infected targets (and for uninfected targets and tumor cell lines) has become the subject of intensive investigation, with the demonstration that the activity of natural killer cells can apparently be augmented by interferon. this topic, as it pertains to virus-infected cells, has again been reviewed for the murine (welsh, ) and human (santoli and koprowski, ) systems. serum antibody, produced in response to virus infections, is of major importance in preventing the spread of infection b y virtue of neutralizing free virus in extracellular fluids (reviewed by daniels, ; burns and allison, ) . virus neutralization by antibody is enhanced by complement; antibody and complement can lyse enveloped virions, and complement may lyse or neutralize some viruses in the absence of antibody (reviewed by cooper, ; cooper and welsh, ) . antibody can also act in a number of ways on virus-infected cells, and this review is concerned with those effects of the immune response on virus-infected cells that are mediated by antibody. we deal with the actions of antibody alone, of antibody and complement, and of antibody interacting with cytotoxic cells, on virus-infected target cells. it should become apparent that, despite the foregoing emphasis on the role of antibody in neutralizing free virus and of cytotoxic t cells in killing virus-infected cells, antibody can profoundly affect virus-infected cells in vitro; there is little reason for thinking these actions may be less important in vivo than other cytotoxic mechanisms demonstrated in vitro. furthermore, as with cytotoxic t cells, antibody-mediated effects on virus-infected cells can occur prior to re-lease of progeny virus, eliminating a source of continuing virus production. in surveying this subject, emphasis is given to the effect of antibody on virus-infected cells in homologous human systems and, particularly in sections i and iv, to work from this laboratory. some aspects of the mechanism by which viral proteins are expressed on the surfaces of infected cells merit discussion, as these are the target antigens whose recognition initiates immune lysis. all enveloped rna viruses, which acquire an outer lipid envelope by budding from the host cell plasma membrane, express viral glycoproteins on the cell surface. this group comprises, in part, togaviruses, negative strand rna viruses (arena-, rhabdo-, orthomyxo-, and paramyxoviruses) and retroviruses. dna viruses, such as herpes viruses, which bud from the nuclear membrane, and poxviruses, which manufacture their own envelope, also express structural viral glycoproteins on the cell surface. in addition, virus infection may cause expression of nonstructural viral proteins (e.g., tumor-specific transplantation antigens) or alter expression of host membrane proteins. it is worthwhile briefly to summarize the mechanism for assembly of enveloped viruses. after viral entry into a cell and uncoating, transcription of viral mrna and its translation into viral proteins on host cell ribosomes follow. enveloped viruses have three main classes of structural proteins: nucleocapsid proteins, matrix (m) proteins, and envelope glycoproteins. the nucleocapsid protein is associated with the viral nucleic acid, the m protein (not present in togaviruses) is a peripheral membrane protein associated with the cytoplasmic surface of the cell membrane (see choppin and compans, ) . the glycoproteins are inserted into the lipid bilayer of the plasma membrane, probably as transmembrane proteins, with a hydrophobic region in (vsv) glycoprotein provides a model for this process (rothman and lodish, ) . briefly, the nascent polypeptide chain synthesized on membrane-associated polyribosomes, is inserted through the membrane of the endoplasmic reticulum, insertion apparently being mediated by a specific "signal" peptide sequence in the chain in accordance with the "signal hypothesis"(blobe and dobberstein, ). glycosylation then occurs on the noncytoplasmic side of the membrane utilizing the host cell glycosylating enzymes, and the protein is transported to the plasma membrane in a vesicle that fuses with the membrane. viral glycoproteins appear to be mobile in the lipid bilayer, as are host cell integral membrane proteins (see section ), and become distributed at random over the surface of the cell. prior to the process of budding, glycoproteins locate in the area of budding; m protein aligns under the glycoproteins on the cytoplasmic side of the membrane, the nucleocapsids also align under the m protein, and the whole complex is then enveloped in a bud of plasma membrane. in the virion, as in the cell, the glycoproteins are thus the exposed viral proteins. the lipid envelope reflects the composition of the cell plasma membrane from which it is derived (lenard and compans, ) . most evidence indicates that host cell membrane proteins are almost totally excluded from the viral envelopes during budding (see holland and kiehn, ) . there is some circumstantial evidence that h- antigens can be incorporated into friend leukemia virus (flv) (bubbers and lilly, ) . this was based on adsorption of antisera to h- by flv purified from vireniic mice. however, virus had to be disrupted to demonstrate this adsorption, and there is as yet no firm confirmatory biochemical evidence demonstrating incorporation of gene products of the major histocompatibility complex (mhc) into enveloped viruses. it does seem definite that cellular actin, a peripheral membrane protein, can be incorporated into some enveloped viruses, e.g., measles virus, vsv, and influenza virus, as demonstrated by its presence in purified virions. however, this is probably a passive association, as exposure of infected cells to cytochalasin b does not prevent incorporation of actin into vsv or influenza virions, or inhibit budding (griffin and compans, ) . hence, contractile protein function is apparently not required for the budding process. viruses may in some circumstances bud from particular domains of the plasma membrane. studying two different cultured lines of epithelial cells grown as monolayers, boulan and sabatini ( ) found that influenza virus, simian virus , and sendai virus budded exclusively from the apical (free) surface whereas vsv budded only from the basolateral plasma membrane. this implies that certain viruses might be less susceptible to immune recognition at the cell surface because they bud from areas of the cell removed from direct contact with blood. a point worth making is that these viruses must express virus-specific proteins on the cell membrane before release of free virions occurs. it is also apparent that lysis of a cell infected with a budding virus would be unlikely to result in release of infectious virus, as the nucleocapsids would be devoid of the envelope glycoproteins that mediate virion attachment to the cell membrane prior to entry. the herpes simplex viruses (hsv), which have been widely used in in uitro cytotoxicity studies, are structurally much more complex, coding for some viral polypeptides (roizman and furlong, ) . viral proteins are inserted in cytoplasmic and plasma membranes, and the viral cores can gain envelopes by budding from any of these mem- branes. an intriguing feature of hsv is their ability to induce the appearance of an fc receptor for human or rabbit igg on the membrane of infected cells (watkins, ; westmoreland and watkins, ) . although the structural relationship of this receptor to the "endogenous" fc receptors on lymphocytes and phagocytic cells is unknown, it seems possible that this is a virus-encoded gene product, and it can reportedly be blocked by a f(ab'), antibody to hsv (mctaggart et al., ; adler et al., ) . it has also been reported that cytomegalovirus can induce fc receptors on infected cells (rahman et al., ; westmoreland et al., ) . it remains to be seen whether this novel surface structure participates in any definite way in immunologic reactions. some preliminary evidence indicating that this may be so is summarized in section . unlike other dna viruses, poxviruses replicate in the cytoplasm rather than the nucleus and, unlike budding viruses, synthesize their own lipid membranes, but nevertheless express viral antigens on the cell surface, which can appear as early as hour after infection (ada et al., ) . studies with poliovirus in rabbits (svehag and mandel, ) and humans (ogra et al., ) show a similar sequence in the development of antibody responses to that described for other viral and nonviral antigens. after immunization the level of neutralizing igm antibody attains maximal titers in - weeks and is undetectable by months, igg titers rise in parallel but continue to increase for several months and persist at lower levels for years. serum iga antibody is first detectable several weeks after immunization, and titers rise for several months. secretory iga responses occur at local sites of infection of mucosal surfaces with live virus. subsequent challenge with virus elicits a further transient igm, in addition to a continuing igg, response. studies with a number of other viruses show a similar pattern of response. many viral antigens appear to be thymus dependent, as indicated by their eliciting no antibody or only small amounts of igm antibody in nude mice (burns et al., ; reviewed in oldstone, ) . although in all these studies of the immune response antibody is measured as neutralizing antibody, it is very likely that the same antibodies also recognize antigens on infected cells; neutralizing antibodies are directed against external proteins on the virion, which are likely to be those expressed also on infected cell membrane s. much of the work on the relative importance of responses to individual viral antigens has been done with influenza virus. this is largely because the biochemistry of influenza virus, including detailed knowledge of the structure of the glycoproteins, is better understood than for other viruses. passive transfer of antibody to the m or nucleoprotein (both internal) polypeptides did not protect infected mice, whereas transfer of antibody to the hemagglutinin (ha) or, to a lesser extent, the neuraminidase, was protective (see virelizier et al., ) . direct visualization of the bromelain-extracted influenza ha and its reaction with igg antibody, by electron microscopy, shows that antibody reacts with the tip of the molecule-the end of the spike farthest from the membrane of the virion or cell membrane (wrigley et nl., ) , and the only site on the ha likely to be sterically accessible to antibody on the virion. in paramyxoviruses the relative response to the two envelope proteins may be important in effective immunity. norrby and associates (norrby et al., ; norrby and penttinen, ) showed that formalin-inactivated mumps vaccine or tween -ether-inactivated measles vaccine induce antibodies only to the viral ha, not to the fusion (f) protein (or hemolysin), compared to immunization with live attenuated viruses, which induces antibodies against the f protein. in the case of measles virus, exposure to wild-type virus after immunization with the inactivated virus sometimes produces the clinical syndrome of "atypical measles" with prominent pulmonary infiltrates and severe rash, and the inactivated vaccines generally produce a poor level of immunity. the extensive literature on the subject of humoral immune responses and viral antigens is reviewed by ogra et d. ( ) and bums and allison ( ) . in the absence of any effector system, such as complement or antibody-dependent cytotoxic k cells, antibody itself can act on virus-infected cells to affect the release of progeny virus and expression of viral antigens. these actions appear to be at least partially reversible and, in some circumstances, capable of protecting the infected cell from subsequent immunologic attack. a. antibody-induced redistribution of surface viral antigens the phenomenon of capping of integral membrane proteins by antibodies or lectins is well described. binding of antibody initially causes patching of the antigen, followed by polar redistribution of the cross-linked membrane proteins as a cap. the initial patching is energy independent, whereas capping is temperature and energy dependent and involves the participation of the cell contractile proteins. divalent antibody is required to produce capping. binding of antibody appears to induce transmembrane linkages of integral membrane proteins to intracellular actomyosin-containing filaments at the patching stage for cells in suspension (bourgignon and singer, ) and in monolayers (ash et al., ) . most, if not all, integral membrane proteins can behave in this way. the subject as it applies to b lymphocytes has been reviewed in this series by schreiner and unanue ( ) . viral glycoproteins expressed on cell membranes can also be capped by antibody as shown independently for measles (joseph and oldstone, ; lampert et al., ; ehrnst and sundquist, ) and influenza (rutter and mannweiler, ) and subsequently for other viruses. capping of measles viral glycoproteins required active cell metabolism, a functioning cytoskeleton, membrane atp, and divalent antibody (joseph and oldstone, ) (see table i ). these glycoproteins are thus capable of lateral diffusion in the membrane and behave in this respect as do host cell integral membrane proteins (fig. ) . electron microscopy studies of measles virus-infected cells showed that the nucleocapsids move in concert with the glycoproteins on the cytoplasmic side of the membrane as the latter cap (lampert et al., ) , indicating that a transmembrane connection exists between the surface and internal polypeptides. in studies using fluoresceinated monospecific antisera, low concentrations of cytochalasin b are reported to prevent this associated movement of nucleocapsids (tyrell and ehrnst, ) . more-detailed studies are needed to deter- joseph and oldstone, ) . cells were preincubated with these inhibitors for minutes at °c and then with fitc conjugated antibody to measles for hour at °c. mine whether separate viral envelope glycoprotein species expressed on the membrane cocap, or whether they can behave as independent molecules. the evidence on whether host cell membrane proteins, particularly products of the mhc genes, cocap with viral proteins is somewhat contradictory, partly owing to studies with different systems and antisera. rauscher virus gp was initially reported to cocap with h- on tumor cells (schrader et al., ) . later, using well defined antisera, it was found that retroviral gp / cocapped with h- and with tl on a mouse thymoma line (bourgignon et al., ) . however, in this same study, capping of t , which is a host cell surface glycoprotein, also induced cocapping of h- , and of the thy and tl antigens. this suggested that h- antigens may cocap with a variety of independent cell surface molecules, possibly, as postulated in this report, because they are all linked to the actomyosin filaments (bourgignon et al., oldstone, unpublished observations) . furthermore, in a recent careful biochemical study using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), no evidence for molecular association of mhc molecules and moloney virus gp could be found on a moloney virus lymphoma line (fox and weissman, ) . one major interest of these observations is their relevance to the debate on whether cytotoxic t cells recognize mhc products and viral antigen as a complex, or as two independent molecules (see zinkernagel and doherty, ) . in this context additional evidence arguing against a direct molecular association of h- antigens and viral glycoproteins, at least occurring during transcription or translation, is provided b y experiments mixing independently synthesized proteins. thus, secondary in uitro cytotoxic t cell responses can be generated by liposomes containing isolated h- antigens and sendai viral glycoproteins (finberget al., ) ; and heterokaryons formed by fusing cells bearing sv antigens, but inappropriate h- antigens, with uninfected cells of appropriate h- haplotype, are reported to act as targets for h- restricted cytotoxic t cells (watt and gooding, ) . this could be explained by divalent antibody binding to ha sterically hindering the access of antibody to f. (from sissons et al., b.) gent solubilized, after which the material binding to staphylococcal protein a was analyzed by sds-page. igg antibody bound to both viral glycoproteins on cells infected hours, but at later stages of infection antiviral igc or f(ab'), bound only to the ha glycoprotein whereas the same amount of fab' bound to both ha and f glycoproteins (fig. ) . these findings were interpreted as showing that, at high densities of antigen, divalent antibody to ha could sterically block the access of antibody to f at the cell surface. studies of actual binding sites of antibody at the infected cell surface would be of interest in regard to other viruses. antigenic modulation is a term originally introduced by boyse et al. ( , ) to describe the reversible loss of the tl+ (thymic leukemia antigen) phenotype from tl+ leukemia cells passaged in immunized mice. these cells lost their sensitivity to lysis when a new source of anti-tl antibody and guinea pig complement was added, but the tl+ phenotype and susceptibility to lysis returned after culture in the absence of antibody or passage in unimmunized mice. antigenic modulation of tl, and in other tumor systems, is reviewed in detail by stackpole and jacobson ( ) . it now seems that a similar phenomenon can occur when virus-infected cells are exposed to antibody, although the mechanism may not necessarily be precisely identical to the tl system. joseph and oldstone ( ) found that culture of measles virus-infected hela cells in the presence of antibody to measles virus rendered them resistant to lysis when exposed to a fresh source of human antibody and human complement. the authors termed this "antibodyinduced viral antigenic modulation." antibody-induced measles virus antigenic modulation has subsequently been studied in detail. hela cells persistently or acutely infected with measles virus were incubated in heat-inactivated human serum containing antibody to measles virus and then maintained in suspension culture with % of the same heat-inactivated human serum. at timed points cells were removed from culture and washed. the infected cells' susceptibility to lysis by human antiviral antibody and complement underwent a progressive decline, which was maximal by hours. this correlated with loss of accessible viral antigens from the cell surface as detected by surface staining with fluoresceinated anti-measles igg or by binding of radiolabeled antibody (see fig. ) . after the initial few hours of culture in the presence of antibody, only minimal degrees of cap formation were evident on the measles-infected cells. acutely infected cells maintained in culture regained their susceptibility to lysis by hours, whereas persistently infected hela cells regained susceptibility only if washed and recultured in the absence of antibody. this difference probably reflects the differing rate and density of viral glycoprotein synthesis between the two cell types, the acutely infected cell synthesizing enough viral antigen to eventually bind all the antibody in the experimental system used and escape modulation. under these , .) same conditions, cells persistently infected with measles virus completely lost their susceptibility to lysis by peripheral blood lymphocytes from immune subjects, within days in culture (oldstone and tishon, ) . culture of cells infected with measles virus for longer periods in the presence of antibody can result in the selection of attenuated virus (rustigian, ; gould and almeida, ) , although the biochemical structure of such selected viruses has not been examined carefully. the fate of the virus antigen-antibody complex on the infected cell is important to any understanding of the mechanism of antigenic modulation. in general, the approach used to study ligand interactions with cell surface molecules has been to label one or the other component of the complex and follow its fate. when lz i- abeled igg antibody was bound to the surface of measles virus-infected cells at the start of the culture period, under the conditions described above for antigenic modulation, about % of the radioactivity was still cell associated at hours and nearly all was protein bound perrin and oldstone, ) . the fate of the antibody was followed in detail by perrin and oldstone ( ) . after hours in culture % of the [' gg was present in the supernatant, and analysis on linear sucrose density gradients showed that a third of this labeled igg sedimented in a peak heavier than s igg. this peak of heavier igg also contained measles virus ha but not nucleocapsid, as detected by immunoprecipitation with specific antibodies. material from this peak bound to rheumatoid factor and clq, and to raji cells, indicating the presence of immune complexes. thus, these findings suggested that a significant amount of complexed antibody and viral glycoprotein was shed from the cell surface, and the absence of nucleocapsids indicated that the antibody was unlikely to be complexed with whole virus. in contrast, only a small amount of antibody appeared to be endocytosed. this was shown by studying cell lysates in sds in which most of the [' gg was s, but some non-tca precipitable material was present, suggesting intracellular degradation in lysosomes (perrin and oldstone, ) . immunoelectron microscopy studies of measles-infected cells capped with igg antibody to measles and then labeled with peroxidase conjugated anti-igg rarely showed any internalized peroxidase (lampert et al., ) . hence, endocytosis of the viral antigen-antibody complex appears to be unusual in the -minute duration of these latter experiments and accounts for only a minority of the complex during the longer interval of modulating conditions studied by perrin and oldstone ( ) . some endocytosis of peroxidase label was seen in electron microscopy studies of antibody-treated measles-infected syncytial cells (p. w. lampert however, the use of the second peroxidase-coupled antibody has been shown to favor endocytosis of membrane proteins, at least in b lymphocytes (schreiner and unanue, ) and makes the system more artificial compared to the situation in uiuo. in recent experiments the fate of viral cell surface antigens was followed by surface radioiodination of measles virus-infected cells and maintenance of the cells in culture with or without antibody. the rate of disappearance of lz i-labeled viral glycoproteins from the cell was then determined by running cell lysates on sds-page and excising and counting the viral glycoprotein bands. after an initial acceleration, the turnover rate of the labeled viral glycoprotein in cells cultured with antiviral antibody was no greater than in cells cultured in the absence of antibody . this suggests that, after initial shedding, antibody does not subsequently enhance the net rate of viral glycoprotein loss from the cell under the culture conditions used. in contrast to the fate of viral antigen-antibody complexes on the plasma membrane, ligand interaction with surface ig or fc receptors on b lymphocytes results primarily in their endocytosis (reviewed schreiner and unanue, ) . the fate of other cell surface molecules complexed with antibody is less defined, although it appears that mhc molecules on b cells are less likely to be endocytosed and may be shed (unanue et al., ; reviewed b y schreiner and unanue, ). the only other viral systems in which antigenic modulation has been studied involve oncogenic murine retroviruses. gross virus leukemia antigens can be modulated in vivo in immunized mice (aoki and johnson, ) and in vitro (ioachim and sabbath, ) , as shown by resistance to antibody and complement-dependent lysis. there is also evidence for antigenic modulation in flv-induced leukemia. this was demonstrated in vitro with flv-infected cells by genovesi et al. ( ) and is likely to play a role in uiuo (doig and cheseboro, ) . presence of antiviral antibody, loss of flv antigens from the leukemic cell surface, and recovery from viremia were all associated with a single genetic locus, named rfv- , which is not linked to the mhc. it was suggested that rfv- acts as an immunoregulatory gene influencing production of cytotoxic anti-flv antibodies. nevertheless, despite this evidence for antigenic modulation, progressive fatal leukemia occurred unless appropriate separate h- linked genes, influencing recovery from leukemia, were associated with the rfv- locus (doig and cheseboro, ). mouse mammary tumor virus antigens can also be specifically modulated in vitro (calafat et al., ) . shedding of viral antigen-antibody complexes was also prominent in this study, as assessed by immunoelectron microscopy. these studies are further reviewed by antibody in the medium of infected cultured cells results in a decrease of detectable virus in the medium. the cause is in part complexing of antibody with free virus released from the cell, but also cross-linking of viral glycoproteins on the cell surface by antibody, thereby inhibiting release of virus, probably by interfering with the synthesis and expression of viral proteins budding process. the release of influenza virus was inhibited by antibody to the ha and, less effectively, by antibody to neuraminidase (dowdle et uz., ) ; fab fragments of antiviral igg had earlier been reported not to inhibit release of virus (becht et ul., ). in addition to these effects of antibody on the release of virus, there is evidence to suggest that antibody may also affect the synthesis and/or expression of viral proteins inside the cell. studied and compared to controls. it was found that expression of the f protein was diminished as detected by decreased incorporation of ["slmethionine. since turnover of f protein at the cell surface was not appreciably accelerated (fujinami et a ., ) , this observation most likely represents diminished synthesis of f protein. interestingly, expression of a cytoplasmic viral polypeptide, the measles virus phosphoprotein (p) was also diminished (as assessed by [ s]methionine and p incorporation) (see fig. ). p protein is associated with the transcriptive complex and is probably necessary for replication of the viral genome (mountcastle and choppin, ) . the effects were specific for antiviral antibody, as they could be produced exclusively by purified antiviral igg, but not by incubation with antibody to the hela cell surface or nonimmune igg. hence, regulation of an internal viral polypeptide by antibody initially present outside the cell can occur. whether a transmembrane signal is produced by antibody binding to the plasma membrane or whether the effect is mediated by pinocytosed antibody acting inside the cell is not known. yagi et al. ( ) observed changes in the polypeptide profile of mouse mammary tumor virus when tumor cells were cultured in the presence of antibody in vitro, although these changes were not analyzed in detail with regard to individual proteins. there is evidence that antibodies can inhibit viral transcription and translation in cell-free systems. thus, antibody to the vsv viral transcriptase immediately inhibited rna transcription by vsv nucleocapsids (imblum and wagner, ) , and antibody to newcastle disease virus nucleocapsid had the same effect (miller and stone, ) . these results, presumably produced by direct binding of antibody to viral enzymes, may be useful tools in molecular virology. however, the observation that antibody can affect viral polypeptide expression prior to labeling, cells were incubated with or without antibody for hours. p protein and f (seen here as its major reduction fragment f ) are reduced in cells cultured with antibody. peak found in uninfected cells migrating just before f probably represents cellular actin (p is found inside the cell; f is expressed on the cell's surface). (from fujinami and oldstone, , .) in intact cultured cells suggests that the synthesis, transport, or assembly of viral polypeptides could be significantly affected by antibody in uiuo. both hsv and cytomegalovirus can induce formation of an fc receptor for igg on the surface of infected cells. costa et al. ( ) cultured vero cells infected with hsv- in the presence of nonimmune rabbit igg or its fc fragment at concentrations of - mg/ml and found a marked reduction in virus production. since the igg had no neutralizing effect on free virus or cytotoxic effect on infected cells, it was suggested that binding to the fc receptor inhibited the growth of virus-induced fc recptor hsv. costa et al. did not record the effect of f(ab'), fragments or of the same igg preparation on other virus-infected cells. adler et al. ( ) reported that preincubation of hsv- infected rat neuroma cells with aggregated nonimmune igg resulted in loss of susceptibility to lysis by antibody and complement. the effect of native nonaggregated igg was not reported; although this report mentions that no capping was observed when fitc-aggregated igg was used, there have been no systematic studies of the fate of hsv fc receptors binding igg. in contrast, rager-zisman et al. ( ) reported that aggregated nonimmune igg could mediate cytotoxicity of hsv-infected cells by normal mouse peritoneal macrophages. the authors postulated that the cause was cross-linking of fc receptors on the hsv-infected targets and the effector cytotoxic cells by the aggregated igg. these reports thus suggest that the presence of the hsv fc receptor may inhibit viral replication on the one hand, and might interfere with or enhance immune cytolysis on the other. clearly, more detailed studies in this system are warranted, and the presence of the hsv fc receptor must be taken into account in any assessment of immune cytolysis of herpes virus-infected cells. antibody binding to the surface of virus-infected cells can affect virus production and release in the absence of an effector system. a complex and dynamic interaction exists between antibody and the viral polypeptides expressed on the plasma membrane. clearly, antibody can induce redistribution and capping of viral proteins on the cell surface. most of the measles viral antigen and antibody complexed on the plasma membrane is subsequently shed rather than endocytosed; however, it is difficult to know whether shedding of complexes is more likely to occur with viral membrane proteins on the cell surface than with other membrane proteins. although igg-anti-igg complexes on the b lymphocyte membrane are predominantly endocytosed, this may be peculiar to signaling in the b cell. while other host membrane proteins complexed with ligands may not be as rapidly endocytosed, endocytosis, with subsequent degradation in lysosomes, is believed to be the normal route of metabolism for host cell membrane proteins as indicated by the studies of surface radioiodinated mouse l cells by hubbard and cohn ( ) . hormone receptors complexed with their specific ligands probably are endocytosed and degraded in the same way (catt et ol., ) . viral glycoproteins, which are destined for export from the membrane, may be more sus-ceptible to shedding; when they are actually in segments of the membrane where virus budding has commenced, this might be even more likely. electron microscopic studies on measles virus-infected cells undergoing capping show that budding virus is visible in the microvilli forming the cap (lampert s t ul., ) . antibody-induced viral antigenic modulation as described for measles virus-infected cells has some similarities to the original tl system of boyse et al. ( , ) . hence, capping is not essential and some antibody and viral antigen is still present on the cell at the time of maximal resistance to antibody-and complement-mediated lysis. however, yu and cohen ( ) could not detect any change in tl synthesis in tl+ cells undergoing modulation, but showed by surface radioiodination that there was accelerated removal of t l from the cell surface, at least during the early time course of modulation. in contrast, diminished synthesis may contribute to the loss of surface viral antigen in the presence of antibody in measles virus-infected cells. there is no suggestion of any requirement for c to produce modulation of measles virus-infected cells, whereas there is one in the tl system . it may be concluded that the loss of surface viral antigen induced by antibody and leading to antigenic modulation could result from a combination of ( u ) shedding of antigen -antibody complexes; ( b ) blocking of antigenic sites by igg remaining on the membrane; and ( c ) diminution of synthesis of surface viral polypeptides. because cells can generally be rendered resistant to lysis by antibody and complement via relatively modest reduction of surface antigens, it is important to note that, in addition to being resistant to antibody-dependent complenient-mediated lysis, modulated measles virus-infected cells are also resistant to antibody-dependent cell-mediated cytotoxicity (oldstone and tishon, ) . antibody binding to intact cells can affect the intracellular expression of viral polypeptides as shown in measles virus-infected hela cells (fujinami and oldstone, ) . this area has just begun to be studied at a biochemical level, and it will be interesting to see the parallels and contrasts that arise with other membrane receptor-ligand interactions, such as those between hormones and their receptors. the major interest in these observations lies in the possibility that such effects of antibody may represent mechanisms whereby virus persistence could be established in vivo. the consequence would be a cell rendered resistant to immune injury because of lost surface viral markers, but still retaining the potential to shed virus once antibody is removed or to infect adjacent cells by cell-to-cell spread. this possibility is further discussed in section vi. virus-infected cells can be lysed by specific antibody and complement. the mechanism of this reaction is discussed in detail in this section. emphasis is given to work with homologous human systems, and with measles virus-infected cells in particular. enveloped viruses, as free virions, can also serve as targets for antibody-and complementmediated lysis; this topic has been reviewed by and by cooper and welsh ( ) . a. complement the human complement system is composed of some plasma proteins, including its intrinsic components and the proteins that regulate activation of the pathways. the mechanisms by which the system is activated, produces membrane lesions and mediates some of its biological effects are now relatively well understood. the mechanism of activation of the classical pathway (miiller-eberhard, ) and the mechanism by which the membrane attack pathway (c - ) produces membrane lesions (podack and miiller-eberhard, ) have been reviewed recently. the alternative pathway of complement activation is the subject of a review in this volume by schreiber and miiller-eber- hard. only points that are relevant to the present discussion are mentioned here. activation of the classical pathway is usually dependent on the interaction of the fc portion of igg or igm complexed to antigen with the c l q subunit of c . c is activated and enzymically catalyzes assembly of the classical pathway c convertase c b a by sequential proteolytic cleavage of c and c . after cleavage of c , association of c b with c b a gives the classical pathway c convertase c b a b. the alternative pathway of complement activation consists of six proteins: c , factors b and d, p l h , c b inactivator, and properdin. current evidence indicates that initiation of the alternative pathway on the surface of known activators, such as rabbit erythrocytes (fearon and austen, a; pangbum and miiller-eberhard, ) , zymosan (fearon and austen, b) , and certain strains of escherichiu coli , occurs because c b from the fluid phase is bound there and relatively protected from its serum inactivators. there is probably a low level of background c b generated from the interaction of native c , factor b, and factor d. in the fluid phase or on the surface of nonactivators, p h and c b inactivator rapidly inactivate this c b, whereas the access of p h to c b bound to the surface of an activator is restricted: factor b can then bind to c b and is cleaved by factor d generating the alternative pathway c convertase csbbb, the active site being on the bb fragment of factor b. amplification of c turnover with further surface deposition of c b and acquisition of c cleaving activity by c bbb then occur. native properdin binds to c b and retards the otherwise rapid decay of enzymic activity of c bbb. properdin is thus a regulatory protein and is not required for initiation of the pathway [see schreiber and miiller-eberhard (this volume) for further details]. consequent upon activation of either pathway, c is cleaved and the membrane attack complex is assembled from c - . this complex is inserted into the lipid bilayer of cell membranes as a dimer of c b- (biesecker et al., ) , giving the typical electron microscopic complement membrane lesion. osmotic lysis of the cell then results, either because a hydrophilic protein channel is created by insertion of the membrane attack complex, or because its insertion results in rearrangement of the phospholipids and creation of a lipid channel in the bilayer (podack and miiller-eberhard, ). table , and by rawls and tompkins ( ) . these reports demonstrate convincingly that cells infected with a variety of rna and dna viruses are susceptible to lysis by antibody and complement. however, heterologous sera, particularly rabbit serum, which is frequently used as a complement source, may contain natural antibodies reacting with heterologous cell surface or viral antigens. for example, rawls and tompkins ( ) found that rabbit serum, as a complement source, lysed certain hsv-infected cells in the presence of antibody, whereas guinea pig or human serum did not. a prozone phenomenon is often observed with rabbit or guinea pig serum as a complement source (ehrnst, ; hicks et al., ) . these are not particular problems if the principal purpose is to assay potentially cytotoxic antiviral antibodies, as it was in a number of the studies in table . for instance, in cytotoxicity testing for h- or hla typ- summary of earlier studies of antibody-and complement-dependent lysis of virusinfected cells, in which heterologous sources of antibody and complement were used. most of these studies were designed primarily to detect cell surface viral antigens or antibody to them, rather than to analyze the mechanism of cell lysis. sspe, subacute sclerosing panencephalitis. ing, the presence of natural antibodies in the complement source is often used to "rig" the system to produce maximum lysis (ferrone et al., ) . however, the presence of such natural antibodies makes it difficult to analyze the mechanism of antibody-and complement-mediated lysis of virus-infected cells. thus, use of heterologous systems provides results that are of doubtful relevance to the situation in uiuo. the lysis of virus-infected human cells by human serum has been studied in detail in a homologous system by oldstone, cooper, and colleagues perrin et al., ; sissons et al., a) . in addition to having obvious direct relevance to human infection, the use of a homologous system avoids the problems inherent in the use of heterologous sera as a complement source referred to above. lysis by human serum of cells infected with hsv types and , influenza a, parainfluenza , , , and , mumps, and measles viruses was dependent on the presence in serum of igg antibody specific for the relevant virus and of complement (reviewed by oldstone and lampert, ) . in addition to infected human epithelioid cell lines (hela, hep ), virus-infected lymphoblastoid, neural and glial cell lines of human origin were also lysed. no lysis occurred in serum from agammaglobulinemic children, despite an intact complement system. serum lacking antibody to a particular virus would not lyse cells infected with that virus, but would do so after addition of igg containing specific antibody to that virus . acquisition by serum of the ability to lyse virus-infected cells has been demonstrated after immunization with mumps virus and measles virus in individuals without preexisting antibody. sera obtained before immunization and days after it would not lyse infected cells, but became able to produce specific lysis of mumps virus or measles virus-infected cells days afterward, maximum lytic ability being attained days after immunization and unpublished observations) . a consistent finding in these studies was the requirement for an intact alternative pathway of complement activation in human serum for lysis to occur perrin et al., ; sissons et al., a) . thus the ability of serum to lyse virus-infected cells was extremely sensitive to dilution, % of the cytolytic activity being lost at a : dilution, and was abrogated by heating serum at °c (which inactivates factor b). in contrast, lysis still occurred in serum with . m egta and mm mg +, which selectively chelates ca + and thus inactivates the classical pathway . this dependence on an intact alternative pathway was confirmed by using human sera that were immunochemically depleted of specific complement components by affinity column chromatography with monospecific antisera coupled to sepharose b (perrin et az., ) . lysis of cells infected with mumps, herpes simplex, influenza, or measles viruses " human sera containing igg antibody to measles virus were immunochemically depleted of various complement components by affinity chromatography, except for c , which was obtained from a patient with genetic deficiency of c . depleted sera were reconstituted with a physiologic concentration of the purified depleted component. lysis was assessed by the eosin microcytotoxicity assay ( ). data represent the results of three experiments. spontaneous lysis of infected cells in mem was ? %; lysis of uninfected hela cells in reagents was % (mean sd). in similar additional experiments cells infected with mumps, influenza, parainfluenza, and hsv types and were lysed by antibody and c -depleted, but not factor b-depleted, serum. reconstitution with factor b restored lysis (see perrin et al., ; sissons et al., a). was completely abrogated in serum thus depleted of factor b or of factor d and diminished by % in properdin-depleted serum. reconstitution of the depleted component with physiologic concentrations of the purified protein restored the lytic ability in each case (see table ) sissons et al., a) . in contrast, serum immunochemically depleted of c or genetically deficient in c , both components necessary for formation of the classical pathway c convertase ca, was as efficient as whole serum in lysing measles virus-infected cells . furthermore, dose related lysis curves showed that c depleted serum with no measles virus antibody was quantitatively equal to the original whole serum in its ability to lyse antibody coated measles virus-infected cells (fig. ) . the kinetics of lysis in whole and c -depleted serum were also identical ( fig. ) (sissons et al., b) . these observations thus showed that the lysis of virus-infected cells by human serum depends on antibody and the alternative comple- ( ); by the same serum immunochemically depleted of c ( ); or by the serum imniunochemically depleted of factor b ( ). uninfected cells or infected cells without antibody were not lysed. (from sissons et al., b.) ment pathway. the lack of any requirement for c or c showed that activation of the classical pathway is not necessary for lysis, and that the alternative pathway must therefore be initiated by a mechanism other than recruitment in consequence of classical pathway activation. there are very few other reported studies of lysis of virus-infected cells by human serum. hicks et al. ( ) reported lysis of measles virus-infected monkey (vero) cells by antibody and human serum. specific lysis was observed with both c -deficient and mgz+ egtatreated serum and in serum heated at °c or adsorbed with zymosan, although none of these was as effective as whole serum. these authors therefore concluded that lysis could be mediated by alternative and classical pathways, but that neither pathway alone was as effective as ( ) and c depleted ( ) serum; p of serum was used for each time point. (from sissons et al., b.) both combined. again, human serum produced no lysis in the absence of antibody in these studies. the finding of some classical pathway mediated lysis, in contrast to the findings discussed above, is probably related to the use of a different cell line and a heterologous system. the absolute requirement for antibody in complement-mediated lysis of virus-infected cells by serum is documented above. the amount of cell bound antibody required to induce lysis was determined by using measles virus-infected hela cells as targets and employing a binding assay with radiolabeled igg. n o lysis occurred until a mean of greater than x lo to x lo molecules of igg had bound per infected cell perrin et al., ; sissons et al., a) (fig. ). this is a large amount of igg, although the measles virus infected hela cell has a large surface area. the f(ab'), fragment of igg can also induce lysis of mumps virusand measles virus-infected cells , and the dose related lysis curves for whole igg and f(ab'), in whole and c depleted serum are near-identical (sissons et al., b) . however, fab' fragments are ineffective in inducing lysis. at least times more fab' than f(ab'), or igg molecules bound per infected cell were required to produce % lysis, which was the maximum attainable with fab' in these experiments. at this high density it is difficult to exclude the possibility that fab' fragments reassociated despite being initially alkylated (see fig. ) (sissons et al., b) . these experiments thus show a requirement for divalent antibody, but no requirement for the fc portion, for igg to induce lysis of virus-infected cells by the alternative pathway. the fact that f(ab'), (which cannot bind c l q ) is as effective as whole igg is further proof of the lack of requirement for classical pathway activation for lysis. fab' and fab in addition to f(ab'), fragments were reported as able to induce lysis of measles virus-infected cells in one other study, but rabbit or guinea pig sera were used as complement sources . these findings are difficult to interpret because a heterologous complement source was used, and no determination was made of the amount of fab bound to the cell. in contrast, fab' fragments blocked the lysis of hsv-infected cells b y igg antibody and rabbit complement in a study by brier et ul. ( ) . this requirement for divalency in the homologous human system suggests that antibody-induced redistribution of viral antigens on the cell membrane may be required for production of lysis. prevention of capping with cytochalasin d or sodium azide does not abrogate complement-mediated lysis . however, these inhibitors of actin polymerization do not prevent local patching of viral antigen on the cell membrane b y antibody. antibody directed against different viral polypeptides expressed on the cell surface can mediate lysis of measles virus-infected cells. measles virus has two surface glycoproteins, ha and f. using a persistently infected cell line and a heterologous complement source, ehrnst ( ) reported that antibodies to ha induced lysis by the alternative pathway, whereas antibody to f induced lysis by the classical pathway. again, the amount of antibody bound per cell was not determined in these experiments; by immunoprecipitation and sds-page analysis of infected cells, the antisera used by ehrnst were not monospecific (j. g. p. sissons and m. b. a. oldstone, unpublished observations), making these results at best difficult to interpret. in contrast, using cell surface labeling and immunoprecipitation to assess the site of antibody binding on acutely infected cells, sissons et ul. ( b) found that human igg antibodies that bound specifically only to ha or only to f protein were equally effective in inducing lysis by the alternative pathway (in c -depleted serum) and that neither antibody would induce lysis by the classical pathway alone (in factor b-depleted serum). an equivalent amount of antibody bound to either viral polypeptide induced an equal degree of lysis. the subclass of igg seems unlikely to be of direct importance in the induction of lysis, especially since the fc region is not required. although some experiments indicate that iggl is the major subclass in- there is virtually no information on whether other human immunoglobulin classes can induce complement-dependent lysis of virus-infected cells. this would be of interest insofar as most viruses induce an early igm response on primary infection or immunization and may also produce a serum iga response . joseph et al. ( ) reported that, at amounts equivalent to or greater than cytolytic igg, iga, and igm isolated from convalescent sera of patients with measles or after vaccination (unpublished observations) did not induce lysis of measles virus-infected cells. however, as might be expected, measles virus antibody titers in the igm and iga preparations were very low compared to titers in the igg fraction. rabbit igm antibody appears to be relatively ineffective in inducing lysis of virusinfected cells when compared to igg (brier et al., ; ehrnst, ). the precise molecular mechanism by which antibody produces lysis of virus-infected cells in conjunction with complement remains to be determined. however, the findings described in the next section show that igg is not required for initiation of the alternative pathway by measles virus-infected cells, although it is required for cell lysis. recent work has further confirmed the role of the alternative pathway in lysing virus-infected cells. these experiments have again used measles virus-infected hela cells as a representative model. the alternative pathway of complement activation has recently been assembled in vitro from its isolated constituent proteins in highly purified form. a mixture composed of c , factors b and d, native properdin, and the two control proteins, p h and c b ina, all at physiologic concentrations, can mediate deposition of c b onto activators of the alternative pathway, such as rabbit erythrocytes, with an efficiency equal to serum (schreiberet al., ) . addition to this mixture of the five purified proteins of the membrane attack pathway, c - , creates a system with cytolytic capacity. this purified cytolytic alternative pathway can lyse rabbit erythrocytes and (with lysosyme) e . coli as efficiently as ccdepleted serum (schreiber and muller-eberhard, ; schreiber et al., ) . in recent experiments it was shown that the purified cytolytic alternative pathway could lyse antibody-coated measles virus-infected cells with an efficiency comparable to that of ccdepleted or whole human serum. as in serum, no lysis , a.) occurred in the absence of antibody, and f(ab')z and igg gave near identical dose related lysis curves, but fab' produced no lysis. approximately x lo molecules of igg or f(ab'), bound per infected cell were required to induce % lysis (sissons et al., a ) (see fig. ). omission of properdin from the purified cytolytic alternative pathway totally abrogated lysis of measles virus-infected cells. this is noteworthy insofar as properdin was not essential for the lysis of rabbit erythrocytes or e. coli by the purified cytolytic alternative pathway (schreiber and miiller-eberhard, ; schreiber et al., ) . this difference may well reflect the fact that nucleated cells require more complement-mediated membrane damage to produce lysis. the use of this system, composed of highly purified complement proteins, provided conclusive evidence that the known proteins of the alternative and membrane attack pathways with igg antibody are sufficient for lysis of the measles virus-infected cell, without other serum factors. these findings and the efficacy of f(ab') also excluded any possibility that alternative pathway activation by antibody-coated virusinfected cells could be occurring by the "c bypass activation pathway" described by may and frank ( ) , in which sheep erythrocytes bearing rabbit antibody apparently initiate the alterna-tive pathway by a mechanism involving c . hence, a dominant role is established for the alternative pathway in the complement-dependent lysis of virus-infected cells by human serum. the requirement for both specific antibody and an intact alternative pathway for lysis of virus-infected cells at first suggested that antibody might be required for activation of the alternative pathway in this system. this would be distinctly unusual, as activation of the alternative pathway by known particulate activators (rabbit erythrocytes, zymo- ( ) found that measles virus-infected hela cells incubated in a mixture of highly purified c , factors b and d, native properdin, p l h , and c b ina (the purified alternative pathway of complement activation, without c - ) showed progressive linear uptake of [lz i]c b onto the cell surface (fig. ). this c b uptake, which averaged to . x lo c b molecules per cell after minutes, was specific in that it was not shown by uninfected hela cells and was blocked by . m edta. measles virus-infected hela cells also showed progressive uptake of [lz i]c b from human serum immunochemically depleted of c and igg. in addition to being independent of igg, [lz i]c b uptake from the purified alternative pathway onto measles-infected cells in the absence of antibody was also independent of properdin, the rate of uptake being the same in the absence and in the presence of properdin, and similar to the rate of [ ]c uptake by infected cells in the presence of igg but the absence of properdin. these experiments are summarized in fig. . thus, the rate of [ ]c b uptake by infected cells was increased only in the presence of igg and properdin together. because both igg and properdin are also required for lysis, this suggests that some interaction between the two, possibly affecting the rate of c b uptake, is important in the mediation of lysis. in these experiments, significant [lz i]c uptake by measles virusinfected cells was first demonstrable - hours after infection, by this time all cells were expressing viral polypeptides on their surface, indicating that viral replication must occur before alternative pathway activation becomes detectable. in all the above experiments the absolute amount of c b ultimately bound by infected cells alone was similar or identical to the amount bound by infected cells coated with igg, only the rate of uptake being influenced by igg. further work is needed to determine whether this ability to activate sissons et al., .) the alternative pathway independent of antibody is a general property of cells infected with vivses. however, it should be noted that a number of human lymphoblastoid cell lines can activate the alternative pathway in human serum (budzko et al., ; theofilopoulos and perrin, ; mcconnell and lachmann, ; mcconnell et al., ) . mcconnell et al. ( ) have reported that this ability correlates with transformation by epstein-barr (eb) virus. they found that cell lines positive for eb virus-determined nuclear antigen (ebna) activated the pathway, as shown by c conversion in mg-egta serum and deposition of c on cells detected by immunofluorescence. ebna negative lines did not deposit c on their membranes but acquired the ability to do so after eb virus transformation and ac-quisition of ebna. eb virus-transformed cells are mostly nonproducing and do not express detectable structural viral antigens on their surfaces. there presumably must be changes in the membrane as shown by the presence of the uncharacterized lymphocyte-determined niembrane antigen (svedmyr and jondal, ) . despite the ability of raji cells, one of these ebna-positive lymphoblastoid lines, to activate the alternative pathway, lysis of the cells occurred only after prolonged incubation in serum for - hours and was independent of antibody (theofilopoulos and perrin, ) . in contrast, measles virus-infected hela cells did not lyse (as measured by specific cr release) even after hours of incubation in serum without antibody to the virus (j. g. p. sissons, unpuhlished observations) . there is supportive but less direct evidence suggesting that ineasles virus-infected cells (hicks et d., ) and sendai virus-infected cells (okada et d., ) can activate the alternative pathway. this is implied by their lysis in c -deficient guinea pig serum independent of antibody. it was also suggested that retrovirus-infected cells can activate the alternative pathway, as evidenced hy using mg-egta serum and a rosetting assay to detect cell-bound c (okada and baba, ) . however, it seems equally likely that retrovirus-infected cells might activate the classical pathway, since it is already known that free retrovirus activates the classical pathway independent of antibody b y binding c l q via its surface glycoproteins (welsh et al., ; cooper et al., ; bartholomew et az., ) . the principal aspects of target cells affecting lysis are the density and presentation of viral antigen expression on the cell surface. any factor decreasing surface antigen expression tends to decrease susceptibility to lysis. thus, events that cause the virus-infected cell to produce mutants defective for surface glycoproteins or defective interfering virus, which suppresses replication of the standard virus, result in diminished viral antigen expression on the cell surface. welsh and oldstone ( ) found that the susceptibility of cultured neuroblastoma cells infected with lcmv to lysis by antiviral antibody and heterologous complement declined with increasing time in culture. this was related to diminished lcmv surface antigen expression, which resulted from generation of defective interfering lcmv. the resistance to lysis was specific for lcmv in that both acutely and persistently infected cells were still susceptible to lysis by anti-hi- and complement. antigenic modulation of measles virus-infected cells by antibody, as described in section , also renders cells resistant to human antibody-and complement-mediated lysis. joseph and oldstone ( ) found that after hours of culture in the presence of antibody, cells both acutely and persistently infected with measles virus lost their susceptibility to antibody-and complement-dependent lysis. at certain doses addition of interferon to cultured cells expressing lcmv antigens is followed by a reduction in the expression of viral antigens on the cell surface and susceptibility to immune lysis (m. b. a. oldstone and t. merigan, unpublished observations). a further mechanism whereby infected target cells could escape lysis by antibody and complement is the expression of new membrane proteins that block access of antibody to surface antigens. celis et al. ( ) showed that murine myeloma lpc- cells grown as an ascites tumor express increased amounts of a surface glycoprotein (gp oo) that seemed able to block binding of cytotoxic antibodies to h- antigens. removal of this protein with proteases restored susceptibility to immune lysis by anti-h- and complement or cytotoxic lymphocytes. whether similar events can occur with viral antigens is unknown. the cell cycle can also influence susceptibility to lysis. cikes and friberg ( ) found that moloney virus-transformed lymphocytes were susceptible to lysis by antibody and complement only in the g phase, not during s, g , or m. yet viral antigen was present and accessible to antibody, and complement was activated throughout the cell cycle (lemer et al., ) . complement membrane lesions were also present on the cells at different phases of the cell cycle, although immune lysis was again restricted to g . consequently, the plasma membrane may vary in its ability to withstand complement-mediated damage at various phases of the cell cycle, presumably because of factors affecting membrane repair. susceptibility of nucleated cells in general to complement-mediated lysis is probably related in part to their ability to effect membrane repair. for instance, complex changes in the synthesis and release of several lipid classes have been reported in tumor cell lines upon their exposure to antibody and complement (schlager et al., ) . however, there is no direct information on how virus infection can affect the cell's ability to repair its membrane. in summary, cells infected with a number of different rna and dna budding viruses are lysed by human serum, and this lysis is dependent on igg antibody and the alternative pathway of complement. more detailed studies using measles virus have shown that f(ab'), is as effective as whole igg in inducing lysis but that fab' is ineffective. furthermore, igg is not required for activation of the alternative pathway, which occurs on the surface of measles virus-infected cells independently of antibody. properdin is not required for activation of the alternative pathway, but is required with igg for lysis of infected cells. the major remaining questions concerning the mechanism of this reaction are as follows: do cells infected with other viruses activate the alternative pathway? how do virus-infected cells activate the alternative pathway? what is the mechanism by which igg induces lysis? the mechanism by which measles virus-infected cells activate the alternative pathway may be similar to that described for other activators, activator-bound c b being protected from p h. it has been proposed that specific surface molecules on activating particles might act as "plh antagonists," restricting access of p h to c b (pangburn and muller-eberhard, ; pangburn et al., ) . it is conceivable that the viral glycoproteins, which are inserted in the membrane as integral membrane proteins, could function in this way. alternatively, some other virus-induced change in the membrane may be responsible. for example, virus infection could result in expression or release of membrane proteases that might cleave c . immunoelectron microscopy studies in which measles virus-infected cells were incubated in human serum containing antibody to measles, and then stained with ferritin-conjugated antisera to c , factor b, or properdin, showed that the antisera bound to the viral antigen-antibody complex on the cell membrane. adjacent sites on the membrane, where viral spikes were not apparent, showed no labeled antibody binding. this suggests that membrane deposition of c occurs at the sites of insertion of viral glycoproteins (oldstone and lampert, ) . the fact that properdin is not required for activation of the pathway by measles virus-infected cells accords with its known function, which is to delay intrinsic decay of c bbb. properdin can be recruited only when multiple c b molecules are deposited on a surface in close spatial association, and activation of the pathway must therefore precede its recruitment (fearon and austen, ; medicus et al., ) . the requirement for properdin in complement-dependent lysis of measles virus-infected cells, in contrast to its nonessential role in lysing other activators, may reflect the fact that a nucleated cell with the capacity for membrane repair requires more extensive complementmediated membrane damage before lysis results. properdin would be expected to enhance c and c turnover and membrane deposition. the fact that virus-infected cells are not lysed by antibody and the classical pathway alone in human serum requires explanation. the classical pathway is actiuated by igg antibody bound to virus-infected cells, as shown by uptake of c and c (assessed b y immunofluorescence) from factor b-depleted serum ) and binding of [ ]c following assembly of c;f on the surface of igg bearing measles-infected cells (m. k. pangburn and j. g. p. sissons, unpublished observations); however, it is not known whether membrane lesions result. it is possible that, despite activation of the classical pathway, insufficient membrane damage occurs to achieve lysis in the absence of the amplification provided by the alternative pathway c b-dependent feedback mechanism. the mechanism whereby igg induces lysis by the alternative pathway remains to be determined. the requirement for divalent antibody suggests that patching of viral polypeptides on the membrane may be important. the fact that the rate of c uptake onto measles-infected cells is enhanced only when igg and properdin are both present suggests that igg may facilitate the uptake of properdin. further experiments are needed to test these possibilities. there are a few other instances in which antibody and the alternative pathway are both required to lyse cells. human lymphocytes coated with human alloantisera can be lysed by the alternative pathway in human serum, although not all the alloantisera used in this study had this ability and some were effective in inducing lysis only by the classical pathway (ferrone et al., ) . trypanosornu cruzii are also lysed by igg antibody and the alternative pathway (krettli and nussenzweig, ) . additionally, there is some evidence to suggest that a serum factor, probably igg, enhances the alternative pathway-dependent lysis of rabbit erythrocytes by human serum (polhill et al., ; nelson and ruddy, ) , although igg is not essential for this reaction. it is apparent that considerably more surface-bound igg is required to induce complement-dependent lysis of virus-infected cells than is required for antigenic modulation or antibody-dependent cell-mediated cytotoxicity. the possible implications of this are discussed in section v. further work showing that cells infected with other viruses can activate the alternative pathway independent of antibody would strengthen the evidence for viewing the alternative pathway as a means of nonspecific host defense against microorganisms. it is also important to determine whether free viruses can activate the pathway. the observations of wedgewood et al. ( ) and of welsh ( ) suggest that newcastle disease virus may be inactivated by the alternative pathway independent of antibody in human serum (reviewed by cooper and welsh, ) . the main emphasis in this review is directed toward the effect of antibody alone or antibody and complement on virus-infected cells. now we discuss antibody-dependent cell-mediated cytotoxicity (adcc) briefly, both to illustrate the range of actions that antibody may have on virus-infected cells and to place all these actions of antibody in a comparative context. this section deals only with adcc as applied to virus-infected cells, with emphasis on human systems and the role of antibody, rather than on detailed discussion of the effector cells involved. adcc has been studied in a number of different systems (reviewed by mclennan, ; perlmann et al., ; lovchik and hong, ; perlmann and cerottini, ) . although the lysis of antibody-coated erythrocytes can be mediated by phagocytic cells-polymorphonuclear leukocytes (pml) and macrophages-the lysis of nucleated cells sensitized with antibody by nonimmune, human peripheral blood lymphocytes (pbl) is mediated predominantly by lymphocytes that have fc receptors. these cells do not have conventional b or t lymphocyte markers (surface ig and sheep erythrocyte (e) rosette negative) and thus fall within the "null cell" population. however, there is evidence that some pbl able to mediate adcc can be found in the subpopulation of t cells with fc receptors for igg (see perlmann and cerottini, ) as has also been reported for virus-infected targets . this t cell subset also contains cells able to suppress antibody production by b cells (moretta et al., ) . although cells that can mediate adcc are often referred to as k cells, this is a functional definition and is not based on the presence of unique surface markers. adcc is usually rapid; membrane damage can be detected minutes after the reaction's inception, and cell lysis is usually completed by - hours. there is evidence to suggest that the effector cell is inactivated by interaction with its targets (ziegler and henney, ) , which may result from modulation of the fc receptors on the effector cell surface (perlmann and cerottini, ). in assaying for adcc, exogenous antibody is conventionally added to the cytotoxicity assay in the presence of pbl. however, pbl can exhibit cytotoxicity for a range of target cells in the absence of added antibody, and this activity is mediated, in part, by a class of cells func-tionally designated as natural killer (nk) cells. there has been some uncertainty over whether or not this spontaneous or natural cytotoxicity could be dependent on small amounts of antibody present in the assay system. for this reason, recent observations concerning human nk cells are summarized below. the spontaneous cytotoxicity exhibited by unsensitized human pbl for various cultured cell lines and virus-infected cells in uitro has received increasing attention. this lysis, mediated by nk cells, has been recently reviewed in human (herberman et al., ; saksela et al., ; perlmann and cerottini, ) and murine (herberman et al., ; kiessling et al., ; welsh, ) systems. the bulk of evidence indicates that human nk cells cannot be easily separated from cells mediating adcc, both being chiefly null cells with fc receptors. depletion of nk cells by target binding and cross competition experiments suggests that the same cell population mediates both activities. however, the mechanism of target recognition differs, and a functional distinction between cells mediating adcc and nk activity can be made on this basis. the available evidence (summarized in the reviews above) suggests that nk cells do not recognize and bind to target cells via their fc receptors or depend on antibody for cytotoxicity. furthermore, nk cells do not exhibit conventional immunologic specificity but can lyse a wide range of infected or uninfected target cells. in contrast, cells mediating adcc recognize and bind to target cells sensitized with antibody via their fc receptors and require antibody for cytotoxicity, which thus confers immunologic specificity on the reaction. an obvious and important question is whether small amounts of igg antibody secreted during the course of a cytotoxicity assay, or bound to cytotoxic cells via their fc herberman et al., ) and in viuo (hudd-lestone et al., ) . however, it is not clear whether interferon can also enhance the ability of pbl to mediate a d c c herberman et al., ) . virus-infected cells (and tumor-and virus-transformed cell lines) can induce interferon release from pbl. interferon can then enhance the activity of nk cells within the pbl population and contribute to lysis of target cells in the course of a cytotoxicity assay (santoli et al., ) . thus, distinguishing nk cell activity from a d c c presents difficulties. unless carefully defined reagents are used to block igg-fc receptor interactions with uninfected and infected cell targets, it is difficult to interpret their relative importance during lysis of virus-infected cells by pbl. when nk activity is induced in vitro by interferon released during a cytotoxicity assay, cytotoxicity is maximal at - hours, whereas a d c c in the presence of exogeneously added antibody is usually maximal by - hours. however, killing by nk cells already induced by preexposure to interferon in vivo or in vitro is maximal at - hours, whereas adcc dependent on antibody secreted during an in vitro assay might be expected to have slower kinetics. hence, clearly distinguishing between nk cytotoxicity and adcc on the basis of kinetics is also difficult. it is possible that if the same cell is involved in both nk cell killing and adcc, as current evidence suggests, then attachment to target cells sensitized by antibody via its fc receptor may induce a more efficient cytotoxic mechanism than binding via the putative nk cell receptor. the mechanism of a d c c appears to be the same for different cells infected with a variety of viruses and is unlikely to differ from that involved in a d c c of other nucleated cells. although there are numerous descriptions of virus-infected cells killed by human pbl, only those reports in which a d c c activity was deliberately assayed by addition of exogenous antibody are discussed below. the first examples concerned a d c c of hsv-infected cells by human pbl (shore et al., (shore et al., , rager-zisman and bloom, ) as well as mouse peritoneal exudate cells (rager-zisman and bloom, ) . both reports emphasized that a d c c could be produced by antibody concentrations several hundredfold less than those needed to produce antibody and complement dependent lysis of the same virusinfected cells. extending these findings, shore et al. ( ) and kohl et al. ( ) noted that the effector cell in the human pbl population could be either an adherent cell or a nonadherent fc receptor-positive lymphocyte. the adherent cell required higher antibody concentra-tion and displayed slower kinetics of killing than the nonadherent cell. this is the only report of an adherent mononuclear cell mediating adcc of virus-infected cells in a human system; others have found that eliminating adherent cells did not diminish or might enhance adcc. other studies with hsv (moller-larssen et d., ) suggested that trace amounts of antibody present in the medium used for washing pbl could mediate adcc by pbl in the absence of added igg. the added complication of the hsv-induced fc receptor (discussed in section ) should be recalled. lysis of hsv-infected cells may occur in part by interaction involving this receptor. rager-zisman et al. ( ) suggested that even traces of aggregated igg in fetal bovine serum could cross-link target and mouse peritoneal effector cell fc receptors to produce apparent adcc of hsv infected cells, although the possibility of nk cell killing was not considered. in studies with mumps virus-infected cells (anderson et al., ; harfast et al., ) , high concentrations of rabbit antibody to mumps virus inhibited, but lower concentrations enhanced, pbl-mediated cytotoxicity. antibody independent but virus specific cytotoxicity mediated by fc receptor bearing pbl was also noted in these studies (harfast et al., ) . several laboratories have independently assayed pbl-mediated killing of measles virus-infected targets. antibody to measles virus enhanced the killing of measles virus-infected cells by pbl harvested from normal immune subjects (perrin et al., a; kreth and ter meulen, ; ewan and lachmann, ) . antibody from normal immune subjects or patients with subacute sclerosing panencephalitis (sspe) also enhanced the cytotoxicity of pbl obtained from patients with sspe (kreth and ter meulen, ; perrin et al., a; a. tishon, j. h. huddlestone, and m. b. a. oldstone, unpublished observations) . since patients' sera enhanced cytotoxicity, these observations are at variance with the earlier report of ahmed et a,?. ( ) of a "blocking factor" in the sera of sspe patients that inhibited lymphocyte-mediated activity (migration inhibition, proliferation) against measles virus-infected cells, although ahmed et al. did not assay cytotoxicity. perrin et al. ( a) calculated that an average of to x antibody molecules bound per measles virus-infected target (hela) cell was required to induce adcc. this contrasts with the much greater (at least -fold) amount of cell-bound antibody required to induce antibody-and complement-dependent lysis in the same system (see section iv). respiratory syncytial virus-infected cells were lysed by adcc (scott et al., ) using colostrum as well as serum as sources of igg antibody. adcc has also been used to detect igg antibodies to e b virus (jondal, ) , but only lytically infected cells that express virusspecific surface antigens, not transformed cells, can be used as targets. c. summary and conclusions it is clear that addition of specific antibody can enhance the cytotoxicity of pbl for virus-infected cells. minimal amounts of antibody are effective for adcc activity. because the kinetics of adcc are usually rapid, increased ionic efflux from target cells being observable at minutes in some systems (ziegler and henney, ) , it seems unlikely that antibody would produce significant modulation during the course of a cytotoxicity assay. nevertheless, it is possible that the inhibitory effects of high doses of antibody on adcc might be related to capping or redistribution of viral antigens by antibody; inhibition might also be produced by modulation and loss of fc receptors on the effector cells by aggregated igg (perlniann and cerottini, ). igg antibody has been responsible for adcc of virus-infected cells in all the human studies cited. at present there is no published work on the relative efficacy of antibodies directed against different viral antigenic determinants on the cell surface in mediating adcc. however, unpublished work from this laboratory (l. h. perrin and m. b. a. oldstone) suggests that there may be differences in the ability of antibody to measles virus ha and antibody to measles virus f protein to produce adcc. if spontaneous cytotoxicity b y n k cells does involve recognition of target antigens by a receptor distinct from the fc receptor on cells within the null cell population, it is conceivable that antiviral antibody on the target cell surface might sterically hinder binding of such cells to their target antigen, whatever that antigen is, on the infected cell. antibody might thus inhibit n k cell binding as it simultaneously facilitated k cell binding. there are a number of reports that evaluate the spontaneous cytotoxicity of human pbl for virus-infected targets without added antibody. some selected reports are discussed that indicate a possible role for antibody. in some cases there is evidence that cytotoxicity is dependent on antibody adsorbed to effector cells as suggested by studies of greenberg et ul. ( ) with influenza and moller-larssen et ( . ( ) with hsv. an alternative explanation would be that antibody is produced by plasma cells in the course of the cytotoxicity assay. after vaccination of human subjects with measles virus or vaccinia virus, perrin et al. ( b perrin et al. ( , found that cytotoxic pbl were generated. enhanced lysis of virus-infected targets b y pbl began at day , peaked by day , and was rapidly dissipated by day after immunization. these induced cytotoxic pbl showed immune specificity. pbl generated after vaccination with measles virus killed measles virusinfected target cells preferentially, but not vaccinia virus-infected targets. similarly, cytotoxic pbl generated after vaccinia vaccination showed enhanced killing of vaccinia-infected targets, but not of measles virus-infected targets. the target cells used were autologous fibroblasts and homologous and xenogeneic cultured cell lines. pbl cytotoxicity induced by measles virus or vaccinia virus vaccines was not mediated by t cells (e-rosetting cells), but by fc receptor positive, e rosette negative cells. the specific cytotoxicity for virus-infected target cells was inhibited % by f(ab'), antihuman igg. further, experiments using different subsets of pbl from immune and nonimmune subjects showed that fc receptor bearing lymphocytes from either immune or nonimmune persons lysed virus-infected targets in the presence of b cells from immune donors. hence, two populations within pbl participated in the killing of virus-infected targets. in addition to the fc receptor positive effector lymphocytes, it is likely that b lymphocytes producing antibody were present. the evidence for antibody playing a role in these circumstances is compelling, but synthesized antibody has not been identified directly. however, pbl from subjects days after immunization or natural infection with influenza virus have recently been shown to secrete specific antibody to the viral hemagglutinin in vitro, coinciding with a peak in cytotoxicity by these pbl against influenza infected target cells (greenberg et al., ) . however, in other instances the enhanced cytotoxicity against virus-infected targets shown by human pbl in the absence of added antibody is likely to result from interferon-induced nk cell activity (santoli and koprowski, ). the abundance of evidence indicates that the effector cells mediating adcc of virus infected targets are the same as those mediating adcc of other nucleated cell targets by human pbl, namely, nonadherent cells with fc receptors that do not rosette with sheep e. the report of an adherent cell mediating adcc against an hsv-infected target cell (kohl et al., ) could relate to special circumstances in the hsv system. however, in most other studies the cytotoxicity of adherent cells had not been studied directly, their ineffectiveness being indirectly deduced from the f x t that their depletion from pbl does not diminish cytotoxicity b y the remaining pbl. human adherent mononuclear cells can reportedly mediate adcc against uninfected sensitized nucleated targets (k and cla- cells) (honvitz et al., ) . rouse igg antibody is highly efficient in its ability to sensitize virus-infected cells for lysis by human pbl. the amount of cell-bound specific antiviral antibody required for adcc is at least to -fold less than that required for antibody-dependent complement-mediated cytotoxicity and the kinetics of adcc are faster than those of modulation. at present, in virus-infected human target models there is no evidence that complement potentiates adcc activity. although precise assessment of the role of adcc in cytotoxicity assays is complicated by the presence of nk cell activity in the same lymphocyte population, adcc appears to be a more efficient cytotoxic mechanism in uitro than nk cell cytotoxicity. the relative role of adcc in vivo in man during acute viral infections, as well as that of nk cells induced by interferon and that of cytotoxic t cells, remains to be determined. there is ample evidence from in vitro studies that, in the absence of any cytotoxic effector system, antibody can inhibit virus production and can prevent infected cells from subsequent immunologic attack. in the presence of complement-or antibody-dependent cytotoxic lymphocytes, antibody can mediate the destruction of virus-infected cells. as with other types of cytotoxicity demonstrated in vitro, it is difficult to be certain about the relative importance of these actions in vivo. complement alone, nk cells, cytotoxic t cells, macrophages and interferon are all potentially able to contribute to the control of virus infection in vivo, in addition to any effects dependent on antibody. there is evidence that antibody-induced viral antigenic modulation occurs in vivo. this is well documented for the tumor virus models alluded to in section , namely for gross leukemia virus (aoki and johnson, ) and flv (doig and cheseboro, ) , as it is for the tl system (boyse et al., (boyse et al., , . the evidence is the loss of surface viral antigens in vivo despite the presence of cytoplasmic viral antigen and the reexpression of surface antigen when leukemic spleen cells from affected animals are transferred to an antibody-free environment. the reason why antigenic modulation, rather than immune jysis, of infected cells should occur is not clear. however, both complement-mediated lysis and adcc are relatively inefficient in the mouse compared to human systems; for instance, the dbn mice with dormant flv erythroleukemia [studied by wheelock et al. ( ) and genovesi et al. ( ) l are c deficient. in addition, antibody might block t cell cytotoxicity (welsh and oldstone, ; effros et al., ) , or the infection could adversely affect the function of cytotoxic effector cells. it is not known whether antigenic modulation of virus-infected cells occurs in vivo in man. however, one situation with possible relevance to the in vitro antigenic modulation of measles-infected cells is the disease subacute sclerosing panencephalitis (sspe). sspe is a rare disease characterized by chronic progressive brain damage afflicting children or young adults. measles virus has been isolated predominantly from the brain and lymphoid tissue by cocultivation techniques, and electron microscopy shows accumulations of measles viral nucleocapsids in brain cells (ter meulen et al., ) . thus, sspe is a persistent measles virus infection in man, but the reasons for the latent period of several years between typical measles virus infection and the insidious onset of sspe as well as for the localization in the brain are unknown. patients with sspe have high titers of antibody to measles in their sera and cerebrospinal fluids (csf), but no evidence of impaired antibody and complement killing joseph et al., ) or adcc (kreth and ter meulen, ; per-rin et al., a) of measles virus-infected target cells. csf has no detectable hemolytic complement, either in normal subjects or in patients with sspe. we have postulated that in the presence of antibody, but absence of complement or paucity of cytotoxic lymphocytes in local areas of the central nervous system, antibody could modulate viral antigens expressed on infected cell surfaces. this could prevent cell fusion and death and initiate a state of viral persistence. these conditions would provide selective pressure favoring the emergence of mutant viruses, such as temperature-sensitive or defective interfering virus (oldstone, ) . there is experimental evidence suggesting that antibody can be associated with modification of measles infection and facilitation of a persistent infection. albrecht et d. ( ) found that an sspe isolate produced a chronic encephalitis in animals with preexisting antibody to measles, compared to an acute fatal disease in animals without antibody. similarly, wear and rapp ( ) found that maternal antibody was required to produce latent brain infection upon inoculation of newborn suckling hamsters with an adapted strain of human measles virus. hsv is commonly maintained in a latent state within nerve ganglia in the central nervous system. it has been suggested that antibody to hsv could maintain hsv latency in infected ganglia, as evidenced by passively transferred antibody preventing reactivation in latently infected ganglia implanted into nonimmune mice (stevens and cook, ) . this has been interpreted as a possible example of antibody-induced antigenic modulation (stevens and cook, ; joseph and oldstone, ) . others have suggested that an additional mechanism may maintain latency, because in their experiments reactivation of hsv occurred despite high titers of hsv antibody both in uitro and in immunosuppressed mice (openshaw et al., ) . allison, ) , although the protection is not invariable. however, the protective effect of antibody could result from neutralization of free virus rather than any cytotoxic effect on infected cells. hicks et al. ( ) showed that decomplementation with cobra venom factor increased the mortality and severity of pneumonia from influenza in mice (disease was also more severe in c -deficient animals) despite serum neutralizing antibody responses equivalent to controls. the mechanism, whether by direct lysis of infected cells or chemotaxis of inflammatory cells, or an effect on free infectious virus, is unknown. there is evidence that tissue injury at sites of virus persistence in chronic lcmv infection in mice or aleutian disease of mink can be mediated by passively transferred antibody (oldstone and dixon, ; porter et al., ) , and that decomplementation with cobra venom factor protects from death adult mice acutely infected with lcmv (oldstone and dixon, ) . igg and c have been demonstrated by immunofluorescence on neuronal cells infected with measles, in perivascular areas of the brains of patients with sspe (vandvik, ; jenis et d., ) . this type of evidence, suggesting that antibody and complement can induce tissue damage by reacting with virus-infected cells, provides the strongest evidence for antibody-me- the evidence provided by human immunodeficiency syndromes suggests that patients with t cell deficiencies are more likely to develop severe infections from common viral agents (e.g., measles virus, herpes virus, vaccinia virus infections). many of the immunodeficiencies characterized by t cell defects are also accompanied by deficiency in antibody production, and many viral antigens are t dependent. hence, antibody deficiency cannot be totally excluded from contributing to the severity of virus infections in these circumstances. patients with hypogammaglobulinemia generally recover normally from virus infections, although whether their small amount of igg effectively mediates adcc is not known. patients with deficiencies of c itself, or of c - , do not seem to be unduly predisposed to virus infections, although c -deficient patients are prone to recurrent pyogenic bacterial infections (lachmann and rosen, ) . nevertheless, passively administered antibody can clearly exert a protective or therapeutic effect in a number of human virus infections. this applies also to immunodeficient patients who contract vaccinia virus or varicella-zoster virus infections (pahwa et al., ) . in this review we have documented, particularly by reference to work in human systems, that antibody exerts a number of significant effects on virus-infected cells. igg antibody can mediate the destruction of virus-infected cells in conjunction with complement or cytotoxic lymphocytes. in addition, at a conceptual level there is evidence to suggest that antibody may enhance and confer specificity on basic nonspecific humoral and cell-mediated defense mechanisms. thus, virus-infected cells can activate the alternative complement pathway independent of igg, but antibody is required for subsequent lysis of the cell. similarly, nk cells can lyse virus-infected target cells independent of antibody and without immunologic specificity, but antibody enhances, and confers specificity on, cytotoxicity by what is probably the same class of effector cells. the importance of these reactions in viuo, particularly in comparison with cytotoxic t cells, remains to be uncovered, as does the possibility that antibody can block cytotoxic t cell function in vivo as it can in vitro (welsh and oldstone, ; effros et al., ; zinkernagel and doherty, ) . because there is limited information on the relative importance of these cytotoxic mechanisms in acute virus infections in humans, future collection and analysis of data in this area are necessary. finally, the ability of antibody to affect the synthesis and intracellular and surface expression of viral proteins has only recently been investigated on a molecular basis. there is already evidence that antibody can act at this level, and such mechanisms may play an important role in the establishment and maintenance of viral persistence. proc. natl. acad. sci proc. natl. acad. sci. u.s.a. , . bourgignon proc. natl. acad. sci. u.s.a. , . brier nature (london) , nature (london) , nature (london) , . daniels clin. e x p . zmrnunol. , proc. natl nature (london) , virol. (submitted for proc. natl. acad. sci proc. soc. e r p symposium on chagas disease , . . , proc. nutl progress in medical virology springer seminars in immunopathology proc. n a f l . acad. sci i n "the antigens nature (london) , proc. natl. acad. sci clin. e x p . im- - proc. natl. acad. sci proc. natl. acad. sci. u.s.a. , nature (london) , . wear key: cord- -jdu h e authors: dubourdieu, dan title: colostrum antibodies, egg antibodies and monoclonal antibodies providing passive immunity for animals date: - - journal: nutraceuticals in veterinary medicine doi: . / - - - - _ sha: doc_id: cord_uid: jdu h e passive immunity can be provided to animals by several sources of antibodies including from colostrum, avian eggs, and monoclonal sources. these antibodies have been shown protect production and companion animals from a number of pathogens. this chapter reviews the immune system for the principles of immune response to antigens and the synthesis of immunoglobulins of the five classes of antibodies in the body. colostrum antibodies are described for passive immunity protection in animals such as calves. chicken egg antibodies are another source of antibodies for passive immunity. therapeutic monoclonal antibodies are also used to provide passive immunity in the veterinary field. the use of antibodies by veterinarians to maintain the health of animals has a long history. fundamentally, when it comes to the immune system health of production and companion animals, there are little absolute differences in the intended purpose of the immune system. mammals have the same basic immune system with minor differences between the species. even the differences between birds and mammals are not so great since the purpose of the immune system is to keep infectious microorganisms, such as certain bacteria, viruses, and fungi, out of the body and to destroy any infectious microorganisms that do invade the body. how veterinarians take advantage of the immune system to maintain health can roughly be defined as taking advantage of the body's inherent method of maintaining health through adaptive immunity provided by vaccinations to generate antibodies inside the animal or by administering preformed antibodies to an animal through a process called passive immunity. evolution has produced an amazing immune system in animals that utilizes various cell types and proteins to protect them from invasive organisms. this system has two broad categories: nonadaptive and adaptive. the nonadaptive immune system is mediated by cells that respond in a nonspecific manner to foreign substances. this response includes phagocytosis by macrophages, secretion of lysozymes by lacrimal cells, and cell lysis by natural killer cells. the adaptive immune response is mediated by lymphocytes that produce a set of proteins called antibodies that are either secreted by or found on the surface of the lymphocyte. when the antibodies themselves are created within the animal following vaccination or from exposure to pathogens, the process is called adaptive immunity. when preformed antibodies from a host animal are given to another recipient animal, such as it offspring or even a completely different species animal, the process is called passive immunity. scientists have long taken advantage of the adaptive immune system by using vaccines. vaccines for animal diseases were the first to result from laboratory-based scientific investigation. french chemist louis pasteur developed a vaccine for chicken cholera in , and one for anthrax of sheep and cattle in . the major goals of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. these diverse aims have led to different approaches in the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked dna injections (meeusen et al. ). it has also resulted in various guidelines for vaccinations of companion animals such as for dogs (ford et al. ) and production animals such as swine (alabama and auburn ) , in poultry (stewart-brown ), cow/calf (missouri ) , and other production animals. successful veterinary vaccines have been produced against viral, bacterial, protozoal, and multicellular pathogens, which in many ways have led the field in the application and adaptation of novel technologies. passive immunity whereas active immunity refers to the process of exposing the individual to an antigen to generate an adaptive immune response, passive immunity refers to the transfer of antibodies from one individual to another (marcotte and hammarström ) . passive immunity provides only short-lived protection, lasting from several weeks to up to - months, but is immediate. nature intended passive immunity to occur when maternal antibodies are transferred to the fetus through the placenta or from breast milk to the gut of the infant. however, it can also be produced artificially when antibody preparations are derived from sera or secretions of immunized donors and are delivered via oral or systemic routes to nonimmune individuals. passive immunization is a new approach to providing protection to animals against pathogens because of the emergence of new and drug-resistant microorganisms, diseases that are unresponsive to drug therapy and individuals with an impaired immune system who are unable to respond to conventional vaccines. the immune system can respond specifically to millions of different molecules and is constantly challenged by huge numbers of antigens. a major feature of the immune system is that it can synthesize a vast number of antibodies. each of these antibodies can bind to a different antigen. this binding is the basis for the molecular specificity of the immune response. antibodies are proteins produced by a type of terminally differentiated b lymphocytes. b cells take the b name from chicken bursa cells where they were first discovered (gitlin and nussenzweig ) . antibodies are produced in response to the presence of foreign molecules in the body. the antibodies circulate throughout the blood and lymph where they bind to the foreign antigens. once bound, these antibody-antigen complexes are removed from circulation, primarily through phagocytosis by macrophages. this is the basis for antibodies protecting the animal against pathogens. antibodies are a large family of glycoproteins that share key structural and functional features. from a structure standpoint, antibodies look like a y-shaped molecule (fig. ) . each y contains four polypeptides. two of the polypeptides are identical and called heavy chain. the other two, also identical, are called light chain and are connected by disulfide bonds. there are five classes of antibodies, igg, igm, iga, ige, and igd, that are classified based on the number of y-like units and the type of heavy-chain polypeptide they contain (fig. ). igm is the largest antibody, and it is the first to appear in response to initial antigen exposure. the spleen, where plasma cells responsible for antibody production reside, is the major site of specific igm production (capolunghi et al. ; marchalonis et al. ) . igg is the main type of antibody found in blood and extracellular fluid allowing it fig. antibody structure as drawn, by protein model and by electron microscopy to control infection within body tissues. approximately % of all antibodies in humans and companion animals are of the igg class. immunoglobulin a (iga) plays a crucial role in the immune function of mucous membranes. the amount of iga produced in association with mucosal membranes is greater than all other types of antibodies combined (fagarasan and honjo ; holmgren and czerkinsky ; snoeck et al. ) . igd was initially thought to be a recently evolved antibody class because it was only detected in primates, mice, rats, and dogs and not guinea pigs, swine, cattle, sheep, and frogs (preud'homme et al. ) . however, recent discoveries of igd in ancient vertebrates suggest that igd has been preserved in evolution from fish to humans for important immunological functions (chen and cerutt ) . immunoglobulin e (ige) has only been identified in mammals. ige's main function is immunity against parasites such as helminths (erb ) like schistosoma mansoni, trichinella spiralis, and fasciola hepatica (watanabe et al. ; pfister et al. ) . ige also has an essential role in type i hypersensitivity (gould et al. ) which manifests in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergies, and specific types of chronic urticaria and atopic dermatitis (mueller et al. ) . antibodies bind antigens at the upper tips of the y molecule. the region of antigen where binding occurs is called the epitope (fig. ). antibodies can bind to a wide range of chemical structures and can discriminate among related compounds. how well the antibody binds to an antigen is known as affinity. this affinity can range from low to high. colostrum and passive immunity mammals are born without a fully functional adaptive immune system even though the basic elements are present. when a mammal is born, it emerges from the sterile uterus into an environment where it is immediately exposed to a host of microorganisms. the gastrointestinal tract (git) acquires a complex microbial flora within hours. if it is to survive, the newborn animal must be able to control this microbial invasion. in practice, the adaptive immune system takes some time to become fully functional, and innate mechanisms are responsible for the initial resistance to infection. in some species with a short gestation period, such as mice, the adaptive immune system may not even be fully developed at birth. in animals with a long gestation period, such as domestic mammals, the adaptive immune system is fully developed at birth but cannot function at adult levels for several weeks. the complete development of active immunity depends on antigenic stimulation. the proper development of b cells and b-cell receptor diversity requires clonal selection and antigen-driven cell multiplication. thus, newborn mammals are vulnerable to infection for the first few weeks of life. they need assistance in defending themselves at this juncture. temporary help is provided by the mother in the form of colostrum, which contains antibodies. the passive transfer of immunity from mother to newborn is essential for survival. calves are born without an active immune system and rely on the consumption of antibodies for protection from disease such as scours and pneumonia. the cow provides its calf with nutrients for growth and development during gestation, but the cow cannot directly provide the calf with antibodies to protect it from diseases. fortunately, immunoglobulins form an important component of the immunological activity found in milk and colostrum. while humans have a large amount of iga in their colostrum, the colostrum from most other animals contains a high percentage of igg (hurley and theil ) (fig. ) . immunoglobulins found in mammary secretions arise from systemic and local sources. in the case igm iga ige igd of igg in milk, the major portion comes from the serum (mayer et al. ) . while plasma cells producing igg may occur within the mammary tissue, their contribution to the igg in colostrum is minor compared with the igg derived from serum. the other major classes of immunoglobulins transported into colostrum and milk are iga and igm. immunoglobulin a (iga) is the major immunoglobulin in human colostrum and milk; however, it is also present in milk of most other species. colostrum and milk iga and igm are found in the form of secretory iga, or siga, and sigm. much of this is produced by plasma cells in the mammary tissue. the plasma cells are part of the gut-associated lymphoid tissue (galt), the largest immune organ of an organism, which includes the peyer's patches, lymphoid and myeloid cells in the lamina propria, and intraepithelial lymphocytes (ishikawa et al. ) . interestingly enough, more than % of the immune system is located in the gastro intestinal tract, the site where many oral pathogens first interact with an animal (vighi et al. ) . galt is a part of the mucosa-associated lymphoid tissue and works in the immune system to protect animals from invasion of pathogens in the gut. one of the physiological functions of the mucosa in the gut is for food absorption. however, of equal importance of the galt is in the body's defense, due to its large population of plasma cells whose number exceeds the number of plasma cells in the spleen, lymph nodes, and bone marrow combined (nagler-anderson ) . lymphocytes from the galt system will move to the mammary gland and provide a direct link between the antigen exposure response in the mother's mucosa system and the secretory immunoglobulins of the mammary gland (brandtzaeg ) . as such, this means that maternal colostrum and milk will contain antibodies specific for pathogens that may be encountered by the neonate's intestine and other mucosal tissues. this provides a rationale for the observations that bovine colostrum from nonimmunized cows may also afford passive immune protection against human pathogens in both humans and animals (li-chan et al. ; yolken et al. ) and opens the door to new technology to provide veterinarians another way to protect animals from pathogens that does not involve antibiotics. antibodies must be obtained by drinking colostrum within the first couple of hours after birth as part of the passive immunization system in order to maximize antibody absorption (pakkanen and aalto ) . like other animals, antibodies are generated by healthy cows as a result of every day exposure to infectious agents. antibodies can also be the result of specific vaccination programs. the cow's natural antibodies to these infectious agents are passed from the cow to the calf through colostrum. the level of antibodies transmitted from the cow through the colostrum can be elevated by a pre-calving vaccination program (thomas ) . when the calf drinks colostrum, the maternal derived antibodies are absorbed from the calf's git into the blood stream. some of the immunoglobulins also remain in the gut where they can neutralize pathogenic bacteria and help prevent the development of diarrhea. the absorption of antibodies from the git into the bloodstream is called passive transfer. failure of passive transfer (fpt) in dairy calves is defined by a blood igg level of < mg/ml - h after birth (stilwell and carvalho ) . calves that experience fpt are more likely to become sick or die in the first months of life than calves with adequate immunity. many factors can contribute to fpt, but colostrum and the management of colostrum feeding are often involved. to successfully obtain passive transfer and provide the calf with protection from diseases, it is thought that the calf needs to consume a minimum of - g of immunoglobulins (meganck et al. ) . the pathway between the gastrointestinal tract and the bloodstream is only open for a short window of time. research shows that this pathway starts to close shortly after birth, and after - h, approximately % of the calf's ability to absorb colostrum antibodies is gone. it has therefore been recommended to feed calves as much colostrum as they want by bottle within - h after birth and at h of age to substantially reduce the probability of fpt (chigerwe et al. ; trotz-williams et al. ) . colostrum also provides the calf with protein, energy in the form of fat and sugar, and vitamins (quigley and drewry ) . some vitamins do not cross the placental barrier, and colostrum is the primary source of these nutrients for the calf after birth. energy is required for all metabolic functions including maintenance of body temperature. one of the leading causes of death in dairy calves is failure to initiate breathing and metabolic processes in the first hours of life. the newborn calf only has a few hours of energy reserves in stored fat and therefore needs the energy from colostrum. research also confirms that the sooner a calf consumes colostrum, the more maternal antibodies it can utilize. the quality of colostrum is a major issue that the dairy industry faces on a regular basis. generally speaking, quality of colostrum is related to the amount of antibody that is present. colostral igg concentration is an important factor that affects whether calves receive sufficient passive immunity (godden et al. ) . unfortunately, the amount of igg in maternal colostrum varies dramatically among cows (< - g/l) with . - . % of samples that do not reach the desired amount of g igg/l (gulliksen et al. ) . colostral quality is difficult to estimate by the farmer based on produced volume or appearance of the colostrum. there are many variables that impact colostrum quality, including nutrition, the time the cow is milked, heat stress, and stage of lactation. a study from iowa state university suggests that a minimum % of dairy calves in the usa are currently being fed colostrum classified below industry standards for igg content and are at a greater risk of fpt, mortality, and morbidity (morrill et al. ) . other production mammals such as piglets face the same issues as calves for colostrum quality. studies have shown that on average % of pigs with a colostrum intake below g usually die. in pigs with a colostrum intake below g, mortality was as high as % (devillers et al. ) . giving spray-dried bovine colostrum to other animals such piglets has been shown (sty et al. ) to help protect against gut dysfunction and inflammation. it may be possible that using bovine colostrum for piglets could help supplement sow colostrum. research has also been done in foals with an enhanced bovine colostrum supplementation (fenger et al. ). since up to % of cows have colostrum with an igg level below mg/ml, which will not prevent fpt, a variety of programs and protocols have been implemented by dairy producers. high-quality maternal colostrum is still the gold standard for feeding newborn calves. however, colostrum supplement and replacer products can be valuable tools to increase calf immunity when colostrum supplies are limited or disease eradication is desired. colostrum products that contain igg are regulated by the usda center for veterinary biologics and are available in bolus, gel, and powder formats. supplement products are unable to raise the blood concentration of igg above the species standard, which is mg/ml for calves. any product that is able to raise serum igg concentration above mg/ml may be called a colostrum replacer (penn state ). supplements do not contain sufficient quantities of antibodies to raise the blood igg level in calves beyond what average quality colostrum will do. colostrum replacer contains greater levels of igg and other nutrients and provides an effective, convenient method of providing passive immunity to calves when maternal colostrum is not available. colostrum supplements available today are made from dried bovine colostrum or serum and contain - g of igg per dose ( - % globulin protein). the fat content of these products ranges from . to %. spray-dried colostrum with high concentrations of immunoglobulin may be produced economically and used as an effective and convenient colostrum replacer in newborn calves (chelack et al. ) . numerous products designed to replace colostrum are now on the market. these products are made from bovine colostrum or serum and contain - g of igg per dose. these products also provide fat, protein, vitamins, and minerals needed by the newborn calf, although the amount varies between products. a summary (pennsylvania state university ) of treatment means from published studies investigating colostrum products indicated that replacer products provided an average of g of igg, with an absorption efficiency of %, and serum igg of mg/ml. supplement products (fed in addition to colostrum) provided g of igg with % absorption efficiency and resulted in serum igg of mg/ml. it has been recognized that while antibodies found in colostrum can certainly reduce diseases in animals via passive immunity when given to a newborn, they will only work if the colostrum donor animal has been exposed either naturally to the disease or given a vaccine to the disease, in order to have specific antibodies produced. bovine colostrum that is typically spray-dried for supplements or replacer will contain only the antibodies that the cow may have encountered naturally. therefore, the colostrum used may not have specific antibodies against particular diseases that a producer might be interested in. the animal industry has recognized this issue and has developed methods to produce specific antibodies in high titer against specific diseases that can be delivered in colostrum products. this is achieved by hyperimmunizing animals such as cows against specific animal diseases, collecting colostrum and processing it into powders such as by freeze-drying methods and then giving to a newborn animal in gels or boluses. a number of diseases including bacterial diseases like e. coli (selim et al. ) and viral diseases such as rotavirus and coronavirus (combs et al. ) or coccidial diseases such as giardia or cryptosporidia (graczyk et al. ; fayer et al. ; naciri et al. ) have been researched for hyperimmunized colostrum efficacy. the hyperimmunized colostrum is collected, processed, and given to newborn calves. to varying degrees of success, these hyperimmunized colostrum antibodies have been proven to be successful in providing passive immunization. besides calves, research has been done in a variety of production animals using bovine or other sources of colostrum. for example, lambs have been supplemented with ewe colostrum as well as hyperimmunized serum from sheep against e. coli (pommer ) . other research has examined vaccination of cows with clostridial antigens and passive transfer of clostridial antibodies from bovine colostrum to lambs (clarkson et al. ) . piglets have also been given passive protection against porcine epidemic diarrhea by hyperimmune bovine colostrum (shibata et al. ) . specific antibodies in a hyperimmunized colostrumderived product can be used while complementing early colostrum feeding and can be delivered at the same time as colostrum. there are some advantages to this strategy as specific immunoglobulins immediately fight at the gut level to protect against diseases that destroy the intestinal lining while also allowing for antibodies to be absorbed into the bloodstream. it's important to protect the intestinal lining because if the cells that line the digestive tract become damaged, milk cannot be digested or absorbed by the calf. besides the quality of the colostrum itself, researchers first believed that calves could not be vaccinated effectively while they had circulating maternal antibodies from the colostrum in their system. preweaning calves can respond to vaccination stimulation as early as month of age. the maternal antibodies absorbed from colostrum, however, cannot distinguish between the antigens of a natural challenge and the antigens in a vaccine. therefore, colostrum antibodies can interfere with the immune response to a vaccination (niewiesk ) . work continues to be done to develop ways to circumvent maternal antibody interference. vaccination of calves in the face of maternal antibodies (ifoma) often does not result in seroconversion as maternally derived immunity interferes with the activation of adequate antibody responses to vaccination. however, it can prime t-and b-cell memory responses that protect calves against clinical disease when maternal immunity has decayed. the activation of b-and t-cell memory responses in calves vaccinated ifoma varies and is affected by several factors, including age, level of maternal immunity, type of vaccine, and route of administration. these factors influence the adequate priming of humoral and cell-mediated immune responses and the outcome of vaccination. failure to adequately prime immune memory after vaccination ifoma could result in lack of clinical protection and an increased risk of viremia and/or virus shedding (chamorro et al. ). there is obviously some controversy about whether newborn calves should be vaccinated (cook et al. ) . it is thought that the process of the calf mounting an immune response to a vaccine requires energy that could better be used to fight off disease and gain weight and the response could actually be detrimental to the early health of that calf. on the other hand, while maternal antibodies can block response to vaccination, sometimes they do not (woolums ) . the exact immunologic outcome in calves vaccinated ifoma can vary, and this variation likely depends on many factors. these factors are not well characterized but likely include the nature of the vaccine administered, number of doses administered, age of the calf and level of maternal antibody present in the calf, and the means by which a protective response is defined. guidelines for vaccinating newborn animals such as calves require additional research to clarify the ifoma vaccination reactions. fortunately, producers also have the option of using antibody products in order to generate immediate protection in situations where colostrum quality is poor. antibody products complement colostrum feeding because they can be fed at the same time. these products are available in bolus, gel, and powder form. they also are included in some colostrum replacer and supplement formulas for added value. typically, the antibody products are from hyperimmunized cows, and the specific antibody is found in the colostrum. the colostrum is processed and typically fractionated into a semi-purified preparation of antibody that is freeze-dried and put into capsules or boluses. because antibody boluses can be fed in conjunction with colostrum, they can be a tool to help the calf not only achieve adequate passive transfer but also provide enough specific antibodies to protect against the most common early calf hood diseases. antibody products do not require the calf to react to a vaccine in order to develop antibodies. rather specific antibodies against various diseases are already present, measured, and verified to be at a high enough level to protect the calf from scour-related diseases, and they can be fed as close to birth as possible. united states department of agriculture (usda)-approved antibody products are available on the market that can be fed in conjunction with colostrum and provide the calf with immediate immunity. these antibodies go to the gut to immediately bind and neutralize diarrhea antigens while also being absorbed into the blood stream for extended protection (combs et al. ; chamorro et al. ) . another advantage of this approach is that providing specific antibody can potentially avoid vaccine stimulation. avoiding vaccine stimulation can allow a calf to conserve its minimal supply of fat and nutrients that are critical to get the calf through its first few days of life. passive immunity by egg antibodies cows pass their immunity to their offspring by colostrum, and various colostrum products are on the market to achieve passive immunity. additional sources of antibody products can be utilized in a similar manner to achieve passive immunity. these are from avian sources such as chickens. in birds, passive transfer of immunity occurs through the egg. by hyperimmunizing chickens over a period of time with inactivated multivalent bacterial or viral vaccines, this procedure results in the production of polyclonal immunoglobulins of the igy class (specific to avians) directed against the stimulating organisms. oral consumption of the "immune" eggs containing specific igy antibodies protects the animal against the specific organism(s) with which the hen was stimulated. unfortunately, the eggs really cannot be cooked since heat dentures the antibodies found in the eggs. other physical parameters such as an acidic ph will also destroy the antibodies to a certain extent. however, enough orally administrated igy may survive passage through the git and, after excretion, still retain a great deal of its antigen-binding ability indicating that orally administrated igys are useful for passive immunity. eggs as a natural source of immunoregulatory factors just as immune protection is transferred in utero in mammals or passively by a lactating mother via colostrum, hens passively transfer protection to their young by secreting immunoglobulin and other immune factors into their eggs for use by the hatching chick. the transfer of chicken immunoglobulins from the hen's serum to the yolk and from the yolk to the chick is analogous to cross-placental transfer of igg from the mammalian mother to its offspring. while igm and iga are found in chicken eggs, the principle immunoglobulin is igy which is found in the yolk of the egg (hamal et al. ) . igy (y stands for yolk) is an immunoglobulin class specific to avians and analogous in function to that of mammalian immunoglobulins. igy has a similar structure as mammalian igg with some minor differences in the heavy chains (fig. ) . both eggs and milk (including breast milk) contain naturally occurring antibodies, and there are reports of immunomodulatory factors in milk as well (li et al. ) . however, immunoglobulin levels in eggs can be significantly higher than levels found in serum or milk (woolley and landon ) . this may not be surprising since mammals have a considerably longer time of weeks or months during which they may passively transfer immunoglobulin and immune factors, while the hen has a single opportunity (the egg) to transfer all necessary survival components to its offspring. all the aspects that the chick needs to survive must be in the egg. because egg products are a common source of protein in human diets and eggs contain antibodies and immune factors, it was obvious to utilize egg antibodies to provide passive immunity to animals. as such, this has resulted in a number of commercial egg antibody products on the market for production animals as well as companion animals to prevent various diseases. with few exceptions, oral consumption of specific antibodies has been reported (diraviyam et al. ) to protect both humans and animals. furthermore, in vitro studies with specific igy antibodies have been found to inhibit processes associated with bacterial growth, adhesion to intestinal cells, and toxin production (sugita-konishi et al. ) . meta-analysis has demonstrated the beneficial effects of igy (diraviyam et al. ) for a variety of animals. this fig. structure of mammalian igg compared to avian igy analysis supports the opinion that igy is useful for prophylaxis and treatment. currently, the oral passive immunization using chicken igy has been focused as an alternative to antibiotics for the treatment and control of diarrhea. igy has been demonstrated to be effective in controlling and preventing diarrhea in humans and in animals including piglets (cui et al. ; vega et al. ) , mice (buragohain et al. ) , poultry (el-ghany ; farooq et al. ) , and calves (cook et al. ; germine et al. ; vega et al. ) . most commercially available chickens are immunized at birth to protect them from avian diseases. the only difference between such supermarket eggs and "immune" eggs is that the latter are from chickens that have received additional proprietary vaccinations with other inactivated pathogens known to be the etiologic agents of animal infections. a wide range of bacteria, viruses, and coccidia have been used in vaccines for producing commercial igy products. a partial list includes shigella dysenteriae, staphylococcus epidermidis, escherichia coli, e. coli k , salmonella enteritidis, and s. typhimurium, pseudomonas aeruginosa, klebsiella pneumoniae, haemophilus influenzae, species of streptococcus, salmonella dublin, salmonella anatum, clostridium perfringens type a and type c toxoids, rotavirus types and , coronavirus, reovirus, parvovirus, and cryptosporidium parvum (schade et al. ) . only the chicken (not the egg) is exposed to inactivated pathogens. immunoglobulins and other immune factors are passively transferred to the egg from the serum for use by the chick and, more importantly, for passive immunity for other animals. after appropriate times following vaccination, eggs are collected from the specially designated chicken flocks, washed and broken, and the yolk and egg white are typically dried to a fine proteinaceous powder. various processes have been developed to help minimize heat damage to egg antibodies and immunoregulatory factors during the spraydrying procedure. however, spray-drying eggs can denature the igy to an extent, due to at least some heating during the process. keeping the egg away from heat is an optimal way of maintaining igy titer levels and efficacy. while freezedrying is an expensive option for maintaining antibody titer levels, simply feeding fresh immunized eggs is another method which exists. this is to keep the immunized eggs in a stabilized liquid (dubourdieu ). this stabilized liquid format is a practical and inexpensive delivery method that allows the specific igy to be delivered in watering systems to production animals or incorporation into other delivery formats such as soft chews that can be readily given to companion animals (fig. ) . the effective mechanism by which avian igy's work is effective is by the same mechanism that mammalian igg antibodies work in the animal to provide passive immunity. for example, bacteria e. coli strain k typically causes problems in production animals that result in diarrhea and possible death. therefore, a vaccination program is used in chickens to create specific anti-e. coli k igy antibodies. these specific antibodies found in the yolk of the egg are given orally to animals to prevent e. coli k from causing disease. this occurs when specific anti-e. coli k igy antibodies bind to the pathogenic bacteria. this binding blocks the ability of the pathogen to bind to the mucin layer obtain egg yolk containing specific igy against antigen spray dry into powder containing specific igy stabilized into liquid containing specific igy fig. vaccination for specific antibodies and processing egg yolk strategies in the gi tract of the animal. the pathogens are flushed out of the gi tract with the feces since they have been rendered unable to bind. the total immunoglobulin content of eggs from hyperimmunized hens is identical to the total level of immunoglobulins found in conventional table eggs. however, the quantities of immunoglobulins to selected antigens are different in the two varieties of eggs. additionally, both the table egg and the "immune" egg contain immunoregulatory factors, but eggs from hyperimmunized chickens may contain many times greater concentrations of individual factors as compared to regular eggs. besides immunoglobulins, eggs contain a number of bioactive components, including phospholipids, cholesterol, lutein, zeaxanthin, and proteins, that possess a variety of proand/or anti-inflammatory properties. two major categories of immune components are found in "hyperimmune" egg: ( ) the immunoglobulins with neutralizing specificities against the stimulating pathogens and ( ) the immunoregulatory factors that modulate cellular functions. the immunoglobulins provide local protection against gastrointestinal intoxication. the immunomodulatory mediators act directly on gastrointestinal surfaces and circulate systemically, affecting every immune, physical, metabolic, and neuroendocrine pathway in the body. these may have important implications for the pathophysiology of numerous chronic diseases and immune responses to acute injury (andersen ) . given the essentiality of pro-inflammatory responses in normal immune defense against pathogens, further research into the role of egg intake on immunity is warranted and may lead to further commercial uses of eggs and igy technology. biological medicine in humans, an intervention pioneered in the last years, is now on the horizon for companion animals. this strategy includes the use of monoclonal antibodies (mabs) to selectively target proteins such as cellular receptors or soluble molecules involved in disease pathogenesis. such treatment holds the potential for targeted therapies of chronic diseases such as osteoarthritis, atopic dermatitis, or lymphomas in dogs and cats. monoclonal antibodies are antibodies that are made by identical immune cells that are all clones of a unique parent cell. monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. in contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell lineages (fig. ) . given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. this has become an important tool in biochemistry, molecular biology, and medicine. therapeutic mabs can be used medically to block disease-relevant proteins (e.g., cytokines or receptors on cells) and cancer and have gained significant use in humans. they can also be used to target viruses or bacteria and aid in their destruction and elimination. veterinary medicine is only now incorporating this tool. despite the success of mabs in human medicine, there are considerably fewer in the pipeline for veterinary medicine. this may be due to uncertain regulatory guidance in both europe and the usa. these regulatory documents are written from the perspective of human medicine-based risk assessment and development. it is recognized that this may present a challenge to veterinary mabs manufacturers to achieve the extent of characterization/quality control testing typically required for human mabs. (ema ) . regardless, the pet medicine industry is making strides to put mabs into the market through basic research. early human therapeutic mabs contained a high proportion of mouse-derived sequences (fully mouse or mouse/ human chimeric mabs) that were recognized by the human immune system as foreign. this immune response triggered production of anti-mabs, leading to reduced therapeutic efficacy. subsequently, the design of humanized and fully human mabs has resulted in a vast reduction in their immunogenicity, although most therapeutic mabs may have some remaining immunogenicity that is not followed by apparent adverse clinical manifestations. these same issues hold true antigen y y polyclonal antibody antigen monoclonal antibody fig. polyclonal antibody compared to monoclonal antibody binding of antigens for making veterinary mabs and utilizing canine mabs for dogs that helps efficacy. it has been found that caninized anti-ige mabs reduce ige hypersensitivity in mite-sensitized beagles (gearing et al. ) . caninized anti-nerve growth factor mab (webster ) significantly reduced pain scores (webster et al. ) in dogs, and mabs that neutralize the pruritogenic cytokine il- in dogs reduced the pruritic response for weeks after injection (dunham et al. ) . one of the aspects regarding commercializing therapeutic antibodies involves regulation from the usda. in , two usda-approved monoclonal antibody treatments for b-cell and t-cell lymphomas in dogs were granted. these mabs fight lymphoma by targeting the protein cd , which is commonly expressed in b-cell lymphoma (ogilvie et al. ; bulman-fleming et al. ) . while these particular mabs were not commercially successful, they are part of a new approach for using therapeutic antibodies in veterinary medicine. other companies are developing veterinary mabs for cancer, allergies, and chronic inflammatory disease such as atopic dermatitis and for the control of pain associated with osteoarthritis in dogs and cats (webster et al. ). the first mab approved for veterinary use was in the european union (bmj ). it treats the clinical signs of atopic dermatitis in dogs, including itch and inflammation, for up to month. the mab treatment works by mimicking the activity of natural antibodies to selectively bind to and neutralize interleukin- (il- ), a key protein involved in cell communication which triggers itching associated with atopic dermatitis in dogs. because it neutralizes il- , it has been demonstrated not to interfere with the immune response, meaning that it does not induce unintended immunosuppression or enhancement. most therapeutic mabs are delivered via intravenous, intramuscular, or subcutaneous injection. this is because antibodies can't be delivered orally because of breakdown in the stomach by acids or other factors found there. various encapsulation methodologies are required in order to overcome this basic issue, and these methods should be forthcoming. once injected, most therapeutic mabs, like natural antibodies, have a long half-life (about days). the absolute half-life for each is unique, depending upon its concentration, distribution of its target, and, if the mab is directed to a cellsurface receptor, clearance and elimination of the target receptor. therapeutic mabs have two main safety advantages: ( ) they have very specific targets, and ( ) they don't have intracellular activity. as a result, there are few anticipated side effects and reactions although they can occur (catapanoab and papadopoulosc ) . mabs are eventually eliminated via intracellular catabolism in the lysosome, where they are broken down into peptides or amino acids that can be either reused for synthesis of new proteins or excreted via the urine. concluding remarks and future directions antibodies for veterinary use have great potential for the future. passive antibody therapy in the treatment of infectious diseases is a concept which dates back more than years, to the s, when the use of serum from immunized animals provided the first effective treatment options against infections with clostridium tetani and corynebacterium diphtheriae (hey ) . however, due to the discovery of penicillin by fleming in , and the subsequent introduction of the much cheaper and safer antibiotics in the s, serum therapy was largely abandoned. but in more recent times, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multiresistant strains of bacteria with limited or no response to existing treatments and thus a need for alternative treatment options. this situation can be partially attributed to the overuse of antibiotics as growth promoters for production animals and other indiscriminate use. the combined specificity and flexibility of antibody-based treatments in providing passive immunity makes them very valuable tools for designing specific antibody treatments to infectious agents. these attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products for human use. however, only very few mabs are approved for veterinary use. mabs therapies are expensive, and this has been a barrier for their development in the presence of inexpensive antibiotics. the use of antibiotics as growth promoters came to an end in in the usa with the rest of the world already limiting their use for this purpose manner (fda ) . this opens the door to new technologies and antibodies from monoclonal sources, chicken eggs, cow colostrum, or other sources to be among the chief contenders for limiting diseases in a safe manner and potentially to act as growth promoters. for that purpose, antibodies and antibodyderived treatments offer very attractive tools and attributes to neutralize infectious agents or modulate the immune system to enable effector cells to escape immunosuppressed conditions and contribute to the elimination of infections. the ability to raise antibodies to any target, and the ability to modulate effector functions, half-life, and size of the treatment units, makes antibodies ideal for tailoring treatments for specific infectious agents. however, more research into the use of antibodies as growth promoters will need to occur. one area that researchers are looking to make better use of antibody treatments for veterinary use includes more use of mabs. it has been predicted that minimal amino acid changes are needed to adapt an antibody from one species to another to avoid immune rejection. using libraries of genetic information and algorithms to make sure that key amino acid sequences are recognized as "self" or "native" by the target species' immune system can reduce the chance of undesirable immune reactions. this will increase the advantages typical of mabs for potency, safety, and a prolonged elimination half-life. therefore, these second-and third-generation mabs will be at the forefront of veterinary antibody technology along with igy technology. molecular targets for therapeutic mabs, igy, and colostrum igg in animals should ( ) be involved in clinical signs or disease mechanism and ( ) not have redundant pathways compensating for blockade of the intended target. the validity of blocking a molecule or eliminating a cell type must also be weighed against the importance of this protein or cell for desirable normal body functions. it is possible to speculate on uses of these antibodies in companion animals. these might include immune-mediated hemolytic anemias/ thrombopenias, myasthenia gravis, and autoimmune blistering diseases such as pemphigus, among other conditions. for cancer, the use of mab or igy therapy targeting b-lymphocytes will be valuable for b-cell lymphomas in dogs and cats. in allergic diseases, the use of mabs to inhibit production of ige via its promoting cytokines such as interleukins il- /il- , their cytokine receptors, or ige itself might be beneficial in dogs and cats with ige-mediated atopic dermatitis or food allergies. the itch sensation itself could be altered, at least theoretically, by antibodies targeting itch-promoting cytokines such as il- , nerve growth factor, thymic stromal lymphopoietin, or neuromediators involved in itch transmission. in arthritis therapeutic mabs that inhibit pro-inflammatory cytokines (tnf-alpha, il , etc.) or their receptors are likely to be of benefit in treating dogs and cats with arthritis. the usefulness of anti-ngf mabs as an analgesic must be confirmed. in autoimmune diseases, the use of mabs specific for b-lymphocyte surface proteins could theoretically lead to reduced production of autoantibodies. veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health, through increasing safe food supplies and preventing animal-to-human transmission of infectious diseases. the continued interaction between animals and human researchers and health professionals will be of major importance for adapting new technologies, providing animal models of disease, and confronting new and emerging infectious diseases. one area of research where more information is needed is on factors that limit efficacy of vaccination ifoma, particularly in calves < month old. this is a complex issue. however, research continues to evolve in the area of newborn calf vaccinations. passive immunity provided by chicken egg antibodies will gain increasing use in production animals. there is no doubt that chicken abs can be produced and used, with minor modifications, in similar ways to mammalian abs. it can also be said that, depending on the circumstances, the use of igy abs often has significant advantage over the use of mammalian abs. however, from a realistic point of view, igy abs probably will not be able to completely replace the use of igg abs in diagnostic systems in the near future (schade et al. ) . chickens have the potential to be used to complete the spectrum of animals that have been used for ab production. however, a prerequisite is to make igy technology more popular and to convince the scientific community of its significant advantages. an interesting possibility for the future is the production of chicken mabs. these would combine the advantages of mabs with the advantages of chicken abs. it is to be expected that studies on the therapeutic or prophylactic use of igy abs will be intensified in the future. in particular, because of the increasing resistance of microorganisms to antibiotics, research on all aspects related to the development of specific igy against pathogenic microorganisms will have to be intensified. in conclusion, the next decade will see continued development of therapeutic mabs, igys, and colostrum antibodies for production and companion animals in both treatment and prevention. these highly specific molecules are likely to prove beneficial to uniquely target disease mechanisms without the side 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colostrum management and fluid therapy programs for the cow/calf operation nation-wide evaluation of quality and composition of colostrum fed to dairy calves in the united states allergens in veterinary medicine treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum man the barrier! strategic defences in the intestinal mucosa maternal antibodies: clinical significance, mechanism of interference with immune responses, and possible vaccination strategies treatment of canine b-cell lymphoma with chemotherapy and a canine anti-cd monoclonal antibody: a prospective double-blind, randomized, placebocontrolled study growth factors and antimicrobial factors of bovine colostrum colostrum and replacer ige production in rat fascioliasis sheep antiserum as an antibody supplement in newborn lambs structural and functional properties of membrane and secreted igd nutrient and immunity transfer from cow to calf pre-and post-calving chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine passive immunotherapy in neonatal calves -i. safety and potency of a j escherichia coli hyperimmune plasma in neonatal calves passive protection against porcine epidemic diarrhea (ped) virus in piglets by colostrum from immunized cows the iga system: a comparison of structure and function in different species vaccination programs in poultry clinical outcome of calves with failure of passive transfer as diagnosed by a commercially available igg quick test kit spray dried, pasteurized bovine colostrum protects against gut dysfunction and inflammation in preterm pigs immune functions of immunoglobulin y isolated from egg yolk of hens immunized with various infectious bacteria precalving vaccination programs for cows passive immunity in ontario dairy calves and investigation of its association with calf management practices egg yolk igy: protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves igy antibodies protect against human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model allergy and the gastrointestinal system ige: a question of protective immunity in trichinella spiralis infection canine brief pain inventory scores for dogs with osteoarthritis before and after administration of a monoclonal antibody against nerve growth factor comparison of antibody production to human interleukin- (il- ) by sheep and chickens vaccinating calves: new information on the effects of maternal iimmunity antibody to human rotavirus in cow's milk key: cord- -a a of authors: gupta, varsha; sengupta, manjistha; prakash, jaya; tripathy, baishnab charan title: molecular diagnostics date: - - journal: basic and applied aspects of biotechnology doi: . / - - - - _ sha: doc_id: cord_uid: a a of effective and early management of diseases requires record of the history, behavioral parameters, and travel information. these are helpful for the diagnosis, prevention, and control of the disease. there have been several advancements in the methods for diagnosing infectious diseases. the wide spectrum of tests such as biochemical evaluation, microbiological tools, immunological and molecular biology techniques, etc., is available. each type of diagnostic technique is strong and reliable in its own sense but poses certain limitations. these limitations may be complemented by using a combination of tests. older techniques such as microscopy and culturing of organisms from clinical specimens are error-free but are very labor intensive and extremely time consuming. there is a need to develop rapid and sensitive tests that can be used in both high- and low-resource settings. molecular diagnostics such as western blot, elisa, pcr, dna, and protein microarrays are revolutionizing the clinical practice of infectious diseases. their effects are significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. effective and early management of diseases requires record of the history, behavioral parameters, and travel information. these are helpful for the diagnosis , prevention, and control of the disease. there have been several advancements in the methods for diagnosing infectious diseases. the wide spectrum of tests such as biochemical evaluation, microbiological tools, immunological and molecular biology tech-niques, etc., is available. each type of diagnostic technique is strong and reliable in its own sense but poses certain limitations . these limitations may be complemented by using a combination of tests. older techniques such as microscopy and culturing of organisms from clinical specimens are error-free but are very labor intensive and extremely time consuming. there is a need to develop rapid and sensitive tests that can be used in both high-and low-resource settings. molecular diagnostics such as western blot , elisa , pcr , dna, and protein microarrays are revolutionizing the clinical practice of infectious diseases. their effects are signifi cant in acutecare settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. the diagnosis of these agents is done by using many tests either alone or in combination. these are serology-based diagnostic tools. they are more sensitive and specifi c than microscopic tests. there are two categories of these diagnostic tools that are based on antigen-detection assays and antibody-detection assays. these assays include the western blotting, enzyme-linked immunosorbent assay (elisa) , and all its derived tests such as the falcon assay screening test-elisa ( fast-elisa ), dot-elisa , hemagglutination (ha) test, indirect or direct immunofl uorescent antibody ( ifa or dfa) tests, complement fi xation (cf) test, and immunoblotting and rapid diagnostic tests (rdts) . in a western blot , the proteins present in a sample are separated according to their molecular weight by gel electrophoresis. a nitrocellulose membrane is placed on the gel, and with the help of electrical current, the proteins are transferred from the gel to the membrane where they adhere. the pattern of protein separation is maintained in the membrane after transfer. the membrane is then probed with specifi c antibodies (primary antibodies) to determine the presence of the protein. often a secondary antibody conjugated to biotin or a reporter enzyme is used to enhance the signal and detect the binding of the primary antibody . this procedure is used mainly to determine the presence of an antigen in biological sample with simultaneous determination of the molecular weight of a protein and measure relative amount of a protein present in different samples ( fig. . ). elisa is a diagnostic tool that is used in medicine and other industries to detect and quantify specifi c antigens . the sample with an unknown amount of antigen is immobilized on a solid support, usually a microtiter plate. this is done either nonspecifi cally by adsorption or specifi cally by capture by another antibody specifi c to the same antigen , in a "sandwich" elisa. after the antigen is immobilized, the detection antibody is added, forming a complex with the antigen . the detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme. the plate is developed by adding an enzymatic substrate to produce a visible signal which indicates the quantity of the antigen in the sample ( fig. . ). both western blot and elisa are used to detect hiv infection in the blood. they are called indirect tests as they measure the immune system's response to an infectious agent rather than looking for the components of the agent itself. since elisa detects hiv antibodies which the body starts to produce between and weeks after becoming infected with hiv, one should wait for at least months to confi rm for hiv aids . western blot is the most common method of testing to confi rm positive results from elisa test. it is used more as a confi rmatory test as it is diffi cult to perform and requires high skills. one advantage of western blot is that it is less likely to give false-positive results as it can effectively distinguish between hiv antibodies and other antibodies. this test uses synthetic and recombinant peptides to evaluate antibody responses to an antigen . however, this technique is subjected to the same drawbacks as most serology-based tests. antibodies raised against a peptide from one protein may cross-react with proteins from other species. moreover, antibodies raised against a antibody specifi c for a particular epitope of antigen are coated on well followed by the addition of the sample containing antigen . this results in antigen-antibody binding. binding of another antigen-specifi c antibody linked with enzyme results in color formation upon addition of the substrate . diagnosis of bacterial, viral, and parasitic diseases peptide may react in some assays but not in others as some regions of a peptide may be more immunogenic than others. in the past, the method has been applied to the study of malaria , fasciolosis, schistosomiasis, and taeniasis. lately it is not used regularly. the main difference between the regular elisa and the dot-elisa lies in the surface used to bind the antigen of choice. in the dot-elisa, the plastic plate is replaced by a nitrocellulose or other paper membrane onto which a small amount of sample volume is applied. the principle is similar to that of immunoblotting. the dotted membrane is incubated fi rst with an antigen-specifi c antibody followed by an enzyme-conjugated anti-antibody (secondary antibody ). the addition of a precipitable, chromogenic substrate causes the formation of a colored dot on the membrane which can be visually read. it is convenient to use, gives rapid results that are fairly easy to interpret, is fast and costeffective, and hence can be used in the fi eld (e.g., as a dipstick ). for all these reasons, the dot-elisa is extensively used in the detection of human and animal parasitic diseases, including amebiasis, babesiosis, fascioliasis, cutaneous and visceral leishmaniasis, cysticercosis, echinococcosis, malaria , schistosomiasis, toxocariasis, toxoplasmosis, trichinosis, and trypanosomiasis [ ] . this test is based on immunochromatographic antigen detection and has been implemented in many diagnostic laboratories as an adjunct to microscopy for the diagnosis of malaria . rdts consist of capturing soluble proteins by complexing them with capture antibodies embedded on a nitrocellulose strip. a drop of blood sample is applied to the strip and eluted from the nitrocellulose strip by the addition of a few drops of bufer containing a labeled antibody . the antigen-antibody complex can then be visualized directly from the membrane. rdts are now rapid, stable at temperatures up to °c, easy to use, and costeffective, thereby providing many advantages over traditional microscopic methods. this is a modifi ed elisa -based assay in which serum containing antigen-specifi c antibodies can be identifi ed by measuring light production. basically, an antigen of choice is fused to the enzyme reporter renilla luciferase (ruc) and expressed as a ruc-fusion in mammalian cell s to allow for mammalian-specifi c posttranslational modifi cations. the crude protein extract is then incubated with the test serum and protein a/g beads. during the incubation, the ruc-antigen fusion becomes immobilized on the a/g beads, which allows the antigen-specifi c antibody to be quantitated by washing the beads and adding coelenterazine substrate and measuring light production. some of the advantages of the lips technology include its rapidity and accuracy in detecting infected patients. sensitivity is improved in part by the use of mammalian cells which produce fusion antigens free of contaminating bacterial proteins. in addition, low backgrounds are produced compared to the elisa. monoclonal antibodies (mab) are derived from identical immune cells that are clones of unique parent cells and can bind to a specifi c epitope (for further details, refer to chap. ). they have been extensively used in biomedical and microbiological research as tools for diagnosis of diseases such as hepatitis , aids , infl uenza, herpes simplex virus infection, chlamydial infection, and treatment of cancer [ , ] . the monoclonal antibodies being directed against single epitopes are homogeneous and highly specifi c and can be produced in unlimited quantities. monoclonal antibodies have tremendous applications in the fi eld of diagnostics, therapeutics, and targeted drug delivery systems , not only for infectious diseases caused by bacteria, viruses, and protozoa but also for cancer and metabolic and hormonal disorders. in , kohler and milstein invented the hybridoma technology . the key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, fuse these cells with healthy antibody-producing b cells, and select for the successfully fused cells. in hybridoma technology , a myeloma cell rendered drug sensitive through mutation in a growth essential gene, hypoxanthine guanine phosphoribosyl transferase ( hgprt ), is chemically fused with immune cells from a host immunized with the antigen of interest, and the resulting cells are grown in medium containing the selective drug. since the immune cells have a short life span in tissue culture and the myeloma cells are drug sensitive, the only cell that will survive are those myeloma cells which obtained a normal hgprt gene from the immune cells. such cells also have a high likelihood of carrying the immune cell's antibody gene resulting in the generation of a hybridoma that can grow continuously in vitro and secrete a single monoclonal antibody ( fig. . ). the diagnosis of any infectious disease often requires the demonstration of the causative organism or presence of a specifi c antibody . specifi c antibody-based tests identify the pathogens associated with the disease. mabs recognizing unique antigenic determinants on pathogens are developed. this restricted reactivity allows for precise identifi cation of the organism of interest which is the major advantage of mabs over polyclonal antisera. in case of a pathogen occurring as subtype defi ned by unique antigenic differences, specifi c mabs can be used, whereas conventional antisera needs laborious absorption to remove cross-reactive antibodies. because of the specifi city, homogeneity, and unlimited availability of the mabs, vast amount of work has been carried out on the production/development the immuno-diagnoses of protozoan and parasitic diseases have signifi cantly been improved by mab technology because the tests involving mab as diagnostic reagents overcome the limitations of polyclonal antibodies. mabs were found to be extremely useful in the rapid outbreak of east coast fever (ecf). mabs of diagnostic value have also been developed against trichomonas vaginalis , leishmania donovani , trypanosoma congolense , and babesia bovis . development of monoclonal antibodies for the detection of mycoplasma pneumonia and plum pox virus has been reported . nucleic acid-mediated tests pcr is the most well-developed molecular technique that has not only been successfully applied for several wide-ranged clinical diagnoses but also has great potential for clinical applications, including specifi c or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profi ling. pcr-based methods may also be cost-effective relative to traditional testing procedures. further advancement of technology is needed to improve automation, optimize detection sensitivity and specifi city, and expand the capacity to detect multiple targets simultaneously (multiplexing). pcr is the most sensitive and rapid method of detecting pathogens in clinical samples. it is very useful as some of the microorganisms are not easily culturable in vitro or has a very long incubation time. under these conditions, the diagnostic value of pcr is very important [ ] . traditional pcr procedure includes amplification of specifi c genes ( fig. . ) of the microorganisms and running the product on a gel. the presence of a microbe is confi rmed by the presence of a band of appropriate size. nested, multi-plexed, and real-time pcr ( rt-pcr ) are used for effi ciency and quantitation. multiplexed pcr allows the detection of multiple sequences in the same reaction tube proving useful in the diagnosis of several infections simultaneously ( fig. . ) . rt-pcr system, unlike conventional pcr, allows for the quantifi cation of the original template's concentration through the use of various fl uorescent dyes and primers. the concentration is measured through comparison to standard curves. this eliminates the need to visualize the amplicons by gel electrophoresis, thereby greatly reducing the time, risk of contamination, and the introduction of false-positives. pcr is used to diagnose the presence of several opportunistic pathogens in the cerebrospinal fl uid of hiv patients or multiple sclerosis patients [ , ] . the viral infections that can be determined by this method are herpes simplex virus (type and ), varicella zoster virus , cytomegalovirus , epstein-barr virus , and japanese encephalitis virus. bacterial infection such as chlamydia pneumoniae is also identifi ed. mycoplasma sp. is very diffi cult to cultivate in laboratory; hence, pcr method is the only reliable method to identify the presence of the samples [ ] . dna probes consisting of cloned ribosomal rna genes, cdna to mycoplasmal rrna, synthetic s rrna oligonucleotide sequences, or cloned mycoplasmal protein genes have been developed and applied as diagnostic tools in a variety of human and animal mycoplasma infections . is a unique amplifi cation method with extremely high specifi city and sensitivity able to discriminate between a single nucleotide differences. it is characterized by the use of four different primers specifi cally designed to recognize six distinct regions on a target gene, with amplifi cation only occurring if all primers bind and form a product ( fig. . ). the reaction occurs at a constant temperature using strand displacement activity of dna polymerase [ ] . amplifi cation and detection takes place in a single step at a constant temperature ( °). it does not require expensive thermo cyclers. the corresponding release of pyrophosphate causes turbidity that is detected visually. sometimes dna-intercalating dye is also used. this has been applied for rapid detection of several dna and rna viruses such as west nile and sars virus. it has also been used for the identifi cation of several parasites. molecular-based approaches based on nucleic acids offer greater sensitivity and specifi city over the existing diagnostic tests. they permit the detection of infections from very low titer samples including those from asymptomatic patients. luminex technology is a bead-based fl owcytometric assay that allows the detection of various targets simultaneously. the microsphere beads can be covalently bound to antigens, anti-bodies, or oligonucleotides that will serve as probes in the assay. up to microspheres are available, each emitting unique fl uorescent signals when excited by laser, therefore allowing the identifi cation of different targets. this method has been successfully used for detecting cryptosporidium species. c. hominis and c. parvum has a single nucleotide difference in the microsatellite- region (ml- ) that can be identifi ed only by sequencing which is very time consuming and labor intensive. they can be detected and distinguished by this technology. however, there are several drawbacks of these methods regarding clinical samples, as pcr is susceptible to inhibitors, contamination, and experimental conditions. the sensitivity and specifi city of a pcr assay is dependent on target genes, primer sequences, pcr techniques, dna extraction procedures, and pcr product detection methods. these might not be optimal in clinical specimens such as blood, urine, sputum, cerebrospinal fl uid (csf), and others. the pcr conditions need to be carefully evaluated and the single nucleotide polymorphisms or snps (pronounced as snips) are tiny variations in an individual's genetic code. snps occur when a single nucleotide (a, t, g, or c) is substituted for another between the members of the same species or between two chromosomes of the same person. when the dna sequence of a gene differs by only one nucleotide between two individuals, they are called as alleles. snp analysis can be done in a single step by using genomic dna and pcr method ( fig. . ) . a single snp analysis can be done by using a specifi c primer attached to a fl uorescence marker, also known as a quenching probe or q-probe. when the primer binds with a specifi c dna sequence, the fl uorescence is quenched due to association with guanine residue. when it disso-ciates, the fl uorescence is acquired. when the primer binds to the wild-type allele, the dissociation occurs at a higher temperature, whereas in a mutant allele, the binding is weak and dissociation takes place at a lower temperature. this change in dissociation curve is analyzed. two different colors can be used for multiplex analysis . snps occur due to mutation, recombination, and natural selection . snps may occur in coding region of genes, in noncoding regions of genes, or in intergenic regions. they are classifi ed into different categories. due to degeneracy of genetic code, the amino acid sequence of the polypeptide might not change. these are known as synonymous polymorphism. when the changes produce different polypeptides, they are known as non-synonymous or replacement polymorphism. this may result in missense mutation, where a different amino acid is produced, or nonsense mutation, where there is a premature stop codon. lot of disease mutations are caused by replacement polymorphism. snps occurring in noncoding region might affect gene splicing, transcription factor binding, m-rna degradation, or mutate noncoding rna. about . % of dna sequences are identical between individuals of same species. out of . % variation, around % is due to snps. thus they bring about diversity among individuals. this trait is used for dna fi ngerprinting in forensic science. several diseases are caused by genetic variations in an individual. genetic factors are responsible for susceptibility and disease progression. snp profi le or haplotype associated with a disease trait may reveal relevant genes associated with a disease state. it provides understanding of many polygenic diseases. in future there are chances that by viewing the snps profi le of an individual, the physicians might be able to fi nd out the risks associated and plan a personalized medicine. snps help in determining the likelihood of a person to develop a particular disease. one of the genes associated with alzheimer's disease in apolipoprotein e or apoe . it contains two snps that result in three possible alleles for this gene: e , e , and e . each allele differs by one dna base, and the protein product of each gene differs by one amino acid. each individual inherits one maternal copy of apoe and one paternal copy of apoe . a person who inherits at least one e allele has a greater chance of developing alzheimer's disease, whereas inheriting the e allele reduces the likelihood of developing alzheimer's. snps are not absolute indicators of disease development. apoe is just one gene that has been linked to alzheimer's. like most common chronic disorders such as heart disease, diabetes , or cancer, alzheimer's is a disease that can be caused by variations in several genes. the polygenic nature of these disorders is what makes genetic testing for them so complicated. protein microarrays are tools that can be used in both translational as well as basic research. protein chips can be used for a variety of applications including identifi cation of protein-protein interactions, protein-phospholipid interactions, and substrates for protein kinase. they are used for clinical diagnosis and disease state progression. they can be used to phenotype leukemia cells, identify new protein-protein interactions, screen entire proteomes, and profi le hundreds of patient samples. several arrays are available for specifi c use. they have been graphically represented in fig. . . some of them are discussed here: these are high-density arrays and are used to identify novel proteins and protein-protein interactions ( fig. . a ). the array library is usually a high-density expression library and the probes are either directly labeled with fl uorophores or are tagged with labeled antibodies. these are used for quantitative profi ling of protein expression in clinical samples and cell culture ( fig. . b ). these are low-density arrays. known antibodies are arrayed to capture antibodies from unknown samples. the antigens are either labeled directly or are attached to a secondary antibody . the latter gives a sandwich assay similar to elisa . it consists of multiple known antibodies arrayed on a solid surface (fig. . c ). it is used to profi le the presence of known antigens from a pool of samples. normal and disease samples are used. they are either labeled directly or with haptens . these are used to detect autoantibodies in clinical and research samples. these are low-density arrays and are probed with serum or plasma ( fig. . d ). reverse arrays are used to probe hundreds of samples to detect the presence of few antibodies. cell lysates, plasma, and serum are arrayed and are probed with few known antibodies. this is an alternative strategy where samples containing several proteins are arrayed on slide and probed with labeled antibodies. level of number of proteins can be measured simultaneously. this is used to identify novel protein-binding motifs and protein-protein interactions (fig. . e ) . engineered proteins and peptides proteomic studies can provide substantial information about clinical state of a disease as they are the fi nal molecular machines of biological processes. they can be used as biomarkers for disease states. diagnostics use protein and peptide biomarkers from body fl uids. all proteomicbased diagnostic efforts seek to identify biomarkers that, alone or in combination, can distinguish between "case" and "control" groups. this can be done in several ways. profi ling and identifi cation of the protein this is a method to identify proteins and peptides in their natural form. here the proteins are resolved in the fi rst dimension based on ph (a process called isoelectric focusing) and in the second dimension by their molecular weight. this technique is labor intensive. this is an analytical technique where mass-tocharge ratios of particles are measured. it is used to determine the composition of peptides. proteins from body fl uids can be proteolytically cut into small pieces. they are ionized usually to cations by removal of electron. these charged particles are then separated according to their charge and mass. the separated ions are measured and displayed. the resulting spectra can be compared with other peptides in the data base ( fig. . ). but in this approach it is diffi cult to quantitate and study the protein modifi cations. it is a relatively novel technique in which a coprecipitate of a uv light-absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. the ionized biomolecules are accelerated in an electric fi eld and enter the fl ight tube. during the fl ight in this tube, different molecules are separated according to their mass-tocharge ratio and reach the detector at different fig. . the usage of proteomics approach for diagnostics or profi ling. ( ) first dimensional isoelectric focusing (ief) gel is used to separate the sample components according to their isoelectric point. ( ) second dimensional sds-page is used which further separates the proteins according to their molecular mass. sample spots obtained are isolated and prepared for application in mass spectrometer (ms). ms consists of ionization device as maldi or esi and mass sorting device as tof or quad and detection is done by a detector. after peptide mass fi ngerprint is obtained, it is analyzed through comparing the experimentally determined peptide mass fi ngerprint with known and virtual mass fi ngerprints using bioinformatics tools times. in this way each molecule yields a distinct signal. the method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides, and oligonucleotides, with molecular masses between and , da. maldi-tof is used for identifying bacterial strains in clinical microbiology laboratories. the development of automated, highthroughput proteomic technologies such as ms and maldi-tof has enabled large numbers of clinical samples to be analyzed simultaneously in a short time. these platforms have made "population-based proteomics" feasible for the fi rst time. with the use of naat , it is now possible to have many copies of target dna and the technique has advantage of being sensitive, specifi c, and rapid. it targets the conserve region of the target species. the naat test may be planned which would be able to detect single species, strain, or resistance-inducing mutation. using broadspectrum probes, the broad categories of the organism may be detected. naat has been successfully used in the diagnosis of infective endocarditis as compared to culture technique even when culture reports were negative. in patients with negative sputum smears, the tests based upon naat were quiet useful in the clinical diagnosis of tuberculosis . [ ] . it might cause potential discrimination regarding social acceptability, job or employment availability, and health insurance coverage. prenatal testing for genetic disorder may lead to abortion of a fetus. carriers of genetic mutations ethically should disclose the fact to their life partner or their siblings. but he or she might face social isolation. he or she might not be able to marry and start a family. similarly if a person is at risk of a late onset of a genetic disorder, the employer might not be willing to hire him or her. the health insurance companies would not want to pay for the medical expenses or might increase the premium [ , ] . one should also keep in mind that genetic testing cannot give all the answers. for example, it cannot tell about the exact time of onset, penetrance, or person to person variation of a disorder. there are several issues regarding the ethical consideration of genetic testing. until and unless there are clear laws to protect the individuals, privacy and confi dentiality of genetic information should always be protected and individuals wish to be tested or not should be respected [ ] . • biotechnology has played a very important role in diagnosis and treatment of various bacterial, fungal, viral, and parasitic diseases. it . chapter end summary has also helped in identifi cation of early stages of cancer. • the advancement in molecular techniques has helped in identifi cation of biomarkers that signifi es early development and progress of a disease. • the various tests like serology-based tests and nucleic acid-based tests are diagnostics, but preliminary data from traditional microbiology-based methods are also helpful. q . how has the advancement in biotechnological techniques helped in diagnosis of the diseases? q . discuss a few serological tests for the diagnosis. q . what is the importance of pcr in pathogen detection? q . how are the proteomic assays helpful in aiding diagnostics? q . discuss dna microarray technology. q . what is maldi-tof? bioethics in india, proceedings of the international bioethics workshop in madras: biomanagement of biogeoresources polymerase chain reaction on cerebrospinal fl uid for diagnosis of virus-associated opportunistic diseases of the central nervous system in hiv-infected patients ethical issues in genetic testing diagnosis of parasitic diseases: old and new approaches. interdisciplinary perspectives on infectious diseases diagnostic testing and the ethics of patenting dna ethics of genetic testing: medical insurance and genetic discrimination monoclonal antibodies in clinical diagnosis: a brief review application dna probes and pcr in diagnosis of mycoplasma infections monoclonal antibodies as diagnostics; an appraisal loopmediated isothermal amplifi cation (lamp) of gene sequences and simple visual detection of products pcr in diagnosis of infection: detection of bacteria in cerebrospinal fl uids pcr-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings key: cord- -bvihkf r authors: hurd, eric r.; dowdle, walter; casey, helen; ziff, morris title: virus antibody levels in systemic lupus erythematosus date: - - journal: arthritis rheum doi: . /art. sha: doc_id: cord_uid: bvihkf r antibody titers to a group of viral antigens have been determined in sera from patients with systemic lupus erythematosus (sle), control groups with inflammatory diseases and normals. mean titers in sle sera for all viruses tested were significantly greater than in four control groups, but not greater than in active tuberculosis, both by the complement‐fixation (cf) and hemagglutination‐inhibition (hi) methods. by the cf method, only measles virus showed significantly higher titers in sle than in all control groups; by the hi method, measles antibody titers were higher in sle than in all groups but tuberculosis. there was no correlation between antibody titers and gammaglobulin levels. the results indicated a moderate though variable overall hypereactivity in sle to the viral antigens tested. lar in appearance to those seen in the brain tissue of patients with subacute sclerosing panencephalitis ( - ). in this condition, elevated measles antibody titers have been consistently demonstrated ( , - ), and measles virus, a myxovirus, has been isolated from brain tissue of patients with this disease ( - ). because of the morphologic similarities of the tubular structures seen in sle to those of the myxoviruses ( ) ( ) ( ) , it seemed appropriate to measure virus antibody levels in patients with sle, with particular emphasis on the myxovirus group. recent studies ( ) ( ) ( ) have reported elevated antibody levels to several viral antigens. in a preliminary study in this laboratory ( ) of viral antibody titers in patients with lupus nephritis and matched normal controls, complement fixing antibody titers were significantly elevated to a number of myxoviruses, coronavirus oc and herpes simplex virus. by the hi technic, significant elevations were observed in parainfluenza and measles viruses, both myxoviruses. t h e overall trend of antibody titers in the sle group as compared with normal controls was highly significant both by the cf and hi tests. in the present study, antibody titers have been studied by the same technics in patients with sle and normal individuals. in addition, control studies have been carried out in groups of patients with a variety of chronic inflammatory diseases. sera were collected from patients who had welldocumented histories of sle with nephritis and who were being followed in the parkland memorial hospital arthritis clinic. the average age of the patients with sle was years ( to ). all had active disease. seventeen were receiving prednisolone; the average daily dosage in the patients was . mg ( to mg). all patients were ambulatory. seventeen were black and white. control sera were obtained from: a) age-and sex-matched hospitalized patients with far-advanced tuberculosis; all were receiving inh, were black (b), white (w) and latin american (la); antinuclear fluorescence tests were negative in all; b) patients with rheumatoid arthritis (ra) ( b, w, la); c) patients with bronchial asthma ( b, w); d) patients with miscellaneous diseases, including with degenerative joint disease, with polymyositis, with chronic alcoholism, with gout and with psoriasis ( b, w, la); and e) normal individuals ( b, w). antibody titers were determined in the laboratories of the virology section of the center for disease control, us public health service, atlanta, ga. the complementfixation microtiter technic ( ) was used to determine antibody titers to the following myxovirus antigens: purified ribonucleoprotein (soluble antigen) of mumps virus and influenza virus types a and b; and unpurified whole-virus preparations of influenza virus type c, parainfluenza virus types , and , mumps virus, measles and respiratory syncytial virus. complement fixation tests were also performed with an rna coronavirus (infectious bronchitis virus-like virus strain oc ) and adeno and herpes simplex viruses, both dna viruses. antibody titers were also determined by the hi technic ( ) to the following an-tigens: parainfluenza virus types , and , mumps virus, measles virus and coronavirus oc . all patients with sle had positive antinuclear fluorescence (anf) and le cell tests. twenty-two of the ra patients had positive sensitized sheep cell agglutination (ssca) tests (range, to ). three of the patients with sle and of the tuberculosis group also had positive ssca tests. the patients in each group were tested for individual viruses, two groups at a time using the following tests: a) to compare patients with sle to those with tuberculosis subjects were matched according to age, and the observed differences for each pair were analyzed using the wilcoxon signed-rank test ( ); b) for comparison of other groups, the wilcoxon rank-sum test ( ) was also used, except the subjects were not matched. in cases where sample values were "tied,"-ie, appeared more than once, a test of equal binomial proportions ( ) or fisher's exact probability test ( ) was applied. correlations between -globulin level and individual virus titers were computed. when the sample size n exceeded , these were tested against the null permutation distribution ( ) . spearman's p was tested when n was . since the discrete nature of the data rendered methods based on normally distributed variables inappropriate, only distribution-free tests were applied. geometric mean viral antibody titers measured by the cf test are shown in table . the average titer for each group is also shown. similar titers measured by the hi test are shown in table . by the cf test (table ) , only measles virus showed significantly higher titers in sle than in all the control groups. respiratory syncytial virus showed elevated titers with respect to of the control groups and in the case of the other viruses the eleva-tions were scattered. when compared with the normal group, patients with sle were higher for parainfluenza , measles, herpes simplex and respiratory syncytial viruses. by the hi test (table ) , none of the six viruses studied showed a significantly higher antibody titer in sle than in all the control groups examined. in the case of four of the vi- statistically significant correlations between these levels and viral antibody titers. when gammaglobulin level in the patient groups was correlated with antibody titers for each of the viruses tested by the c f method, only four of the possible correlations of antibody titer with gammaglobulin level were significant at the . level. among the possible correlations by the h i method, only one was significant at the . level. no instance of a significant correlation between gammaglobulin level and antibody titer occurred in the sle group. in the present study, an attempt has been made to compare viral antibody titers in sle with those of control groups with inflammatory diseases as well as normal individuals. the data obtained demonstrate that patients with sle have elevated antibody titers to a number of viruses when compared with certain control groups. however, with the exception of measles virus, there was no predilection for increased antibody response in this disease to any one specific virus among those tested. of all viral antigens tested by the c f test, only measles antibody levels were significantly higher in sle than in all of the control groups. however, by the h i method, the measles antibody titer was not significantly higher than in the tuberculosis group though it was elevated with respect to the other control groups. when geometric mean titers of all viruses tested were averaged for each patient group, the overall mean titer was higher in the sle group than in of the control groups by both the c f and h i methods. however, it was not significantly higher in the tuberculosis group by either test. except for the comparison with the tuberculosis group, it would appear that there is an overall hyperreactivity to viral antigens in the patients with sle when compared with control groups with inflammatory disease and with normal individuals. it was of considerable interest that patients with far-advanced tuberculosis formed the only group with respect to which the patients with sle did not have an overall increase in viral antibody response. a high frequency of autoantibodies, particularly antinuclear antibodies and rheumatoid factor, has recently been reported by lindquist, coleman and osterland in patients with chronic pulmonary tuberculosis ( ). these authors suggested that factors such as chronicity of inflammation, tissue breakdown and the adjuvant effect of mycobacteria may play a role in the production of these autoantibodies. injection of freund's complete adjuvant ( ) has, in fact, been shown to hasten the development of coombs positive hemolytic anemia in nzb mice. thus, the relatively high viral antibody titers seen in tuberculosis could be secondary to an adjuvant effect of the mycobacterial infection. it is unlikely that inh, which all of these patients were receiving, contributed to the antibody response since antinuclear antibody tests were negative in all of the patients in this group. the relatively elevated viral antibody titers observed in tuberculosis raises the question of the role of an adjuvant effect in the response of patients with sle to viral antigens. there is some evidence that patients with sle may have defective cellular immunity ( - ). several lines of evidence for the presence of diminished cellular immunity have also been described in nzb and nzb-nzw f, hybrid mice. more-over, the development of autoantibodies in mice has been correlated with a deficient thymusdependent cellular immune system ( , ). diminished cellular immunity could facilitate chronic infection with a passenger virus, which might in turn exert an adjuvant effect on viral antibody formation. viral infections have, in fact, been shown to enhance the formation of autoantibodies ( - ). a variety of antibodies to polynucleotides (native dna, single-stranded dna and single and double-stranded rna) have been found in sera from patients with sle ( , ) . this has led to the speculation that these antibodies may have originated in response to viral infec- it should be pointed out that the viral antibody titers of the patients with ra measured in the present studies by the cf test might be falsely low because serum rheumatoid factor may exert a n inhibitory effect on c f ( ). this possibility cannot be ruled out. however, similarly decreased antibody titers were also obtained by the hi method, which is not dependent on the complement system. t h e present study, using conventional serodiagnostic tests and common viral antigens, does not elucidate the nature of the cytoplasmic myxovirus-like tubular structures in the tissues of patients with sle. however, it is possible that the tubular structures are evidence of chronic infection in s l e with a passenger virus of a type which might act as an adjuvant for the overall elevation in viral antibody titer observed. fresco r: tubular (myxovirus-like) structures in glomerular deposits from a case of lupus nephritis systemic lupus erythematosus and myxovirus (correspondence) et all cytoplasmic tubular structures in kidney biopsies in systemic lupus erythematosus endothelial inclusions in active lesions of systemic lupus erythematosus ultrastructural observations regarding their possible viral nature light and electron microscopic observation on the development of viral inclusions of dawson's encephalitis (subacute sclerosing panencephalitis) evidence of a pseudomyxovirus in the brain in subacute sclerosing leucoencephalitis additional evidence of the relation between subacute inclusion-body encephalitis and measles virus myxovirus as a possible etiologic agent in subacute inclusion-body encephalitis measles virus antibody and antigen in subacute : - virus antibodies in subacute sclerosing panencephalitis: study of patients kolar : reflections on the etiology and pathogenesis of subacute sclerosing panencephalitis isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis subacute sclerosing panencephalitis: propagation of measles virus from brain biopsy in tissue culture subacute sclerosing panencephalitis: structures resembling myxovirus nucleocapsids in cells cultured from brains subacute sclerosing panencephalitis: isolation of measles virus from a brain biopsy the structure and composition of the myxovirus. . electron microscope studies of the structure of myxovirus particles by negative staining techniques immunoelectron microscopy of the morphogenesis of mumpsvirus rapid laboratory diagnosis of paramyxovirus infections by electron microscopy myxovirus antibody increases in human connective tissue disease raised antibody titers to eb virus in systemic lupus erythematosus antibodies to viral antigens in systemic lupus erythematosus virus antibody levels in systemic lupus erythematosus united states department of health, education and welfare, united states public health service, washington, dc . viral and rickettsia infections statistical methods in medical research t h e advanced theory of statistics autoantibodies in chronic pulmonary tuberculosis development of coombs positive haemolytic anaemia in nzb mice injected with freund's complete adjuvant immunological reactivity in patients with systemic lupus erythematosus delayed hypersensitivity in systemic lupus erythematosus in vivo and in vitro study of cell-mediated immunity during the onset of systemic lupus erythematosus autoimmune phenomena and renal disease in mice. role of thyrnectomy, aging and involution of immunologic capacity response of nzb and nzb/nzw spleen cells to mitogenic agents t h e effect of induced chronic viral infection on the immunologic diseases of new zealand mice cryoglobulinemia in infectious mononucleosis antinuclear antibodies in infectious mononucleosis immunologic abnormalities induced by postperfusion cytomegalovirus infection inhibition of cellular immunity and enhancement of humoral antibody formation in mice infected with lactic dehydrogenase virus antibodies to polynucleotides in human sera: antigenic specificity and relation to disease antibodies to ribonucleic acid in systemic lupus erythematosus histocompatibility antigens associated with systemic lupus erythematosus the pathogenesis of autoimmunity in new zealand mice induction of antinucleic acid antibodies by polyinosinic-polycytidylic acid tolerance to polyinosinic-polycytidylic acid in nzb/nzw mice comparison of the immune responsiveness of nzb and nzb x nzw f, hybrid mice with that of other strains of mice relationship between anti-viral antibodies and rheumatoid factor in pregnant women we are very grateful to dr campbell read for the statistical analysis of the data, and to dr g . hurst and mr vincent steingrube at the state tuberculosis hospital at tyler, texas, for supplying sera from patients with faradvanced tuberculosis. key: cord- -lagv tp authors: hooft van huijsduijnen, rob; kojima, somei; carter, dee; okabe, hisafumi; sato, akihide; akahata, wataru; wells, timothy n. c.; katsuno, kei title: reassessing therapeutic antibodies for neglected and tropical diseases date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: lagv tp in the past two decades there has been a significant expansion in the number of new therapeutic monoclonal antibodies (mabs) that are approved by regulators. the discovery of these new medicines has been driven primarily by new approaches in inflammatory diseases and oncology, especially in immuno-oncology. other recent successes have included new antibodies for use in viral diseases, including hiv. the perception of very high costs associated with mabs has led to the assumption that they play no role in prophylaxis for diseases of poverty. however, improvements in antibody-expression yields and manufacturing processes indicate this is a cost-effective option for providing protection from many types of infection that should be revisited. recent technology developments also indicate that several months of protection could be achieved with a single dose. moreover, new methods in b cell sorting now enable the systematic identification of high-quality antibodies from humanized mice, or patients. this review discusses the potential for passive immunization against schistosomiasis, fungal infections, dengue, and other neglected diseases. the last three decades have seen a dramatic rise in the use of monoclonal antibodies (mabs) as therapeutics. by , a total of antibodies had been approved by the us food and drug administration (fda) or the european medicines agency (ema) [ ] , with a further approved by the fda in [ , ]. beyond this, over antibodies are in clinical development [ ] . the global mab market reached us$ billion annually in [ ], underscoring the considerable economic importance of these medicines. the success of mabs starts with the general applicability of the technology used to make them. antibodies can be developed that have not only high affinity for their targets but also high selectivity, meaning they are less likely to have unwanted side effects and unexpected safety problems. mabs are particularly good at targeting cell-surface proteins and circulating protein factors; this is in contrast to small molecules, in which cell surface protein-protein a a a a a in infectious disease, the biggest success story has been the use of antibodies to provide prophylaxis against infection by respiratory syncytial virus (rsv). rsv is responsible for over million episodes of new lower respiratory tract infections, particularly targeting children years and younger, resulting in an estimated , to , deaths globally ( estimates) [ ] . palivizumab, dosed at mg/kg, is given as a monthly intramuscular injection throughout the rsv season, which is suitable for small babies but would not be appealing for adults. a follow-on antibody-medi , now in phase ii clinical studies [ ]-is times more potent than palivizumab in vitro. it is being developed as a single mg injection to cover the typical -month season. taken together, these cases illustrate, that after initial proof of concept, it is possible to find second-generation antibodies with more simple routes of delivery and sufficient potency to support relatively infrequent dosing in the field. a major objection to the use of mabs in infectious diseases as a therapeutic class is their high price, which is presumed to be a consequence of a high cost of goods. manufacturing costs are a particularly important attribute for any medication that is developed for diseases of poverty in low-and middle-income countries (lmics). however, production efficiency of mabs has increased dramatically over recent decades, and cell-culture expression levels around g/l or even higher are common. a published analysis from a decade ago already noted that the costs of mab production were dropping from us$ /g to us$ /g when being produced at the -tonne-per-year scale, potentially using large, , l reactors [ ] . a more recent analysis has similar estimates [ ] , which-depending on process and volume-range from us$ /g to us$ /g [ ] . for a public health application against an infectious disease in an lmic, an intramuscular or subcutaneous delivery would be preferable (rather than the intravenous route). the intramuscular route effectively limits the dose; an injection volume of . ml is probably close to the maximum acceptable in small children, and antibodies are typically only soluble to around mg/ml, which gives a limitation on the total dose of around mg, corresponding to a range of to mg/kg [ ] . taken together, the cost of goods of an injection based on current numbers would be in the range of us$ to us$ . benchmarking such costs is difficult, but as a comparison, the recently launched malaria vaccine provides less than % protection for injections at around us$ each. the programmatic cost of protecting a child from malaria for a year in the sahel using existing low-cost oral medicines has been estimated at us$ . by unitaid [ ] . an injectable therapeutic that could protect a child for a season for this price provides a cost-effective option. the other important factor to manage is the reduction of the frequency of administration, to make the prophylaxis useful in resource-poor settings. mabs used in therapy are generally iggs (immunoglobulin gs), which have plasma half-lives of to days in humans [ , ] . such antibodies can be used to provide several months of protection, as in the case of the once-per-season anti-rsv antibody medi (nirsevimab) discussed earlier or the anti-calcitonin gene-related peptide (anti-cgrp) fremanezumab, recently approved for prevention of migraines with a dose of mg every months. an alternative to simply increasing the dose would be increasing the half-life of the circulating mab. several strategies have been proposed here [ ] : mutations can be made in the igg fc (fragment crystallizable) region, which interacts with the receptors responsible for the uptake of antibodies. such mutations, as for medi , can significantly extend an mab's plasma half-life. mutations derived from the neonatal fc receptor (fcrn) increase the strength of the interaction between the fc region and the fc receptors under the acidic ph conditions in lysosomes, and as a result, the receptor-fc complex is recycled back to the cell surface, escaping degradation [ , ] . this approach was followed for bevacizumab and cetuximab, by mutating met to leu and asn to ser. discussion about even longer periods of protection has been stimulated by recent results from virally mediated expression of antibodies targeting hiv [ ] . these technologies hold out the promise of sustained serum titers of antibodies for more than a year from a single injection, although the technological development is more complicated than for the direct injection of antibodies. one final objection to the goal of administering monoclonals is that vaccination would instead provide lifelong coverage. however, for some diseases, the development of long-lasting potent vaccines still remains elusive. many pathogens will not raise an adequate immune response, due to immunosuppression. finally, antibodies give immediate protection, important during rapidly evolving epidemics or pandemics and for individuals being deployed into zones of high transmission. antibodies would thus form part of the overall armamentarium, along with drugs, bed nets and insecticides, and vaccines. first and foremost for the development of mabs is the question of suitable antigen targets. viruses are the simplest pathogens, and their genomes allow limited means to evade host immunity; therefore, passive and active vaccinations have been most successful in this area. therapeutic antibodies have been developed and marketed for respiratory syncytial, varicella zoster, vaccinia, and hepatitis b viruses (hbvs) [ , ] . historically, immune responses against viruses were considered purely cellular and those against other types of pathogens mostly humoral (antibody based) and less effective. however, nowadays the interplay between both systems is better understood, for instance, with therapeutic opportunities against viruses such as rabies that rely on passive immunization. mabs that target the initial stages of an infection may provide prophylaxis, and the repertoire of potential antigens for such approaches is greatly helped by the increased availability of genomic and transcriptional information. passive immunization is especially effective in infections, such as those with the rabies virus, which avoids apoptosis of infected neurons while killing protective t cells so that carriers do not mount an immune response themselves [ ] . a recent review [ ] lists mabs (and a nanobody, a single-chain type of antibody produced by camelids) that are currently in clinical trials in the united states. in addition, polyclonal antibodies have been approved in the european union or the us for cytomegalovirus, hepatitis viruses a and c, and postexposure prophylaxis against measles, rabies, rubella, and tetanus [ ]. these approaches appear most suitable for virus infections and for protection against the consequences of snake venom but might also be applied to other types of disease. there are fewer marketed antibodies that target bacteria, and these target toxins, namely anthrax and clostridium cytotoxin [ ] . this paucity may be due to a lack of discovery efforts, a consequence of the historic availability of cheap and effective antibiotics. however, with the worrying rise of antibiotic resistance, the tide is turning [ ] . immunization against protist parasites and fungi is generally ineffective. one problem is that organisms such as trypanosoma brucei (which causes sleeping sickness) and plasmodium (malarial parasites) encode dozens or even hundreds of different surface antigens that are sequentially exposed at their most abundant stage, outpacing the host's immune system. for some pathogens, clinical trials in infected patients are not possible or desirable. in the case of inhalational anthrax, raxibacumab has been approved using the fda "animal rule" [ ]. this provides a route forward in disease areas in which trials with infected human patients are not possible or not ethical. recently, the case for discovering mabs for neglected tropical and other infectious diseases was reviewed [ ] . this work took a broad, category-wide approach (viruses, bacteria, parasites) and also included venoms. snake bites, and the challenge of envenoming, represent a serious health problem, resulting in , to , deaths yearly [ ] , and were recently included in the world health organization (who) list of neglected tropical diseases. the discovery of mabs in this area logically follows the long-standing therapeutic successes with passive immunization [ ] , and the costs and feasibility of developing mabs were recently reviewed [ , ] [ ], including specific clinical development and regulatory challenges [ ] . in this review, we selected diseases prioritized by the global health innovative technology (ghit) fund, an international collaboration between the public and private sectors, supporting collaborations between japanese and non-japanese entities to advance global health research and development [ , ] . the present vaccine discovery statuses for these diseases are listed in table . human schistosomiasis is caused by infection, mainly with one of species of the blood fluke belonging to schistosomatoidea. it is estimated that there are over million cases annually in africa, with two-thirds being schistosoma haematobium and the remainder s. mansoni. in southeast asia, other species such as s. japonicum and s. mekongi cause smaller numbers of infections [ ] . the mortality associated with schistosomal infections is difficult to calculate. comorbidities linked to anemia are common, and infection with s. haematobium can lead to female genital schistosomiasis, which results in increased susceptibility to hiv infection [ ] . the life cycle of the parasite is complex, involving both a snail and a human host. in the infective stage, cercariae migrate and penetrate the human host skin to become schistosomula, which then move with the venous circulation, initially to the lungs. after further maturation, the parasite migrates via the heart to the portal circulation and the intestines; from there, eggs are excreted with the feces or, passing through the bladder wall, in urine. prophylaxis could therefore either be causal-and prevent entry and survival of the cercariae-or suppressive, by killing the juvenile and adult worms. clearly, as with the malarial parasites, the more stages of the parasite life cycle that can be inhibited, the more effective the protection will be. the molecular targets best addressed by antibodies are still not clear. some life cycle differences can be characterized: schistosomula recovered from the lungs are elongated and more resistant to antibody-dependent, cell-mediated cytotoxicity than newly transformed schistosomula. praziquantel, the standard treatment for schistosomiasis, acts only against adult worms and has no effects on the juveniles, thus repeat treatment is required to eliminate all the worms in a patient. an ideal treatment would therefore kill both adult and juvenile worms at a submicromolar concentration. this is effectively the target candidate profile for a small molecule inhibitor [ ] . no target-product profile (tpp) for prophylaxis against schistosomiasis has been published, but it is possible to start to build one, with the recent characterization of praziquantel for such use in murine models and in pediatrics. clinically, this treatment reduces infection by % to % using doses of to mg/kg [ ] . in a murine model with infection by s. mansoni cercariae, the maximum dose of mg/kg killed % of all adult worms [ ]. however, this murine dose produced a peak exposure of ng/ml, some -fold higher than the exposure observed in children at the recommended regimen of to mg/kg [ , ] . it seems pragmatic to project that an antibody that can reduce the worm burden by % in the mouse model, at a dose that is clinically achievable in humans, could be acceptable. the maximally practicable injection of an antibody in children is to mg/kg, and given the relatively consistent allometry between humans and mice for mabs, this corresponds to a dose of to μg in mice [ ] . target identification for schistosomiasis is not straightforward. unlike malaria, the vaccine field offers no clues as to useful antibody targets. two vaccine candidates are currently being tested in humans. the first is based on the fatty acid-binding protein (fabp) sm- (schistosoma mansoni- ), which is being tested in small, open-label pilot studies in senegal (nct and nct ). a larger -participant trial in uganda, nct , is being planned for a vaccine based on sm-tsp- (schistosoma mansoni-tetraspanin- ) but is not expected to report before . clues about other possible ways forward, in terms of antigen selection, come from a variety of areas.first, schistosoma are helminths (or flatworms), and the initial immune response is a characteristically strong t helper (th ) cytokine reaction, which is not seen in viral or bacterial infections and is less pronounced in protozoan infections. the cytokines that are released include interleukin (il)- , which drives the production of eosinophils to attack the parasite, and il- and il- , which drive the production of immunoglobulin e (ige). epidemiological studies in endemic areas suggest that an age-dependent immunity may develop against infection, or against reinfection after treatment [ - ]. there is a good correlation between this protection and the development of ige antibodies, resulting from th responses [ , ] . a hybridoma that produces a monoclonal ige antibody to s. japonicum, sj ε. , was identified [ ]. this mab was protective in an in vitro, antigen-dependent, cellular cytotoxicity assay with rat macrophages or eosinophils and also in vivo during the early phase of infection second, beyond the cell-surface proteins, schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and an antibody response against those glycans is mounted by the infected host [ ] . in cases, the specific antibodies produced could be identified using b cell cloning [ ] or after infection of mice genetically modified to express human antibody repertoires [ ] . interestingly, there has been no systematic analysis of the antibodies produced that target the various stages of the parasite life cycle, although early work on s. mekongi suggests schistosomula and adult worm extracts would induce a better response [ ] . in addition to stimulating a th response, schistosome infection can suppress t-cell activation [ - ]; s. mansoni uses distinct mechanisms to suppress t-cell activation, resulting in the selective up-regulation of pd-l on the surface of splenic f / + macrophages. the presence of fatigued or anergized t cells opens up the possibility for cotherapy of low doses of mabs against programmed cell death protein- (pd- ) or pd-l with antibodies that reverse the anergy. the treatment of invasive fungal infections (ifis) remains a major challenge worldwide. there are few broad-spectrum antifungal drugs; the most effective have substantial toxicity concerns, and well-tolerated drugs used prophylactically frequently induce resistance. even with the best current treatment, the risk for mortality due to an ifi can be higher than %. for low-income countries, this figure is substantially worse, as many invasive infections are uniformly fatal without treatment, and estimates of global mortality involving fungal infections are as high as . million annually [ - ]. in addition, fungal infections are the cause of significant comorbidity and mortality in hiv patients. modeling studies suggest that optimal therapy could save the lives of . million hiv patients over a -year period [ ]. the discussion here will focus on mabs developed for the following significant fungal pathogens: cryptococcus, pneumocystis and paracoccidioides, and candida. in addition to antibodies that directly target and inhibit the fungal pathogen, mabs can be directed to checkpoints that control the host immune response. this approach may be particularly useful against fungal pathogens for which sustained infection is characterized by a shift from a protective th or th response to a noninflammatory th response. the clinical use of anti-il in rheumatoid arthritis clearly exacerbates fungal infection [ ], although it remains to be seen whether the reverse-stimulating this pathway-has a clinically useful effect. cryptococcus neoformans predominantly causes opportunistic infection in patients with hiv/aids and is responsible for a large burden of aids-related disease and death in sub-saharan africa [ ] . although the rate of infection has decreased in recent years through greatly improved access to antiretroviral therapy (art), mortality in infected patients has not declined, demonstrating the failure of antifungal development to keep pace with improved antiviral treatment. cryptococcosis first manifests as a pulmonary disease but spreads hematogenously to the cerebrospinal fluid and brain to cause meningitis and meningoencephalitis, adding the complexity of crossing the blood-brain barrier to drug development. the ability of mabs to protect against a lethal cryptococcal infection in mice was first demonstrated over three decades ago [ ], with subsequent work demonstrating the efficacy of various mabs that target the polysaccharide capsule, an essential virulence factor and the primary host-pathogen interface of cryptococcus infection [ ] [ ] [ ] . among these, mab b was evaluated in a phase i clinical trial [ ] and was found to produce a modest reduction in circulating cryptococcal antigen. further development has been hampered, however, by difficulties in securing funding for a disease for which financial returns are likely to be low-a common problem for neglected infectious diseases. subsequent studies have examined mabs that target cryptococcal melanin [ ] , β-glucan [ ] , or glucosylceramide ( [ ] [ ] [ ] ; see [ ] for additional in vivo and in vitro examples). host cd has also been targeted to stimulate the immune response [ ] . in addition to highlighting the potential of mabs as therapeutics, these studies have demonstrated the diversity of inhibitory actions that mabs can perform on cryptococcal cells, which can include opsonization and increased phagocytosis, inhibition of fungal growth, capsular polysaccharide release and biofilm formation, antibody-mediated target cleavage, and augmentation of the host response [ ] [ ] [ ] [ ] . pneumocystis, like cryptococcus, is an important opportunistic pathogen in hiv/aids and other immunosuppressed patient populations, with estimates of up to , cases per year [ ]. however, unlike cryptococcus, which is acquired from the environment, pneumocystis spp. are commensals, with different species occurring in the lungs of many mammals. the capacity for endogenous infection and for acquisition from asymptomatic carriers makes pneumocystis an attractive target for immunoprophylaxis. to this end, a variety of programs are aimed at developing a pneumocystis vaccine [ , ] , and passive immunization studies have been initiated using mabs raised to pneumocystis epitopes. intranasal administration of f , an mab that recognizes the pneumocystis kexin-like protein kex , was able to prevent transmission of pneumocystis pneumonia from infected to susceptible cohoused mice, demonstrating the feasibility of this approach [ ] . emerging evidence of the importance of b cellmediated immunity to pneumocystis infection strongly supports further research in this area [ ] . paracoccidioides brasiliensis, while less globally prevalent than cryptococcus or pneumocystis, is the most important cause of ifis in latin america. mabs have been raised against the major p. brasiliensis antigens glycoprotein (gp ) and gp . in an infected mouse model, anti-gp activity, mediated by mab e -enhanced phagocytosis of p. brasiliensis cells, increased interferon-γ production and led to a reduction in the fungal burden [ ] , while anti-gp mabs significantly reduced fungal colony-forming units and almost completely abolished granuloma formation in the lungs [ ] . antibodies raised against mouse cd + regulatory t (t reg ) cells, which control immunity and excessive inflammation, depleted cd + cells, resulting in less severe tissue inflammation, with reduced mortality in susceptible mice [ ] . again, this demonstrates the potential for mabs to exert control of fungal infection, either directly by incapacitating the fungal cells or indirectly via modulation of the host response. candida albicans is the most commonly isolated fungal pathogen globally and is associated with significant morbidity and mortality, particularly in patients with hiv or tuberculosis (tb) infection [ ] . a recent paper demonstrated the cloning of antibody genes from b cell cultures derived from patients infected with c. albicans [ ] . these antibodies were capable of stimulating opsonophagocytic macrophage activity and provided protection in a murine model of disseminated candidiasis. in summary, mabs offer tremendous potential to augment the antifungal arsenal: animal models have provided promising results, there is a wide range of potential targets, they have the capacity to both inhibit the pathogen and augment the host response, and it may be possible to target diverse fungal pathogens with an appropriate mab cocktail or by targeting panfungal antigens. use of mabs as adjuvants to existing antifungal drugs also shows promise [ ] . however, it should be noted that not all mabs are inhibitory; indeed, some may worsen infection, species and strain specificity can be an issue, and the mechanistic basis of inhibition by different mabs can vary dramatically [ ] . to date, only mab treatments have advanced to clinical trials: the previously mentioned b in cryptococcus, and mycograb, a heat shock protein (hsp )-specific antibody fragment, which showed promise for treating candida infections but failed to make it to market due to production difficulties and unresolved safety issues. antifungal immunomodulation is a complex area, and the field is still emerging. these preliminary studies highlight the potential for exciting new advances in mab research and application, both for understanding fungal immunity and for manipulating it to tackle lifethreatening fungal infections. dengue fever is a mosquito-borne viral infection found in tropical and subtropical regions around the world. the dengue virus (denv) is transmitted by female mosquitoes, mainly of the species aedes aegypti and, to a lesser extent, a. albopictus. there are distinct serotypes-denv- to denv- -of the virus, and all serotypes are presently circulating in endemic areas. denv infects cells of the human immune system and other cell types, leading to symptoms that include high fever, severe headache, severe pain behind the eyes, joint pain, muscle and bone pain, rash, and mild bleeding. in severe cases, plasma leaks out of the circulatory system, which can be fatal. the global incidence of dengue has grown dramatically in recent decades. one recent study estimated that approximately million people are infected, of which million manifest clinically each year [ ] . who estimates that, globally, , people with severe dengue require hospitalization each year and that . % of these infections are lethal. antibody-dependent enhancement (ade) is problematic in dengue infection. the presence of subneutralizing levels of flavivirus cross-reactive serum antibodies (acting against one member of the virus family) may result in an increase in infectivity via ade of another virus member or serotype, which is observed particularly after secondary dengue infection [ , ] . despite decades of effort, there is no effective treatment against dengue. currently, dengvaxia is approved by the fda and is the only licensed dengue vaccine in the world. this is also a live attenuated tetravalent dengue vaccine developed by sanofi pasteur [ ] that has been approved in several countries. however, interim results from long-term safety follow-up studies demonstrated an increased risk for hospitalization of vaccine-sensitized individuals [ ] , suggesting that ade-related concerns are relevant. it has been reported that non-neutralizing levels of anti-denv antibody can enhance viral entry into host cells by forming a denvantibody complex [ , ] . there is concern that an incomplete antibody against denvs may cause ade-mediated severe dengue disease. hence, there is a need for a safe and highly efficacious dengue therapy or vaccine that provides immunity against all serotypes simultaneously. mab therapy is an alternative to vaccines and other therapies against dengue. many mabs against dengue from mice and humans have been characterized, and the use of mabs has also been explored as a therapeutic option. antibody sign- c, identified by the singapore immunology network, neutralized all dengue serotypes and decreased viremia of all serotypes in mice when given days after infection [ ] . a humanized mab visterra (vis ), a panserotype anti-denv developed by visterra [ , ] , which binds e protein domain iii (ediii) and neutralizes all serotypes of denv, also showed useful antiviral utility. vis ( mg/kg or mg/kg) was administered at days post infection in nonhuman primates, and no infectious virus could be detected by either plaque assay or virus isolation after treatment, a finding that was, however, not mirrored by reverse transcription pcr (rt-pcr) findings. a human challenge model is now available for clinical research in dengue [ ] . in this model, the efficacy and safety profile of therapeutic antibodies can be evaluated rapidly in small-scale clinical settings, prior to traditional large-scale field studies with naturally infected patients. to develop mabs for dengue therapy, it is important to consider an approach that prevents or reduces ade, and several studies that address this link have been undertaken. a neutralizing human mab, d - b b , that targets the fusion loop in domain ii showed strong neutralizing activity against all denv serotypes [ ] . however, at subneutralizing concentrations, it also elicited ade activity in vitro [ ] . to reduce the ade, injampa and coworkers [ ] modified the d - b b antibody fc domain at position n q. the modified antibody kept the same cross-neutralizing activity to all serotypes as those of wild-type antibody but lacked ade activity against all serotypes at subneutralizing concentrations. in another neutralizing mab, sign- c, leucine to alanine mutations were engineered in the fc part, abrogating binding to fc gamma receptors [ ] . this mutant fc version (sign- c-lala) protected mice, while ade was completely abrogated. similarly, mab /mutated fragment crystallizable region (mutfc) (an mab that is unable to bind to cells with fcγ receptors [fcγr]) and potentiate ade have been used as a prophylactic therapy [ ] . passive immunization with this mab (at mg/kg) reduced viral load and disease progression in nonhuman primates. here again, therapeutic antibodies can also be used for prophylaxis, affording immediate and reliable protection. tb tb is a good example of the gap between a pathogen's prevalence and burden. mycobacterium tuberculosis spreads easily among human populations; presently, about one-third of all humans are infected, and new infections occur in % of the population each year. among these billions of carriers, there are, however, "only" million active tb infections, with . million deaths in . thus, the vast majority of carriers keep the pathogen in check. tb exerts levels of immune evasion: one in which it is maintained in a latent state and one in which it breaks free and causes active disease [ ] . several studies have tested antibody therapy in tb, with varying success (reviewed in [ ] ). even if an mab treatment would not be curative, shortening the standard treatment of patients infected with multidrug-resistant (mdr) and extensively drug-resistant (xdr) strains would represent a major advance. in addition, "checkpoint blockade during chronic tb infection requires further consideration" [ ] . the role of protective antibodies in malaria was demonstrated over years ago with the finding that the passive transfer of sera from mice with radiation-attenuated sporozoites delayed the development of infection in other mice [ ] . the tpps for malaria are well described [ , ] . these include a profile for seasonal malaria chemoprevention, a treatment successfully launched in sub-saharan africa in the last years, consisting of a full treatment course of days of amodiaquine and dose of sulfadoxine/pyrimethamine. it is given monthly to children during the rainy season. antibody therapeutics for malaria could ( ) prevent the entry (initial infection) of the parasite, ( ) block entry of the sporozoite into the liver cells, ( ) block entry of the merozoites into the erythrocytes, or ( ) block the uptake of the gametocytes into the mosquito (breaking the transmission cycle). one difficulty in targeting the merozoites in symptomatic malaria is that the extracellular phase of the pathogen is relatively short-lived and is only a small part of the life cycle. additionally, the number of merozoites invading erythrocytes is very large (up to ), compared with the number of sporozoites invading the liver stages (dozens). as such, the most interesting place to intervene with an mab is at the initial infection of the liver. currently there are mabs published with potent activity against the circumsporozoite protein, csp, and these can reduce the parasitaemia in sporozoite-infected frg (triple mutant fah/raγ /il rγ) mice that carry a human liver implant [ ] . these mabs include mab , cloned from b cells obtained from a patient in the recent rts,s vaccine trial [ ] , cis (circumsporozoite protein [ ]), and a set that included mgu [ ] , cloned from patients vaccinated with irradiated sporozoites. antibodies that block the invasion process of the red blood cells by merozoites represent another possible approach. in studies of the merozoite protein rh (reticulocytebinding protein homologue ) as a potential vaccine, some antibodies were described that could block the cycle of erythrocyte infection [ ] . the specific merozoite epitopes are now being characterized, and this could form the basis of a second-generation antibody [ , ] . a general observation with plasmodium infections is that the human host is unable to mount a sterilizing immune response. one theory is that the parasite can control the t-cell response and induce a state of fatigue or anergy similar to that seen in tumor-invading lymphocytes in immuno-oncology. several studies in mice have demonstrated that blocking pd-l or ctla- (cytotoxic t-lymphocyte-associated protein ) improves clearance in mice infected with plasmodium bergheii [ ] , and from this and similar experiments, a strong argument can be made that reversal of this fatigue by mild checkpoint inhibitor blockade may be a way of facilitating the host response [ ]. hiv mab therapies have also been proposed for hiv. the case for hiv was recently reviewed [ , ] . two such mabs are in phase iii trials: pro and cenicriviroc. pro targets ccr (cysteine-cysteine chemokine receptor type ) and recently entered phase iib/iii trials for weekly subcutaneous dosing for monotherapy maintenance [ ] . cenicriviroc is a dual ccr and ccr antagonist investigated for a number of indications, including hiv infection [ ] . as noted earlier, ibalizumab was recently approved as a second-line treatment for hiv treatment [ ]. for hbv infection, the hypothesis is that high circulating hbsag levels prevent a proper immune response. a novel mab, e f , is being evaluated for reducing hbsag levels in patients [ ] . in addition, the hbv s protein is being targeted for the discovery of therapeutic mabs [ ] . in the case of visceral leishmaniasis (vl), il- and glucocorticoid-induced tnf-receptorrelated protein have been considered as mab targets [ ] , but there has been no systematic analysis of antigens or reported cloning of b cells from infected patients. although mabs have made a massive impact in controlling autoimmune disease, inflammation, and cancer, the relative impact in the world of infectious disease has largely been confined to viral diseases. the use of mabs to protect against rsv infections and the profile of secondgeneration antibodies shows that it is possible to obtain mabs that are sufficiently potent to provide long-term protection with a single intramuscular or subcutaneous injection. with the development of new technologies for cloning antibodies from b cells or plasma cells taken from patients infected with bacteria, viruses, fungi, or even protozoal pathogens, it is possible to quickly obtain fully human antibody collections with potential activity against pathogens in vitro and in vivo. technologies for expressing antibodies are now at the stage in which it is not uncommon to see extremely high levels of expression in cell culture, and taken together with the progress in reducing costs of production, the cost of goods for an antibody injection is starting to enter the range of us$ to us$ , reaching the edge of what is affordable for infectious diseases of neglected populations. studies of mutations in the fc region confirm that mab half-lives can be extended, and the goal of a single injection to cover an entire season for those infections with seasonality is now a possibility. new technologies with viral delivery offer the promise that a single injection could give protection for even longer periods. for many infectious diseases, we are now seeing the buildup of a portfolio of potential antibodies. in cases in which little progress has been made, a systematic attempt is needed to identify the antibodies resulting from successful control of an infection in patients. beyond these basic antibodies, the availability of new monoclonals with anti-infective activity in vivo would open up the door to even more creative options: bispecific antibodies could be used in key immune cells and could effectively support the natural response to infection. studies in animal models of chronic malaria infection led to the observation that this results in a reduction in the impact of cytotoxic t cells and modulates the t reg capacity, leading to an "exhausted" or ineffective t-cell response [ ] . clinical trials are already underway that use immune checkpoint blockers for chronic hiv and hbv infection [ ] . this has important implications for any infectious disease in which a single infection does not drive a sterilizing immune response. for such diseases, which include malaria, one question for the longer term is whether immune checkpoint inhibition, or interfering with interferon-α signaling, could be used [ ] . two decades ago, one of the biggest challenges of working in anti-infective drug discovery was the need to have new medicines with activities against the widest range of pathogens. in more recent times, the tide has changed, and clinical diagnosis now is such that medicines with a high degree of selectivity and specificity are often preferred-as long as they show good clinical activity. this shift to highly specific medicines favors the use of mabs. given the overall rise in interest for new treatments in infectious diseases caused by concerns about antimicrobial resistance, there is a real opportunity now to progress the newly emerging families of mabs to target infectious diseases of neglected populations. because of outdated preconceptions about this class of therapeutics, few research funds are being allocated to their discovery, resulting in an egg-and-chicken problem (the absence of conspicuous success driving additional efforts). one of the goals of the analysis that we present here is to promote making additional funds available to pursue the initial discovery of mabs for neglected diseases. monoclonal antibodies approved by the ema and fda for therapeutic use protective murine monoclonal antibodies to cryptococcus neoformans antibody-mediated protection in mice with lethal intracerebral cryptococcus neoformans infection monoclonal antibodies to cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection protection by anti-betaglucan antibodies is associated with restricted beta- , glucan binding specificity and inhibition of fungal growth and adherence human antibodies against a purified glucosylceramide from cryptococcus neoformans inhibit cell budding and fungal growth monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fungal glucosylceramides: from structural components to biologically active targets of new antimicrobials immunotherapy of cryptococcus infections. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases immunomodulation with cd stimulation and interleukin- protects mice from disseminated cryptococcosis a monoclonal antibody to cryptococcus neoformans glucuronoxylomannan manifests hydrolytic activity for both peptides and polysaccharides antibody to cryptococcus neoformans glucuronoxylomannan inhibits the release of capsular antigen immunoglobulins in defense, pathogenesis, and therapy of fungal diseases the yin and yang of current antifungal therapeutic strategies: how can we harness our natural defenses? frontiers in pharmacology immunization with pneumocystis cross-reactive antigen (pca ) protects mice against pneumocystis pneumonia and generates antibody to pneumocystis jirovecii vaccine-induced immunogenicity and protection against pneumocystis pneumonia in a nonhuman primate model of hiv and pneumocystis coinfection passive intranasal monoclonal antibody prophylaxis against murine pneumocystis carinii pneumonia an emerging role of b cell immunity in susceptibility to pneumocystis pneumonia. american journal of respiratory cell and molecular biology the monoclonal antibody against the major diagnostic antigen of paracoccidioides brasiliensis mediates immune protection in infected balb/c mice challenged intratracheally with the fungus characterization of gp and anti-gp monoclonal antibodies in paracoccidioides brasiliensis pathogenesis candida albicans, a major human fungal pathogen single human b cell-derived monoclonal anti-candida antibodies enhance phagocytosis and protect against disseminated candidiasis immune modulators as adjuncts for the prevention and treatment of invasive fungal infections immunotherapy of fungal infections the global distribution and burden of dengue antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications development of the sanofi pasteur tetravalent dengue vaccine: one more step forward the impact of the newly 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falciparum circumsporozoite protein a public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein human vaccination against rh induces neutralizing antimalarial antibodies that inhibit rh invasion complex interactions kilchip v . : a novel plasmodium falciparum merozoite protein microarray to facilitate malaria vaccine candidate prioritization. frontiers in immunology human antibodies that slow erythrocyte invasion potentiate malaria-neutralizing antibodies programmed death- ligand -mediated regulation of the pd-l to pd- axis is essential for establishing cd (+) t cell immunity antibody-mediated therapy against hiv/aids: where are we standing now? broadly neutralizing anti-hiv- monoclonal antibodies in the clinic the return of pro , a ccr -directed mab apobec g/ a expression in human immunodeficiency virus type -infected individuals following initiation of antiretroviral therapy containing cenicriviroc or efavirenz a human monoclonal antibody against hepatitis b surface antigen with potent neutralizing activity combined immune therapy for the treatment of visceral leishmaniasis immune checkpoints and their inhibition in cancer and infectious diseases we thank dr. kiyoshi kita, dean and professor at the school of tropical medicine and global health, nagasaki university; dr. yoshimasa tanaka, assistant professor at the center for therapeutic innovation, nagasaki university; and dr. simon croft, professor at the london school of hygiene and tropical medicine, all of whom provided insight and expertise that greatly assisted the research. key: cord- -lc onm authors: patel, ami; bah, mamadou a.; weiner, david b. title: in vivo delivery of nucleic acid-encoded monoclonal antibodies date: - - journal: biodrugs doi: . /s - - - sha: doc_id: cord_uid: lc onm antibody immunotherapy is revolutionizing modern medicine. the field has advanced dramatically over the past years, driven in part by major advances in isolation and manufacturing technologies that have brought these important biologics to the forefront of modern medicine. however, the global uptake of monoclonal antibody (mab) biologics is impeded by biophysical and biochemical liabilities, production limitations, the need for cold-chain storage and transport, as well as high costs of manufacturing and distribution. some of these hurdles may be overcome through transient in vivo gene delivery platforms, such as non-viral synthetic plasmid dna and messenger rna vectors that are engineered to encode optimized mab genes. these approaches turn the body into a biological factory for antibody production, eliminating many of the steps involved in bioprocesses and providing several other significant advantages, and differ from traditional gene therapy (permanent delivery) approaches. in this review, we focus on nucleic acid delivery of antibody employing synthetic plasmid dna vector platforms, and rna delivery, these being important approaches that are advancing simple, rapid, in vivo expression and having an impact in animal models of infectious diseases and cancer, among others. monoclonal antibody (mab) therapy has changed the landscape of modern medicine. to date, there are over different mab biologics approved by the food and drug administration (fda) and european medicines agency (ema) for treatment of infectious diseases, cancer, asthma, and autoimmunity, among others. with these successes, the field is expanding into new and exciting related areas for biologics that target multiple specificities. this includes a range of bispecific and trispecific mabs which can bind to the same or multiple antigens. among the newest mab-related biologics are bispecific t-cell engagers (bites), bispecific and trispecific killer cell engagers (bikes and trikes), and dual-affinity re-targeting antibodies (darts), as well as many others (reviewed in ref. [ ] and fig. ). these developments are the direct result of over years of continuous advances in mab isolation approaches, including hybridoma technologies, yeast surface display, phage display, and, more recently, single b-cell sorting strategies that identify paired heavy chain and light chain from single cells, among others (reviewed in refs. [ ] and [ ] ). new structural engineering strategies to improve potency and cell culture production, as well as the parallel development of sophisticated bioprocess manufacturing technologies, have additionally contributed to meet the growing demands for mab biologics [ ] . despite these many advances, large-scale bioprocessing is faced with challenges that currently hamper wider global deployment. the intrinsic biochemical and biophysical properties of antibody sequences are frequent liabilities for large-scale manufacturing and may also lead to post-manufacturing aggregation and stability issues. such limitations may prevent an otherwise highly potent and effective mab from advancing through development and into the clinic [ , ] . delivery challenges must also be overcome as in vivo administration of mab biologics often requires high doses (grams of mab) to achieve therapeutic efficacy, frequently at a high cost (fig. ) . while the cost of raw materials is relatively inexpensive, bioprocess manufacturing and purification can be lengthy and costly (reviewed in ref. [ ] ). the price of mab biologics is driven by a combination of research and development costs, duration of treatment, patient market size, geographic location, private insurance coverage, and availability of biosimilars [ , ] . mabs requiring higher doses need to be administered through slow intravenous (iv) infusions to limit infusion reactions. iv delivery frequently requires hours of clinical monitoring and may involve post-infusion monitoring for allergic or anaphylactic reactions, further increasing the medical personnel required and costs of administration. subcutaneous (sc) delivery has advantages for lower dose antibody delivery, including being less invasive and the possibility for self-administration in several indications, such as rheumatoid arthritis, primary immunodeficiencies, and multiple sclerosis [ , ] . drug autoinjectors have greatly improved the uptake and convenience of sc delivery, also regulating dosing. however, sc delivery is associated with pain related to injection volume and injection site reactions, and absorption is slow due to reliance on the lymphatic system [ ] for biodistribution [ ] . as a result, the mab may be eliminated before reaching systemic circulation (reviewed in ref. [ ] ). nonetheless, the impact and importance of mabs on human disease and the growth in new applications of such technology cannot be understated. with all mab biologics, cold-chain storage and transportation is required to enable reasonable product shelf life. temperature control is important for lengthening mab biologic stability and storage. however, the need for a fig. different types of traditional ig, bispecific ig, and non-ig designs. ig immunoglobulin, bite bispecific t cell engager, bike bispecific natural killer engager, scfv single-chain variable fragment, dart dual affinity retargeting, dvd-igg dual variable domain immunoglobulin cold-chain can limit global distribution to at-need populations in resource-limited areas, and requires additional production runs to resupply patients, due to expiring product. for these reasons and others, novel creative approaches for in vivo gene-encoded mab delivery building on existing biologic technologies are under investigation, with the goals of increasing patient accessibility and providing novel clinical options for this growing class of important biologics. in vivo gene-encoded antibody delivery is an elegant approach that may address many limitations of traditional antibody biologics. in general, the antibody heavy-and light-chain complementary dna (cdna) are examined and then optimized or designed for encoding and expression specifically for the type of vector chosen for in vivo delivery. historically, in the gene therapy arena, viral vectors [adeno-associated virus (aav) is the current favored viral vector] have been promising for in vivo transfection and gene expression. more recently, the newly emerging nonviral synthetic nucleic acids [synthetic dna formulated for facilitated delivery by electroporation, or formulated lipid nanoparticle (lnp) encapsulated messenger rna (mrna)] have started to receive considerable attention. the synthetic antibody is transcribed (in the case of aav and synthetic dna) and translated within the in vivo transfected targeted cells, followed by antibody secretion into the systemic circulation. rna approaches use lnp to allow for delivery in vivo, and then the mrna is directly loaded on ribosomes for translation in the cytoplasm, skipping the nuclear steps used by aav and dna deliveries. the synthetic nucleic acid field is rapidly providing insights into the in vivo biology of genetic expression which suggest novel exciting strategies for drug delivery, with broad implications for the prevention and treatment of disease. a high-level summary of synthetic dna and synthetic mrna and comparison with viral vector delivery platforms for in vivo mab delivery is presented in table . the original studies surrounding in vivo antibody gene delivery focused primarily on gene delivery using recombinant viral vectors such as aav and adenovirus (ad), which were advanced clinically, building on work in the traditional gene therapy-based field. among these choices, aav has been the most studied option for mab delivery, due to its particular advantages. these include the fact that it is more immune silent as a vector compared with other viral vector platforms and can achieve long persistence of the genetic cassette with a single administration (reviewed in ref. [ ] ). viral vector delivery is dependent on viral surface receptor-mediated entry into cells, with different vectors displaying unique tissue tropisms (e.g., liver, muscle, and lung) based on their surface capsid (aav) [ ] or hexon (ad) [ ] proteins. in vivo viral vectors encoding antibody genes have been reported to be administered either locally [via intramuscular (im), intranasal (in), intraocular (io), or intracranial (ic) routes] or systemically via iv or intrathecal (it) routes, leading to viral infection with production of the virus-encoded antibody predominately from liver and lung and perhaps other tissues (reviewed in ref. [ ] ). interestingly, aav vectors take several weeks to reach their peak expression; therefore, to accelerate expression, de et al. evaluated a combination of both an ad vectorencoded antibody followed by an aav-encoded mab targeting anthrax and demonstrated successful rapid and long-lasting expression [ ] . around the same time, skaricic et al. demonstrated delivery of a murine version of the anti-respiratory syncytial virus antibody palivizumab using ad serotype and aavrh. vectors, showing protection and expression for weeks [ ] . with additional vector promoter optimizations, aav vectors are capable of high antibody expression, approaching mg/ ml serum concentration at the highest vector doses in mice [ ] . high systemic aav-mab expression levels have been observed in macaques that have uptake of the transgene, typically in the range of up to μg/ml in serum (reviewed in ref. [ ] ) and as high as μg/ml in a single macaque, with expression for years. however, expression can decrease in animals when suppressed by host development of anti-vector cellular immunity or host anti-drug antibody (ada) immune responses [ ] . in humans, re-administration of aav vectors can be difficult due to rapid development of serotype-specific antibodies. aav-mabs have been studied for their ability to deliver anti-hiv broadly neutralizing antibodies. aav serotypes , , and have been evaluated as vector backbones for delivery of anti-hiv mabs b , vrc , vrc , and - , and immunoadhesins such as ecd ig (reviewed in ref. [ ] ). however, it was observed that these viral vectors were limited to single-use delivery, as natural serology and vectorinduced neutralizing antibodies impaired repeat administration. this reduces the potential applications for long-term, repeat delivery of anti-hiv aav-mabs or delivery of combinations of anti-hiv mabs. development of additional aav vector serotypes targeting different tissues and assessing different routes of delivery is ongoing. many of the initial aav-mab studies were performed utilizing im delivery. iv delivery is less targeted, resulting in viral vector delivery and production in multiple tissues, including liver, lung, and others. limberis et al. have focused on aav delivery to the lungs via in delivery for anti-influenza virus [ ] [ ] [ ] , and laursen et al. demonstrated aav delivery of an anti-influenza multi-domain antibody [ ] . additionally, limberis et al. described the delivery of anti-ebolavirus aav-mabs [ ] . however, a parallel study evaluating aav delivery of the same anti-ebolavirus antibodies by im and in delivery routes demonstrated different results based on the route of administration [ ] . these data support the hypothesis that there may be variations in delivery take of aav vectors [ ] and that further studies investigating different routes of delivery continue to be important. nevertheless, the data with viral vectors, particularly aav-mabs, are promising and supportive of the potential for an in vivo mab delivery approach to administer highly potent antibodies. these vectors have unique advantages and disadvantages, supporting the need for more work in this area. importantly, these studies serve as an important foundation for the development of additional in vivo mab approaches using non-viral delivery platforms. we focus below more specifically on non-viral antibody gene delivery technologies using synthetic mrna or synthetic plasmid dna vectors (fig. ). dna and rna represent the nucleic acid building blocks of life. dna must first be transcribed into mrna before translation to proteins. in vitro-transcribed mrna delivery circumvents in vivo transcription, leading directly to protein translation. although related, there are significant differences between dna-and mrna-vectored antibody delivery approaches. dna is a highly stable, double-stranded regulated nucleic acid with antiparallel nitrogenous bases bonded no by intervening deoxygenated pentameric sugar and a phosphate backbone capable of being read and faithfully transcribed into mrna. this can generate logs of mrna from a single dna expression cassette. the naïve mrna is then processed through enzymatic removal of noncoding introns and subsequent ligation of coding exons with the final addition of a ′ -methyl guanosine and polyadenylated ′ to buffer against nuclease activity, yielding a mature mrna that is translated into protein by ribosomal enzymes [ , ] . initial dna vectors were developed by direct cloning and expression of natural sequence inserts derived from the host. the recent change to synthetic generation of highly designed, synthetic inserts has had a major impact on the platform. synthetic plasmid dna vectors can be engineered to encode large, complex proteins. this is achieved through space-saving plasmid engineering encoding only designed coding sequences with enhanced leader and optimized stop sequence which are required for natural downstream mrna processing and termination, but have improved performance compared to native sequences. the natural temperature stability of pure dna means that synthetic plasmid dna vectors can be stored at ambient temperatures for extended periods of time, as they can withstand significant temperature fluctuations without damage [ ] . synthetic dna is therefore capable of being developed to be cold-chain independent, making it highly attractive for product development and administration in resource-limited settings. one dramatic difference compared with viral delivery is that synthetic dna does not induce anti-vector backbone immune responses and a homologous synthetic dna backbone encoding the same or a different gene can be re-administered over and over [ ] . as a result, dna is a promising platform for in vivo delivery of vaccines, protein replacement, or antibody genes, especially when combined with enhanced delivery, as discussed in sects. , , , and . dna is transiently expressed and can continue to produce protein as a depot until it is lost from cells. in general, as plasmid delivery is non-live, non-replicating, and non-integrating, these deliveries are transient. plasmid dna-encoded mabs (pdna-mabs) are engineered to carry synthetic antibody genes, similar to aav-mab platforms. the literature describes that pdna-mabs can exhibit peak serum concentrations after just weeks and then can express at consistent levels for - months and decline thereafter as the plasmid is lost from cells and then cleared from the serum due to the natural half-life of immunoglobulin g (igg) [ ] [ ] [ ] [ ] [ ] [ ] . long-term small animal studies show the total duration of pdna-mab expression to be at least year [ , ] . however, in contrast to viral vectors, synthetic dna requires an efficient delivery system. by far the most advanced of these are the adaptive electroporation systems, where a consistency of in vivo dna delivery and efficiency has been reported [ ] , representing potential utility for pdna-mab delivery, at least preclinically. in the clinic, vaccine delivery via facilitated electroporation has generated impressive data in people [ ] [ ] [ ] . the delivery technology is discussed in sect. . . mrna offers a platform with the ability to rapidly express protein, bypassing dna to rna processing and directly attaching to ribosomes in the cytoplasm for translation of the protein of interest. this results in a fast and efficient delivery, followed by a single round of expression leading nucleic acid antibody gene delivery using synthetic dna or mrna administration into the cell to bolus protein expression and secretion. rna, with its oxygenated ′ sugar backbone, is inherently unstable, especially in alkaline ph, and prone to enzymatic activity and degradation. this increases the chances of rna integration via reverse polymerase transcription processes. synthetic mrna must encode all the necessary rna processing structures, including a ′ cap in the correct orientation and a polyadenylation tail, limiting its overall coding capacity [ ] . frequent repeat administrations are required for longterm delivery, due to its short half-life [ ] . synthetic in vitro-transcribed (ivt) mrna is more biochemically unstable compared to dna, and this has driven the search for rapid parallel advances in various lnp formulations for packaging the rna. lnps improve overall stability as well as providing a means for ivt mrna to enter target cells. delivery by specific lnp has significantly reduced recognition of mrna by innate sensors; however, mrna must exit the lnp into the cytoplasm in order to be translated, and therefore still encounters sensors. this results in recognition by innate pattern recognition receptors such as toll-like receptors tlr , tlr , tlr , and tlr that respond to mrna by inducing inflammatory responses via endosomal compartments and retinoic acid-inducible genelike receptors (rlrs), rig-i, mda , and lgp , that recognize unmethylated cpg nucleotides (or strands as in plasmid delivery) and single-stranded rna to initiate degradation [ , ] . rna sensors can be particularly inflammatory. for this reason, modified nucleosides such as pseudouridine [ ] and -methylcytidine are being advanced to enhance translation and stability, as well as lower innate inflammation via many of the innate immune sensors (reviewed in ref. [ ] ). the limited studies of mrna in the clinic have shown some adverse events at higher doses and are being watched closely, and work-arounds are in progress [ ] [ ] [ ] . in contrast to mrna, supercoiled plasmid dna drives more limited innate stimulation of cytosolic dna sensors (sting, c-gas, and tbk ) [ ] [ ] [ ] and activation of tlr [ ] , even in the context of advanced electroporation delivery. however, additional investigations and developments in this area are receiving significant attention to further improve mrna delivery and prevent undesirable immune activation in dose escalation studies. understanding the safety of gene delivery platforms is paramount for in vivo antibody gene delivery to progress. the safety data to date for both non-viral and viral vectors have been established from vaccine and gene therapy studies. ongoing studies in humans with gene-encoded mabs will be informative as the field moves forward (nct , nct ). viral vectors have mostly been focused in the area of disease therapy, with few studies in normal healthy humans for gene therapy. in a limited number of studies, adverse events, including lymphopenia and neutropenia, among others, have been reported for ad vectors [ ] , and concerns over genotoxicity are being raised for aav (reviewed in ref. [ ] ). this includes evidence suggesting genome integration by aav vectors [ ] [ ] [ ] [ ] , potentially resulting in permanent rather than transient gene transfer. such integration events are still unpredictable and require additional study to further elucidate. higher doses of viral vector may circumvent preexisting immunity; however, toxicity has been reported in dose escalation studies with ad vectors [ , ] , including one fatality due to high-dose vector delivery [ , ] . similarly, high-dose aav vector delivery in pig and non-human primate studies has also been associated with severe toxicity, under specific conditions [ ] , but most clinical studies which have focused on more moderate doses support that aav delivery is very well-tolerated in the clinic. excitingly, clinical impact has been achieved for specific applications [ ] , underscoring the importance of this approach. however, the utility of aav for systemic delivery has continued to show challenges, with anti-vector immune responses, as well as breaking tolerance to the encoded transgene. this area is receiving additional attention. additional development is important and is likely to eventually pay more dividends in the clinic. the mrna delivery field is still relatively new and there have only been a few mrna gene therapy clinical trials and even fewer for delivery of antigens as vaccines or for in vivo encoded antibody delivery. the few mrna studies reported have described primarily mild-to-moderate injections site reactions [ , ] ; however, these can have a high frequency, and occasionally they have been serious. in a small vaccination study, a serious adverse event was reported following administration of an mrna-rabies vaccine [ ] . the afflicted individual presented with transient bell's palsy, recovering after a second vaccination. vaccine studies with an mrna h n vaccine demonstrated that intravenously delivered mrna can disseminate to different tissues, such as the liver, heart, brain, kidney, and many others [ ] , with many individuals exhibiting less severe fevers and chills. the vaccine was immunogenic and produced serological responses in most vaccinated persons. the long-term impact of antibody production from many of these tissues is only beginning to be followed. as the field is still relatively new, additional studies and monitoring will be informative for the safety of mrna delivery in people. as mrna currently requires lnp formulation, further studies to evaluate the safety of different formulations in the clinic are ongoing. an ongoing important first-in-human trial to evaluate the safety of an anti-chikungunya virus (anti-chikv) mrnaencoded mab (mrna-mab) mrna- is recruiting (nct ). the interim safety results from this study were reported at the oligonucleotide therapy society meeting (october ), with no adverse events reported in the lowest . -and . -mg/kg dose groups. however, grade , , and adverse events were observed in the . -mg/kg group [ ] . importantly, expression was observed of the encoded antibody, which is a very exciting development for the field. microgram levels of antibody were reported after continuous administration of mrna ab for several hours by the iv administration route. these exciting results, while showing some challenges, are very important. continued study and development in this area will provide important guideposts regarding the potency and the safety of this approach. further understanding of potential rna methylation and the impact of epigenetic modifications is still needed. however, our understanding of rna retrotransposons through long interspersed element (line) and short interspersed element (sine) processes has informed much about the potential for dna or rna integration. the risk of integration for either platform is theoretical, which is not the case for many viral vectors. a survey of integration historical events in the human genome is illustrative. for example, specific elements such as lines and sines make up roughly % of the entire sequenced human genome and are distributed at intergenic and intragenic regions, respectively [ ] ( fig. a ) [ , ] . both lines and sines move through the genome via rna intermediates that are converted to dna before integrating into the genome at a different location. these transposons can have either an advantageous or deleterious effect. findings by kaessmann et al. and others have shown that, similar to sines, mrna retrotransposons can also integrate with the help of line-encoded reverse transcriptase (rt) enzymes. these rt enzymes recognize polyadenylated mrna destined to be copied into cdna and lead to host integration of transposed genes with varying mutation and functional capacity [ ] . using retrotransposon capture sequencing (rc-seq), baillie et al. were able to identify three active families of retrotransposons (l , alu, and sva) that are known to have deleterious effects in humans by mobilizing nonspecific integration, which results in the abrogation of genetic functionality in somatic neuronal development [ ] . undoubtedly, these studies highlight the complexity and divergence of mrna processes and the natural challenges and opportunities that exist to enhance a robust safety profile [ , ] . synthetic plasmid dna has been extensively studied and has been administered to thousands of healthy human participants in numerous clinical trials over the past decades. to date, there have been no reports of any serious adverse events related to plasmid dna [ ] . dna delivery has a local biodistribution and stays at the site of initial injection, typically clearing within months of administration [ ] . the injection is well-tolerated, with only mild-to-moderate local (injection site) reactogenicity and no systemic toxicity observed in the thousands of persons studied, irrespective of higher dose administrations, in contrast to mrna. plasmid dna delivery represents almost . % of gene therapy vectors that have been investigated in human clinical trials (fig. b) [ ] . this number does not include the numerous human trials evaluating plasmid dna vaccines against infectious diseases in normal healthy persons. the theoretical possibility for plasmid dna integration is often cited as a risk factor for this platform. however, in experimental studies, plasmid integration events occur at rates below the statistical rate for dna integration observed in nature [ ] , or the background mutation rate of normal exposure to the fig. a elements in the human genome (adapted from ref. [ ] ), b breakdown of clinical trials by platform (adapted from ref. [ ] ). ltr long terminal repeat sun. to date, there have been no integration events in the preclinical studies in the tens of thousands of individuals receiving plasmid dna, supporting the safety of synthetic dna plasmid-based technologies like pdna-mabs. dna transposons exist as part of < % of the genome; however, they are considered ancestral, and there is no evidence for new dna transpositional activity for at least million years [ , ] , again supporting the rare nature of such an event. much of the activity of dna transposons occurred during early primate evolution and appears to have ceased. double-stranded dna requires an additional integrase for genome insertion. this machinery is unlikely to be available in adult human cells. this is clearly unlike the line and sine rna intermediary retrotransposons that continue to exert mobile transpositional activity in the genome [ ] , suggesting additional study of the rna in such events could be informative. as nucleic acid-delivered antibody technologies begin to demonstrate interesting preclinical results, questions have arisen concerning how to control the magnitude and duration of in vivo expression. while long-lasting expression of antibodies targeting infectious pathogens is unlikely to be problematic, prolonged expression of checkpoint inhibitors could have lasting immune impact. therefore, strategies that serve as on/off switches would be useful. gene therapy studies with viral vectors have previously considered herpes simplex virus thymidine kinase (hsv-tk) as a potential "suicide" gene in combination with the antiviral ganciclovir. however, hsv-tk can be immunogenic [ ] and is not active in non-dividing cells such as skeletal muscle [ , ] . alternatively, the inducible caspase- system (icasp ) has also been studied. the icasp system employs a modified caspase , combined with a homodimerization domain that is activated in combination with a chemical inducer of dimerization (cid), leading to apoptosis [ , ] . unlike hsv-tk, icasp is active in non-dividing cells. icasp -based systems are being evaluated as safety switches for chimeric antigen receptor t-cell (car-t) engineering and therapy [ , ] . in recent studies from the car-t field, mestermann et al. employed the tyrosine kinase inhibitor dasatinib to suppress t-cell receptor signaling [ ] . however, dasatinib has been shown to be toxic in skeletal muscle cells [ ] . furthermore, dasatinib does not completely remove car-t from circulation, rather it suppresses activity. in addition, the ligand-inducible rheoswitch therapeutic system has been evaluated in animal models [ ] and people for regulation of interleukin- delivered by an ad vector [ , ] . building on these studies from the gene therapy and car-t fields, additional study of gene safety switches in skeletal muscle would be useful for pdna-mab platforms. recently, the first phase i human trial evaluating the safety, tolerability, and pharmacokinetics of an anti-zika virus dna-encoded mab (dmab) (nct , estimated to complete in early ) has opened. this study will provide additional important data in regard to safety, the initial pharmacokinetics of particular formulations, and the delivery of dmab in the clinic. plasmid dna is administered by a local injection. to date, this has been performed focusing on im delivery strategies; however, other routes such as intradermal (id) may be important as the platform advances. early studies used needle and syringe delivery or older delivery devices, inducing very low in vivo expression levels [ , ] . more recently, through engineering the inserts for increased in vivo expression and using more advanced systems like adaptive cel-lectra™ electroporation, improved levels of in vivo antibody expression have been reported in many animal studies. the adaptive electroporation utilizes a direction-changing field, with depth-sensing novel technology and integrated resistance sensing. this greatly improves dna delivery tolerability, as well as enhances in vivo transfection efficiency approximately -to -fold, leading to a direct increase in protein expression [ ] . pdna-mabs delivered using cellectra™ technology are referred to as dmabs. other clinical systems, such as the ichor trigrid system [ ] and the igea cliniporator (igea) [ ] , are also being employed for pdna-mab delivery, with demonstration of in vivo expression and impact in small animal studies. advancements such as hyaluronidase pre-treatment of muscles to transiently facilitate dispersion through the extracellular matrix (ecm), coupled with updated electroporation, have greatly enhanced this approach [ , ] . in the early pdna-mab studies, yamazaki et al. observed -ng/ ml expression in mice before hyaluronidase pre-treatment and as high as > μg/ml following hyaluronidase pretreatment in mice [ ] . studies by several other groups displayed similar increases in in vivo expression following hyaluronidase pre-treatment [ , , ] . a recent study by schommer et al. utilizing chondroitinase abc to degrade chondroitin, also present in the ecm, describes this enzyme as an alternative to hyaluronidase [ ] . more recent studies have addressed the need for hyaluronidase pre-treatment and are utilizing co-formulation with recombinant human hyaluronidase (hylenex ® ) in non-human primates [ ] . the zika dmab phase i trial is utilizing this co-formulated approach. the most studied promoters for dna-mab delivery are associated with the use of the human cytomegalovirus (cmv) promoter [ - , , - ] or the chicken beta-actin (cag) promoter [ , ] . learning from the mrna field, further optimizations in the ′ and ′ untranslated regions (utrs), polyadenylation signals, and other post-translational response elements should also provide added benefits. other strategies include evaluating heavy chain (hc) and light chain (lc) gene delivery on one versus two plasmids. in several early studies, it was demonstrated that two-plasmid delivery affords superior expression to single-plasmid delivery [ , ] . additional study is necessary in order to understand the biological reason for these expression differences and improve on strategies to develop single-plasmid pdna-mabs as well as other modifications. as the pdna-mab platforms advance, considerations such as shrinking plasmid backbones, including studies of closed linear dna and minicircles, as well as linear deliveries represent interesting alternative options that are worth exploration. analysis of antibody sequence liabilities and evaluation of "developability" criteria are essential for recombinant protein manufacturing [ , ] . interestingly, many of the rules that have been thoroughly studied over decades to establish traditional antibody manufacturing are unique with regard to in vivo mab expression [ , ] . patel et al. made the first observation that in vitro pdna-mab expression data did not correlate with in vivo expression levels using the dmab platform [ ] . for example, recombinant anti-ebola glycoprotein (gp) mab g is a challenging antibody to manufacture in vitro; however, it is feasible through modified cell lines [ ] . however, the original version of dmab- g had no expression in vivo as a human igg pdna-mab. patel et al. introduced single amino acid modifications into the fab framework region to optimize dmab- g to restore in vivo expression [ ] . similar modifications were introduced into the framework of an anti-ctla dmab [ ] and anti-hiv dmabs [ ] to achieve highly optimized in vivo expression [ ] . therefore, sequence optimizations have clearly played a critical role in in vivo expression of synthetic pdna-mabs. further studies evaluating the contribution of amino acid modifications at the cellular level will provide highly valuable insights into the biology of in vivo-delivered antibodies. like traditional antibody studies, approaches that reduce the immunogenicity of amino acid substitutions will also be valuable. taken together, these modifications introduce a new approach to selectively modify pdna-mabs that would otherwise be discarded by traditional sequence liability screening methods, opening new avenues for delivery of highly potent and important antibodies. additionally, many of the fc modifications identified in traditional antibody research can be applied to synthetic pdna-mabs. introduction of the l a, l a (lala) mutation to the pdna-mab fc can abrogate fc receptor (fcr) interactions, similarly to recombinant antibody [ , ] . other strategies to increase the duration of biologic antibodies in vivo include modifications in the fc region that will increase interactions with the neonatal fc receptor (fcrn) [ ] . these modifications include m y/s t/ t e (yte) [ ] in the fc ch regions and m l/ n s (ls) [ ] and n h [ ] in the ch region. new modifications including ytw/kf [ ] have been identified, and further study to determine potential benefits for nucleic acid-encoded mabs will be highly informative to further extend expression in vivo. overall, sequence engineering has been key to the success of synthetic dmabs, and although similar modifications have not yet been studied for other pdna-mabs or mrna-mabs, such modifications will likely be important for diverse gene-encoded platforms. pdna-mab studies with species-matched fc, such as studies with fully mouse (fab and fc) antibodies [ , ] or fully sheep antibodies (fab and fc) [ ] , show long antibody expression in circulation. not surprisingly, human antibody evaluation is challenging in animal models, as they develop a rapid immune response against foreign human fc. it is also well-described that fully human antibodies have the potential to develop ada responses in people. these immune responses can also be directed against the complementarity determining regions (cdrs) [ ] . additional approaches to assess fully human biologics in non-clinical and preclinical models are therefore needed. patel et al. showed that the anti-dmab antibody response is mhc class ii-dependent and can be overcome in mouse models using t-cell depletion [ ] . the t-cell-depleted animal is an important model, as a fully functional immune system returns in - days by normal thymopoiesis, allowing for complex immune and challenge studies to be performed. other pdna-mab and mrna-mab studies utilized immunodeficient animal models such as athymic nude mice and rag knockout, scid, or nsg mice as alternative models to evaluate human igg antibody expression and functionality. however, immunedeficient mice are unable to model the intricacies of a functional immune system and should be considered carefully as preclinical models. a recent study observed that nsg mice cleared recombinant mab faster than other mouse models, presenting a significant challenge when evaluating biologics in this model [ ] . additional studies to help evaluate fully human pdna-mabs in mouse models would be highly informative for both non-viral and viral delivery and provide an important path forward for preclinical evaluation of in vivo-delivered antibodies [ ] . the first clinically relevant mab approved by the fda was orthoclone okt , an anti-cd antibody targeting t cells, in , as a prophylaxis for organ transplant patients [ ] . as the field evolved, the steady progression towards harnessing and arming the immune system to combat transformed cells intensified, leading to the discovery of rituximab, an anti-cd b-cell receptor antibody and the first successful mab therapy approved for the treatment of hematological b-cell lymphoma [ ] . nivolumab (anti-pd ) and ipilimumab (anti-ctla ) are two of the most successful checkpoint inhibitors currently on the market and have clinically been shown to increase overall survival and tumor progression for inoperable metastatic melanoma. according to the first-quarter average sales prices for from the centers for medicare and medicaid services (cms), the mg/kg price for nivolumab and ipilimulmab was us$ and us$ , respectively [ ] . for an -kg, stage iii or higher melanoma patient, a combination regimen of nivolumab and ipilimumab prescribes a -and -min iv injection every weeks for four cycles, followed by nivolumab every weeks until the patient presents a good progress report or can no longer tolerate treatment. this combination treatment alone amounts to about us$ , a year in total overall treatment, not accounting for other point-of-care measure costs such as post-infusion monitoring, which places a significant financial burden on the patient and their family as well as the overall healthcare system [ ] . genetic platforms such as pdna-mabs have the potential to address the significant challenge of cost and delivery time, without compromising safety and efficacy. muthumani et al. were the first to demonstrate a novel dmab approach to target cancer, developing an anti-prostate-specific membrane antigen (anti-psma) encoded dna in a mouse prostate cancer model [ ] . in vivo, their study showed expression for this dmab delivery of over days post-injection, with important anti-tumor functionality, as tumor-challenged mice exhibited tumor regression and greater than % survival days post-challenge compared to % survival in the vector control mice. the study also highlighted the potential for dmab inducing antibodydependent cellular cytotoxicity (adcc) and phagocytosis (adcp) with the recruitment of other immune cells such as natural killer (nk) cells and macrophages in identifying and clearing antigen-specific tumors. nk-cell-depleted mice injected with anti-psma dmab and challenged were significantly less able to clear tumor and had less than % viability after a -day challenge compared to nkexpressing mice. although in early stages of development, this treatment has the potential to offer an attractive clinical alternative/adjuvant therapy that can be coupled with current standard-of-care treatment options. pdna-mabs have demonstrated that they can not only target and shrink specific tumors and those in hematological cancer models in vivo, but they can have a survival advantage and are being generated from a likely cost-effective platform. duperret et al. reported on in vivo intermuscular dmab delivery of mouse anti-ctla , and showed serum concentrations of up to μg/ml, with a detectable level of expression of μg/ml for over a year [ ] . mice challenged with tumors and subsequently administered anti-ctla dmab showed clearance in eight out of ten animals, with all demonstrating immunological memory after re-challenge by clearing % of the tumor. investigators next designed and studied the functionality of dmabencoded anti-human ctla ipilimumab and tremelimumab in vitro using donor-derived peripheral blood mononuclear cells (pbmcs) co-incubated with two different luciferaseexpressing lymphoblast cancer models and showed a dose-dependent blockage of ctla , leading to increase luciferase expression. perales-puchalt et al. extended this work by engineering another important immune checkpoint inhibitor, anti-pd , and showed expression and functionality supporting the possibility of administering both anti-ctla and pd checkpoint as combination therapy [ ] . a similar approach is currently being evaluated in the clinic with recombinant nivolumab and ipilimumab and is demonstrating greater efficacy in combination, prolonging overall survival in melanoma patients [ ] . therefore, pdna-mab delivery of checkpoint inhibitors is important, and the data from early mouse studies are supportive for continued development of high cancer value targets. such developments provide opportunities to broaden the populations that receive such high-cost therapies. as the pdna-mab cancer approach continues through preclinical studies, new complementary applications are also being investigated. bites are another parallel approach that is demonstrating promise in the clinic for bringing t cells into proximity with tumor surface antigens. blinatumomab is the first bite antibody approved by the fda. it targets hematological acute lymphoblastic leukemia by binding to both cd t-cell coreceptor and the malignant b-cell cd antigen. this antibody allows for contact-dependent activation of t cells and initiation of cytotoxicity against lymphoblastic cells. although blinatumomab is highly promising in people, the bite has a short half-life of approximately h. the current regimen requires iv delivery for consecutive days and costs about us$ , per -day course of treatment for patients kg and above [ , ] . therefore, a plasmid-encoded delivered bite would undoubtedly improve upon the relative short expression time length and could significantly lower the cost of such an important treatment. building on previous dmab studies, perales-puchalt et al. engineered a dna-encoded bispecific t-cell engager (dbte) that binds to cd t cells and the her tumor target [ ] . this study demonstrated consistent in vivo expression and cytotoxic activity for approximately months, with potent functional activity showing / tumor regression/ clearance in mice > days after tumor challenge. stadler et al. encoded a similar bispecific antibody in mrna, encoding the tumor-associated antigen claudin, a tight junction protein and epithelial cell adhesion molecule (epcam), which is overexpressed in colorectal, prostate, ovarian, and endometrial cancers [ ] . they detected the translated mrna bispecific antibody, with cd and claudin or epcam-binding capability, in as little as h following intravenous delivery; plasma at the -h time point showed in vitro cytotoxicity at above %, with expression still present at the % level through day post-injection of mrna. in vivo data demonstrated three mrna doses of µg/mouse by iv injection compared to ten doses of µg/ kg of recombinant cd and claudin protein had comparable median tumor volume. the mrna-encoded bispecific antibody showed earlier and rapid tumor regression, with fewer overall doses. overall, both mrna-encoded bispecific antibodies and dbte pdna-mab approaches offer the ability to harness t-cell functionality in blocking tumor antigen function and initiating potent cytotoxicity, but vary in length of expression, stability, and dosing regimen, with dna having longer and more durable expression from a single injection compared to mrna. these are interesting and promising and likely complementary strategies for cancer immunotherapy that deserve significant attention. there is tremendous potential for mab delivery to have an impact in the infectious diseases arena. through advancements in antibody discovery technologies, highly specific mabs are being isolated directly from convalescent humans with activity against anti-microbial resistant bacteria, emerging and re-emerging viral pathogens, and parasitic and fungal diseases. yet, palivizumab (synagis), an anti-respiratory syncytical virus mab, is the only commercially successful mab approved as a standalone intervention to prevent an infectious disease. synagis costs can be > us$ , for a five-dose ( mg/kg/dose) regimen, administered at -month intervals [ ] . bezlotoxumab (zinplava), an antibody targeting treatment of clostridium difficile, was approved in and is indicated to reduce recurrence of c. difficile infection (cdi) in patients years of age or older who are receiving antibacterial drug treatment of cdi and are at a high risk for recurrence. according to the cms, zinplava currently costs > us$ /g [ ] and has a recommended dose of mg/kg iv over min [ ] . two additional mabs, raxibacumab and obiltoxaximab, have been approved for treatment of inhalational anthrax via the fda animal efficacy rule. the animal rule serves as a mechanism to gain approval for drugs and biologics using animal data in situations where it is not feasible or ethical to perform human studies. this mechanism is therefore reserved for unique circumstances such as evaluation of countermeasures against biological select agents. although many antibodies are currently under preclinical and clinical trial evaluation, in vivo mab potency, dosage, delivery, and patient cost remain important obstacles that impede administration for the treatment of infectious diseases. current antibody limitations impede widespread delivery for seasonal outbreaks and epidemics for influenza viruses. repeat doses are required to lower hiv viral loads. the development and manufacturing timeline for rapid response to emerging infectious disease outbreaks, for example, ebolavirus, is long and requires cold-chain storage, which may limit deployment in resource-limited areas. therefore, in vivo nucleic acidencoded antibody delivery represents an attractive approach for antibody administration targeting infectious diseases. both dna-and mrna-mabs represent potential rapid delivery platforms for emerging infectious disease control. both platforms start with initial antigen-specific mab sequence identification. for pdna-mabs, antibody sequences are nucleotide and amino acid optimized using in silico methods, followed by rapid gene synthesis and insertion into the dna vector backbone. pdna-mab dna can then be amplified in small or large batches, and is ready for in vivo delivery within a short timeline (fig. ) . the process for mrna-mab development is similar, but requires additional manufacturing steps. antibody sequences are engineered into a dna plasmid that includes a ′utr, ′utr, and polyadenylation signal. this is followed by plasmid dna amplification in bacteria. mrna-mabs must then be transcribed from the dna and formulated and then repurified and stored for administration in an lnp. this last step is critical for mrna stability and extends the timeline for delivery in vivo. several studies have demonstrated the protective efficacy in mice for pdna-mabs and mrna-mabs targeting dengue virus [ ] , influenza a and b viruses [ , , ] , ebolavirus [ , ] , zika virus [ ] , chikv [ ] , rabies [ ] , and hiv [ ] . close to billion people are estimated to be at risk for dengue virus infection worldwide, mostly in developing countries and in resource-poor settings [ ] . flingai et al. demonstrated the first generation of dmab engineering and delivery of anti-dengue virus antibodies [ ] . they evaluated both a wild-type (wt) and variant dmab incorporating the lala mutation into the fc region to mitigate dengue secondary infection. the anti-dengue virus dmab achieved peak expression levels of around μg/ml for at least weeks in mice and demonstrated neutralization of dengue virus serotypes - . both the anti-dengue virus wt-dmab and lala-dmab demonstrated protection against primary dengue infection. the lala-dmab demonstrated protection against dengue virus primary infection, as well as protection in a mouse model of antibody-dependent enhancement that models dengue secondary infection. overall, this demonstrates the portability of traditional mab engineering strategies to pdna-mab platforms for unique strategies against emerging viral infections. an estimated million people in the usa were infected by influenza viruses during the - influenza season, resulting in , hospitalizations and , deaths [ ] . advancing mab delivery could have an important impact on lowering overall influenza disease burden during seasonal influenza epidemics and against potential pandemic viruses. yamazaki et al. were one of the first to show in vivo expression of a pdna-mab anti-influenza a virus hemagglutinin (ha) mouse igg antibody [ ] . they demonstrated initial expression of approximately μg/ml anti-ha antibody in mice. utilizing an early generation electroporation (ep) device, they showed the benefit of delivery and hyaluronidase pre-treatment of the muscle to increase muscle uptake of dna, resulting in serum levels of > μg/ ml in mice and expression for days. the pdna-mab expressed anti-ha antibody was detectable in lung bronchioalveolar lavage and protected mice against lethal a/ h n /puerto rico/ virus challenge. following these studies, elliott et al. engineered human igg dmabs targeting pan-influenza a virus and pan-influenza b virus ha proteins (a/ha and b/ha, respectively) [ ] . using an advanced ep device and hyaluronidase pre-treatment, the anti-ha dmabs were expressed to levels of > μg/ml against a/ha and > μg/ml against b/ha, with expression for days. dose titration of both a/ha and b/ha dmabs was performed and evaluated in parallel with recombinant antibody to demonstrate equivalency to protein antibody. the anti-a/ha dmabs demonstrated protection against fig. nucleic acid-encoded mab timeline. antibodies are identified from infected individuals and sequences are confirmed to binding and/or neutralize the target antigen. the mab gene is then synthesized and cloned into a dna plasmid, followed by amplification and dna scale up. at this point, all pdna-mabs are ready for in vivo administration. mrna requires additional processing, including dna linearization, followed by mrna transcription. rna stability must be evaluated and formulated into lnps before an mrna-mab is ready for in vivo delivery. lnp lipid nanoparticle, mab monoclonal antibody, mrna messenger rna, mrna-mab mrna-encoded mab, pdna-mab plasmid dna-encoded mab seasonal h and h viruses, as well as neutralization of several group and group subtypes. similarly, anti-b/ha dmabs protected against both b/yamagata and b/victoria lineage virus infection in mice. additionally, a combination of both anti-a/ha and anti-b/ha dmabs protected against both a/california/ / and b/florida/ / virus challenges, demonstrating the potential for co-delivery, and protected against homologous re-challenge. in a parallel study, andrews et al. monitored the delivery of anti-influenza a/ h /ha or a/h /ha mouse igg a pdna-mabs long term in mice [ ] , observing similar pharmacokinetics as elliot et al., supporting the data generated with human igg dmabs in mice. they reported a maximum expression with one a/h /ha pdna-mab of μg/ml in balb/c mice, with expression lasting for weeks. a combination of all three a/ha pdna-mabs protected mice against lethal a/ h n /wsn/ and a/h n /aichi/ / challenges and protected against virus re-challenge. taken together, these three studies highlight mouse igg pdna-mabs as in vivo antibody development tools and the potential to evaluate fully human igg pdna-mabs in the mouse model to demonstrate preclinical protection against seasonal and pandemic influenza a and b viruses. the - ebolavirus disease (evd) epidemic was the largest to date, with over , confirmed cases and over , deaths. there is an ongoing outbreak in the democratic republic of congo, and additional gene-encoded alternatives could be useful for evd infection control to support post-exposure vaccines and therapeutics. antibodies against the zaire ebolavirus gp are demonstrating promising therapeutic protection against evd. the three-mab zmapp cocktail was the first antibody combination to demonstrate promise in an evd outbreak setting [ ] . andrews et al. encoded the zmapp cocktail antibodies into their pdna-mab platform [ ] , showing delivery of each individual mouse igg a antibody to a maximum of μg/ml and μg/ml for combined delivery and weeks of in vivo expression. the pdna-zmapp was delivered to mice, followed by challenge days later with mouse-adapted ebolavirus, demonstrating protection against lethal infection. in a separate study, patel et al. engineered different anti-gp dmabs, three mouse-human chimeras, and fully human igg dmabs [ ] . they performed a series of sequence, formulation, and delivery optimizations to achieve peak expression of dmab- targeting the gp fusion loop of > μg/ml and > μg/ml for dmab- , expressing for days following administration. they compared anti-gp dmab delivery in parallel with recombinant antibody. these studies demonstrated functional equivalency to bind antigen and that dmabs bind the same molecular epitope as the parent recombinant mab. both anti-gp dmab- and dmab- protected mice against lethal mouse-adapted ebolavirus challenge as single candidates as well as when delivered together. these studies also showed that anti-ebolavirus gp dmabs are protective in mice when delivered only days before lethal challenge and also provide long-term partial protection when challenged days postadministration. these studies are the first demonstration of the potential for prophylactic delivery of gene-encoded mabs to prevent ebolavirus infection. over billion people are at risk for zika virus infection [ ] . building on the early studies with the anti-dengue dmab, esquivel et al. engineered anti-zika virus wt-dmab and lala-dmab targeting the virus e protein [ ] . they showed average expression of μg/ml for the wt-dmab and μg/ml for the lala-dmab, with days of expression. both anti-zika virus wt-dmab and lala-dmab protected ag mice against zika virus challenge and, in addition, protected against zika-related damage to the testes. the investigators next delivered the anti-zika wt-dmab to rhesus macaques using a sequential administration strategy on days − , − , and − before challenge with zika virus prvabc on day . macaques expressed on average μg/ ml of wt-dmab at the time of challenge, and four out of five animals controlled zika virus infection. this was the first demonstration that a nucleic acid-encoded antibody can control an infectious disease challenge in a larger animal model, further supporting translation of the dmab platform for administration in people. this zika virus dmab candidate is now in a human trial (nct ), which we will discuss in more detail later in this review (sect. ). there are approximately million people living with hiv, with the envelope gp (env) being the primary target for immune responses. due to the high diversity of env proteins, vaccine development and therapy have been challenging. several env-targeted mabs have been identified and shown to broadly neutralize multiple circulating hiv- strains (reviewed in ref. [ ] ). wise et al. engineered a panel of broadly neutralizing hiv- antibodies as dmabs and delivered them in mice and non-human primates [ ] . the dmabs, delivered alone or in combination, demonstrated long expression for days and neutralized against the global panel viruses in an in vitro neutralization assay. the data highlight how the combination of sequence optimizations, formulation, delivery, and animal model development can lead to a significant in vivo expression level using pdna-mab platforms. to date, mrna-mabs, formulated in lnp, have been evaluated targeting rabies [ ] , hiv [ ] , and chikv [ ] . human anti-rabies immunoglobulin and the rabies vaccine must be delivered immediately following potential rabies exposure. thran et al. evaluated an mrna-mab platform for rapid expression of an anti-rabies antibody. they observed peak human igg titers of μg/ml within - h postadministration and expression for days, before it declined [ ] . this anti-rabies mrna-mab provided both pre-exposure (day − ) and post-exposure ( h post-infection) protection against lethal rabies virus challenge in mice. in this study, an anti-influenza b mrna-mab was also described as a control, expressing at similar levels to the anti-rabies mrna-mab. several promising broadly neutralizing antibodies have been identified targeting the hiv env protein and are demonstrating promising results for hiv treatment (reviewed in ref. [ ] ). pardi et al. [ ] encoded the hiv-vrc antibody into a nucleoside modified mrna. they achieved peak expression levels of > μg/ml day post-administration, which lasted for days. eight weeks of expression were achieved with weekly mrna-vrc delivery for weeks. protection against sf and jr-csf hiv- challenges was observed in humanized mice receiving the mrna-vrc , similar to recombinant mab. recently, kose et al. described an anti-chikv mrna-mab that was delivered by iv injection to ag mice that were then challenged with chikv. they demonstrated dose-dependent protection against the virus, with no viral load and protection against arthritis and muscoskeletal disease in competent c bl mice [ ] . they further evaluated expression in cynomolgus macaques, indicating rapid expression of mrna, and repeat dosing at a -day interval. however, the infusion duration was over min, which a significant time for field delivery. additional studies to reduce mrna-mab infusion times would greatly improve the platform to make it more accessible in low-resource settings. antibodies are promising for control of antimicrobial resistant organisms and other serious bacterial infections. each year, at least two million people acquire antimicrobial resistant infections, including multi-drug resistant pseudomonas aeruginosa. first-line antimicrobials are rapidly becoming ineffective, and alternative biologics are urgently needed. patel et al. described the first delivery of an engineered, non-natural bispecific immunoglobulin using the dmab platform [ ] , achieving expression levels of μg/ml for days. this dmab targeted two p. aeruginosa proteins (pcrv type iii secretion system and psl exopolysaccharide), protecting against lethal pneumonia in a mouse challenge model. lower levels of pro-inflammatory cytokines, organ colonization, and lung pathology were observed following both monospecific and bispecific dmab delivery, similar to the recombinant mab positive control. additionally, the bispecific dmab demonstrated adjunctive activity when delivered in conjunction with a carbapenem-family antibiotic. this demonstrates the potential for dmab delivery as a standalone intervention or alongside first-line antimicrobials. new-generation pdna-mab development to protect against eskape (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, enterobacter species) pathogens and other resistant bacteria have the potential to dramatically impact infection control and the spread of anti-microbial resistance in both hospital and community settings. around , cases of lyme disease are reported each year to the us centers for disease control (cdc); however, it is likely that the actual number of cases each year is closer to , in the usa [ ] . wang et al. engineered an anti-borrelia burgdorferi dmab candidate expressing a transmission-blocking antibody targeting ospa [ ] . initial expression of the wt-dmab averaged . μg/ml, and sequence optimizations further increased expression to . μg/ml. the modified dmab retained bactericidal activity comparable to the original dmab and recombinant parent mab. both the wt-dmab and modified dmab were protective against b. burgdorferi challenge in mice. this supports the potential for a dmab approach to protect against lyme disease transmission from tick to host. to date, the only mrna-mab targeting a bacterial pathogen is against botulinum toxin a [ ] . botulism is caused by the toxin from clostridium botulinum and requires immediate delivery of anti-toxin to prevent paralysis. thran et al. encoded a camelid vhh antibody targeting botulinum neurotoxin serotype a (bont/a), with levels reaching > μg/ ml in less than h post-administration. the mrna-mab successfully protected against bont/a toxin challenge when delivered h post-exposure. additional development of mrna-mabs for rapid delivery against bacterial toxins that require protection within hours of exposure has the potential to be important. pdna-mab platforms can be utilized to rapidly evaluate potential mab candidates. antibodies isolated from infected or convalescent individuals can be directly engineered into pdna-mab for in vivo evaluation in a biologically relevant environment. the top candidates from such a pipeline can then be rapidly tested in animal models. the simplicity and flexibility of synthetic dna plasmid enables rapid scaling for administration in a person. this could open a new paradigm for rapid antibody administration in response to an outbreak (fig. ) . several funding agencies have expressed interest in this process for the rapid development of nucleic acid biologics, including the national institutes of health, defense advanced research projects agency (darpa), biomedical advanced research and development authority (barda), and the bill and melinda gates foundation (bmgf). the darpa pandemic prevention program (p ) was initiated in , with the goal of pandemic prevention in less than days through rapid deployment of nucleic acid-vectored synthetic dna or mrna technologies delivering protective antibody. similar to what is shown in figs. and , the program is supporting rapid isolation, nucleic acid development, and delivery approaches to achieve protective in vivo expression levels within days of administration. in the final stage of the darpa p program, the dna and mrna technologies will be tested in a capability test with an unknown pathogen. importantly, in an earlier study, muthumani et al. delivered an anti-chikv dmab in combination with a chikv dna vaccine, demonstrating both immediate and persistent protection against lethal challenge in mice [ ] . this study supports the concept that pdna-mabs have the potential to be delivered in conjunction with protective vaccination, providing rapid protection, with the pdna-mab to cover the period needed to establish vaccine-induced immunity. the concept of immediate and persistent protection introduced by muthumani et al. is intriguing. since pdna-mabs express for months, they have the potential to afford a new way for both pre-exposure and post-exposure prevention that has previously been unachievable with recombinant biologics. additional studies to confirm immunogenicity and protective efficacy with combined pdna-mab-vaccine delivery would be informative. by comparison, there are much fewer studies with mrna-mab delivery against infectious diseases. as the field advances, it will be important to utilize the information coming out of these studies to advance new approaches and to better define the pathway for development. large animal studies and data from human clinical trials will be highly informative for gene-encoded mab delivery platforms. an older study by tjelle et al. demonstrated in vivo expression of a mouse igg a pdna-mab in sheep, observing expression levels as high as ng/ml before development of sheep anti-mouse antibodies [ ] . more recently, hollevoet et al. describe delivery of fully sheep antibodies, achieving expression levels up to . µg/ml [ ] . this is the first study to demonstrate microgram expression levels in larger animals more similar in mass to people. previously, timmerman et al. evaluated plasmid dna delivery of an anti-idiotype antibody targeting b-cell lymphoma, observing modest anti-tumor activity [ ] . however, this study did not measure in vivo expression levels. building on these previous studies, significant advancements in formulation and delivery are now leading to higher expression levels in monkeys (fig. , not previously published) . at the start of these studies, animals received four to six im injections (animals in black); however, only - ng/ml human igg expression levels were detectable in rhesus macaques following dmab delivery. following a series of optimizations, expression increased to a - µg/ml (summary of data from nonhuman primates receiving - mg total dna in - injection sites) (fig. , magenta) , and additional device/delivery optimizations, including hyaluronidase treatment, have led to further increases, now to levels > µg/ml ( mg total dna injection, six injections), which express for days before elimination via the development of macaque antihuman antibodies (fig. , teal) . wise et al. demonstrated that using an optimized delivery strategy with a -mg dose, divided across six injection sites, anti-hiv dmab delivery can achieve in vivo expression levels as high as . µg/ ml [ ] . these expression levels parallel mrna expression levels, which are at > μg/ml in rhesus macaques [ ] . only aav can surpass these levels in macaques; however, virus take in animals is reported to be not consistent with aav, and there were wide variations in delivery observed, ranging from to µg/ml across multiple studies. dose scaling is an important consideration for nonviral and viral vector-encoded antibodies. plasmid dna is transiently transfected into muscle and may persist in the tissue for a few weeks. likewise, viral vectors typically persist in tissues for a few months, or permanently if integrated. this is quite different to a biologic, for which the maximum amount of drug is at the time of infusion. mrna is most similar to traditional protein biologics, as it is rapidly expressed and degraded. therefore, initial empiric dose escalation studies will be important to set the bar and move the field to optimal doses in the clinic. additional optimizations including sequential administration [ ] , formulation, and delivery technology are rapidly developing and will likely contribute to increases in delivery in vivo. recently, priddy et al. evaluated in vivo administration of an aav-vector encoding the anti-hiv pg (aav-pg ) antibody in people [ ] . no expression of pg was detected in participants' sera, although pg was detected in muscle biopsies. one possibility for the lack of detection is the high enzyme-linked immunosorbent assay (elisa) limit of detection ( . µg/ml), and more sensitive assays are likely needed as development continues. furthermore, ada was observed against both the aav vector and pg , suggesting that it did induce an immune response. more studies are needed in this regard to build on this initial study. a first-in-human phase i trial evaluating the safety, tolerability, and pharmacokinetic expression of the anti-e protein zika virus dmab (ino-a ) is currently underway (nct ; trial start date: february , ; estimated study completion: february ). in this study, dose escalation starting at . mg up to mg of ino-a is being evaluated in healthy human volunteers. supported by the preclinical data in mice and macaques, the human study will deliver dmab in formulation with recombinant human hyaluronidase to improve drug dispersion. in parallel, a first-in-human mrna-mab phase i trial evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of an mrna-encoded anti-chikv antibody (mrna- ) is also underway led by moderna (nct ; trial start date: january , ; estimated study completion: september ). this study will evaluate dose escalations starting with . up to mg/kg via iv administration. at a recent presentation at the oligonucleotide therapy society ( ), moderna presented mrna- data demonstrating expression in people at levels > µg/ml even in the lowest dose group [ ] . following iv infusion with mrna- , peak expression was reached within h, declining afterwards, with detectable levels for at least days, with the . mg/kg dose. patients were treated with anti-histamine, and at . -mg/kg doses there were multiple adverse events observed. however, this trial is a very positive development for nucleic acid biologics. while much more work is needed, this study is an important advance for the field. together, these trials will be informative to help understand additional optimizations that must be undertaken to improve human translation of nucleic acid-encoded antibodies and will be significant for the field. the clinical use of mabs has had a dramatic impact on improving human health and affording a better quality of life for many individuals. however, use has been restricted to disease populations with high commercial value, due in part to the high cost of development of these biologics. the future for pdna-mabs and gene-encoded antibodies is promising and presents interesting opportunities to make life-saving biologics more accessible. pdna-mabs build on existing antibody technologies and can be engineered to deliver new types of biologics, such as bispecifics [ ] , trispecifics, and alternative forms and isotypes. other modifications including half-life extension to improve interactions with the neonatal fcrs and glycosylation profiles would be highly informative for modulation of in vivo duration and effector functions. further in vivo modification may be accomplished through co-delivery of different enzymes to direct post-translational processing. pdna-mabs and mrna are opening a new 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lyme disease: data and surveillance. centers for disease control and prevention anti-ospa dna-encoded monoclonal antibody prevents transmission of spirochetes in tick challenge providing sterilizing immunity in mice immunogenicity of a plasmid dna vaccine encoding chimeric idiotype in patients with b-cell lymphoma adeno-associated virus vectored immunoprophylaxis to prevent hiv in healthy adults: a phase randomised controlled trial the authors would like to thank alex t. bebbington for the drawing of the cell (fig. ). funding the authors are grateful for funding from the bill and melinda gates foundation (opp and opp award to david b. weiner) and national institutes of health (r ai award to david b. weiner and u ai subaward to ami patel). the research described in this manuscript was funded by the bill and melinda gates foundation (opp and opp award to david b. weiner). conflict of interest m. b. declares no competing interests. a. p. is listed as an inventor on patents related to dmabs. d. b. w. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board service. remuneration received by d. b. w. includes direct payments or stock or stock options, and, in the interest of disclosure, he notes potential conflicts associated with this work with inovio and possibly others. in addition, he has patents on dmabs and dna vaccine delivery.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -ytv dwr authors: casadevall, arturo; scharff, matthew d. title: return to the past: the case for antibody-based therapies in infectious diseases date: - - journal: clin infect dis doi: . /clinids/ . . sha: doc_id: cord_uid: ytv dwr in the preantibiotic era, passive antibody administration (serum therapy) was useful for the treatment of many infectious diseases. the introduction of antimicrobial chemotherapy in the s led to the rapid abandonment of many forms of passive antibody therapy. chemotherapy was more effective and less toxic than antibody therapy. in this last decade of the th century the efficacy of antimicrobial chemotherapy is diminishing because of the rapidly escalating number of immunocompromised individuals, the emergence of new pathogens, the reemergence of old pathogens, and widespread development of resistance to antimicrobial drugs. this diminishment in the effectiveness of chemotherapy has been paralleled by advances in monoclonal antibody technology that have made feasible the generation of human antibodies. this combination of factors makes passive antibody therapy an option worthy of serious consideration. we propose that for every pathogen there exists an antibody that will modify the infection to the benefit of the host. such antibodies are potential antimicrobial agents. antibody-based therapies have significant advantages and disadvantages relative to standard chemotherapy. the reintroduction of antibody-based therapy would require major changes in the practices of infectious disease specialists. the antibiotic era is about years old. as we approach the st century, the progress made in controlling infections is threatened by the growing numbers of immunosuppressed individuals; the emergence of new pathogens; the reemergence of old pathogens; and the widespread resistance of organisms to antimicrobial drugs. each development poses its own set of challenges; in combination, these developments are already undermining the efficacy of antimicrobial chemotherapy. there is a worldwide epidemic of immunosuppression due to malnutrition, aids, medical therapies for cancer and autoimmune diseases, and organ transplantation. immunosuppressed hosts provide ecological niches for low-virulence microorganisms [ ] . in addition, antimicrobial drugs are less effective and sometimes are unable to eradicate infection in individuals with impaired immunity. classical pathogens such as mycobacterium tuberculosis and treponema pallidum have reemerged and are difficult to treat in immunosuppressed individuals [ , ] . the increasing frequency of drug-resistant organisms is an equally alarming development, and resistance may be developing faster than new antibiotics can be introduced [ ] . for example, in one new york city hospital, the percentage of vancomycinsusceptible enterococcus faecium decreased from % to % in years [ ] . the problem of resistance is not limited to bacteria; there are numerous reports of fluconazole-resistant candida albicans [ ] . we briefly review the use of antibody-based therapy in the early th century and make the case for reintroducing passive antibody administration for the treatment ofinfectious diseases. advances in monoclonal antibody (mab) technology provide new opportunities for developing antibody-based therapies. in the case of pathogens for which antimicrobial chemotherapy already exists, antibody-based therapies could be used with existing chemotherapy to improve outcome and possibly reduce the development of resistance. in the case of pathogens for which no effective chemotherapy exists, antibody-based therapies could be used for primary therapy. given the diminishing efficacy of existing antimicrobials because of widespread resistance and the difficulties of treating infections in immunosuppressed individuals, the reintroduction of antibody-based therapies is an option that should be given serious consideration. this reintroduction would require major changes in the practice of infectious disease medicine. the administration of immune serum (usually animal) for the treatment of infections was called serum therapy and was introduced in the s for the treatment of diphtheria. by the s, serum therapy was widely used for the treatment of table . infectious diseases that were treated with antibody-based therapies in the preantibiotic era. bacterial and viral infections (table ) . for example, in the late s, % of patients with type i pneumococcal pneumonia admitted to boston city hospital were treated with type-specific serum [ ] . from to at bellevue hospital (new york), > , patients with erysipelas were treated with serum [ ] (figure ). the efficacy of serum therapy varied with the type or severity of infection. several large controlled studies revealed that type-specific serum reduced the mortality ofpneumococcal pneumonia [ ] . serum therapy also appears to have significantly reduced mortality due to meningococcal meningitis in some epidemics [ , ] . serum therapy reduced mortality due to haemophilus irif/uenzae meningitis, but the effect was small [ ] [ ] [ ] . serum therapy for erysipelas reduced mortality in comparison to historical controls [ , ] . serum therapy reduced mortality in diphtheria, and antibody therapy continues to be used today to treat this disease [ ] . the efficacy of serum therapy for whooping cough, anthrax, dysentery (shigella dysenteriae), and gas gangrene was uncertain. human convalescent serum was effective for prophylaxis ofmeasles, which had a mortality rate of %- % in some populations [ ] [ ] [ ] . the effectiveness of serum in the pre-paralytic stage of poliomyelitis was uncertain [ , ] . no consistently effective sera were developed against many pathogens, including staphylococcus [ ] , mycobacterium, and salmonella species [ ] . the historical use of serum therapy provides lessons for the effective use and development of antibody-based therapies. accurate microbiological diagnosis was essential for successful treatment, and serum therapy was most effective when used for prophylaxis or for therapy early in the course of infection. note. this is not a complete list. [ ] [ ] [ ] [ ] [ ] nevertheless, it was possible to treat established bacterial infections such as meningococcal meningitis and pneumococcal pneumonia if antibody was administered shortly after symptoms began [ ] . antibodies functioned as direct antibacterial agents (e.g., pneumococcal antisera) or antitoxins (e.g., diphtherial antisera). nonphysiological and nontherapeutic animal models of infection allowed investigators to successfully identify clinically useful antibody reagents [ ] . a considerable amount of basic immunologic and microbiological research was required to develop each serum, and many fundamental discoveries were made in the search for better sera. (the contributions of pneumococcal research to basic science are discussed in [ ] .) sulfonamides were introduced in and rapidly became the standard therapy for many infections [ ] . because antimicrobial chemotherapy had significant advantages over serum therapy, the latter was largely abandoned. systemic administration of animal sera caused fevers, chills, and allergic reactions [ , ] ; "serum sickness," a self-limited syndrome characterized by rash, proteinuria, and arthralgias, occurred in %- % of patients because of immune complex disease. in addition, strain typing was necessary for choosing pneumococcal antisera; there was significant lot-to-lot variation in serum efficacy [ ] ; and dosing was based on clinical experience. inadequate dosage, delays in treatment, errors in typing, mixed infections, and complicated infections (e.g., empyema) could result in failure of serum therapy [ ] . serum was expensive because of the costs of animal husbandry, antibody purification, refrigeration, and reliance on mouse protection tests for standardization. chemotherapy was less toxic and more effective than serum therapy. treatment with type-specific serum reduced the mortality of pneumococcal pneumonia from %- % to %- % [ ] , whereas the mortality rate among patients treated with sulfonamide was % [ ] . serum therapy reduced the mortality rate of erysipelas from % to % [ ] , whereas the mortality rate for patients treated with sulfonamides was %- % [ ] . for meningococcal meningitis, the efficacy of serum depended on the epidemic. in the mid- s, waghelstein [ ] reported that the mortality rate for patients with meningococcal meningitis who were treated at sydenham hospital (baltimore, md) with sulfonamide was . % and for those treated with serum it was %. in controlled trials, the superiority of chemotherapy over serum therapy was lessevident. for example, the mortality among patients with meningococcal meningitis who were treated early with adequate serum therapy was . % vs. . % for sulfanilamide therapy [ ] . the mortality among patients with pneumococcal pneumonia treated with antimicrobial drugs or serum was % and . %, respectively, but there was no difference in the mortality rates among younger .. literature sent on request. is used at bellevue hospital. it is supplied in packages of one therapeutic dose of approximately ten cubic centimeters. the data cited were published in [ ] . patients, and younger patients who received serum recovered faster than those who received antimicrobials [ ] . in the early days of the antibiotic era there was interest in using combination therapies with antibiotics and serum. sulfonamides and serum had a synergistic or additive effect against streptococcus pneumoniae [ ] [ ] [ ] [ ] , streptococci [ ] , and meningococci [ ] . the finding that sulfonamides made pneumococci more susceptible to antibody-mediated phagocytosis supported the idea of combination therapy [ ] . it was similarly recognized that sulfonamides, unlike antibody, did not neutralize bacterial toxins or modify the course of disease caused by diphtheria or tetanus toxins [ ] . combination therapy was recommended for scarlet fever [ ] , pneumococcal pneumonia [ , , ] , whooping cough [ ] and meningitis due to pneumococcus [ , ] , meningococcus [ , , , ] , or h. influenzae [ ] . however, the side effects of serum made the potential benefits of combination therapy marginal [ ] . in summary, serum therapy was abandoned because of toxicity, difficulty in administration, narrow specificity, lot-to-lot variation, and expense. chemotherapy was less toxic and easier to use, lots were more consistent, and it was more effective in eradicating infection. in addition, there was no need for strain typing with chemotherapy. however, it is ironic that the broad antimicrobial activity of these drugs used for chemotherapy might have contributed to widespread resistance, and today susceptibility testing is essential to the selection of appropriate antimicrobial drugs. although antibodies are no longer used directly as antibacterial drugs, they continue to be used in infectious diseases (for recent reviews see [ ] [ ] [ ] ). the majority of antibody preparations are now human. antibody administration reduces infection in individuals with immunodeficiencies [ ] [ ] [ ] [ ] and is effective for postexposure prophylaxis against measles, hepatitis a and b, rabies, and varicella viruses. specific antibody is used for the treatment of botulism [ ] , diphtheria [ ] , tetanus [ ] , snake bites [ ] , and spider stings [ ] . passive antibody administration has been used for prophylaxis and/or therapy of several viral infections, including cytomegalovirus (cmv) [ , ] , rotavirus [ ] , lassa virus [ ] , varicella [ ] , enterovirus [ , ] , and parvovirus b infections [ ] . in recent years, there has been renewed interest in the use of antibody preparations to prevent infection in high-risk groups. intravenous administration of polyclonal antibody has been reported to reduce the number of infections in patients with aids [ ] [ ] [ ] , patients in surgical intensive care units [ ] , organ transplant recipients [ ] , and neonates [ , ] (for critical reviews and commentary on this practice see [ ] [ ] [ ] [ ] ). antibody therapy is used for a variety of illnesses that may or may not be infectious, including autoimmune thrombocytopenia, kawasaki disease, and autoimmune neuromuscular diseases [ , ] . passive antibody administration is used for prevention of rh-hemolytic disease [ ] . equine lymphocyte antiserum [ ] and murine mabs to lymphocytes are used to prevent organ rejection [ ] . digitalis-binding antibodies are useful for the treatment of digoxin toxicity [ ] . thus, antibody therapy is still widely used in medicine, but its role in the treatment of infections is limited largely to viral and toxin neutralization and replacement therapy in patients with immunoglobulin deficiencies. in reconsidering the antibody option it is clear that heterologous antibody (i.e., serum therapy) is a poor choice because of toxicity. human immune sera have fewer side effects, but there are concerns about availability, potency, and consistency. a major disadvantage of all immune sera is that specific antibodies make up a small minority of the antibody present. there is significant variation in pathogen-specific opsonic activity of commercially available immunoglobulin preparations [ ] . there is also concern about the potential of human sera to transmit infectious agents [ ] . the discovery of hybridoma technology in [ ] and recent advances in mab technology, including the generation of humanized antibodies [ ] , make mab-based therapies a more attractive therapeutic option. in contrast to polyclonal sera, mabs are homogeneous and reproducible reagents that can be generated in large amounts. generation of mabs from mice and rats is still easier and more efficient than production of human mabs. however, several technologies are available for the humanization of murine mabs and generation of human mabs [ ] . mouse-human chimeric antibodies can be constructed by linking the genes expressing the mouse variable region to human constant region genes [ ] . the result is a molecule that is mostly human and has a longer half-life than the murine precursor [ ] . as an alternative, the antigen binding regions of murine mabs can be grafted into human antibody frameworks by molecular techniques [ ] , resulting in molecules that are almost completely human in origin. other strategies to generate human antibodies are the transformation of human b cells, the generation ofrecombinant antibody libraries from human b cells, and the use of transgenic mice that have the human immunoglobulin locus. recent setbacks for mab-based therapies: historical perspective clinical trials of antiendotoxin mabs for gram-negative sepsis have produced inconclusive results [ , ] . when the difficulties encountered in developing antiendotoxin mab strategies are considered in the context of the development of serum therapy, they do not seem unusual or unexpected. for example, passive protection with pneumococcal antisera was demonstrated in by the klemperers [ ] , but - years elapsed before serum therapy for pneumococcal pneumonia was widely accepted. for pneumococcal antiserum therapy to be consistently successful, several developments were necessary. these included the appreciation of antigenic variation in pneumococci [ ] , the need for type-specific sera [ ] , the development of rapid in vitro assays to establish antigenic type (i.e., capsular swelling and agglutination reactions) [ ] , the development of methods to standardize potency (mouse protection test) [ ] , and improved purification techniques that would reduce the toxicity of serum. for meningococcal antisera the situation was reversed; flexner's serum significantly reduced mortality in early epidemics of meningitis [ ] but was subsequently less effective, possibly as a result of antigenic changes in neisseria meningitidis [ ] . research into better meningococcal antisera was then hampered by the lack of useful animal models, loss of strain virulence in vitro, and poor understanding of the antigenic diversity of n meningitidis [ , ] . a lesson from the preantibiotic era is that a considerable amount of basic research on the immunology and pathogenesis of each infection is usually necessary before antibody therapy can be developed that will be successful in clinical practice. spectrum ofactivity. antibodies can modify bacterial, fungal, parasitic, and viral infections and are a class of biological agents with broad antimicrobial activity against diverse pathogens. antibody-based therapies are usually pathogen-specific and have the theoretical advantage that they should not affect the normal flora of the patient or select for resistance in nontargeted microbes. nevertheless, narrow specificity is a disadvantage because mixed infections may not be treated by a single antibody preparation. pneumonia due to s. pneumoniae with more than one serotype was recognized as a reason for the failure of type-specific serum therapy [ ] . one solution is to use cocktails of mabs active against common antigenic types. cocktails may be designed to include mabs to multiple serotypes and mabs with multiple isotypes to enhance antibody effector function. pathogen-specific drugs (such as mabs) are less attractive to the pharmaceutical industry because the narrow specificity reduces the potential market for the drug. however, the increasing incidence of resistant organisms resulting from the use of broad-spectrum antibiotic regimens could make pathogen-specific drugs attractive to drug companies. precedents for the development of pathogen-specific drugs already exist in the area of antiviral drug research. mechanisms ofaction. antibodies mediate protection by a variety of mechanisms. direct antibody mechanisms of action include inhibition of attachment, agglutination (and immobili-cm ; (july) zation), viral neutralization, toxin neutralization, antibodydirected cellular cytotoxicity, complement activation, and opsonization [ ] . antibody therapy has the potential to enhance immune function in immunosuppressed hosts. however, since the mechanism of action of many antibodies involves promoting microbial clearance through nonspecific cellular immunity, antibody-based therapies may be less effective in individuals with defective macrophage, neutrophil, and natural killer cell function. in this regard, it is encouraging that antibodies are effective against pseudomonas aeruginosa in neutropenic mice [ ] and that they can reduce the number of infections in pediatric patients with aids [ ] . antibodies are usually considered to be "protective" effector molecules of the immune system. however, not all antibody responses to pathogens are protective and some may be deleterious to the host. for example, some viral-specific antibodies are capable of enhancing infection [ ] . thus, antibody molecules being considered for clinical development will require extensive testing in vitro and in vivo. studies of the mechanisms of antibody action are important for understanding the mode of protection and for designing clinical trials. pharmacokinetics. the pharmacokinetics of human igg suggests several useful characteristics for its role as an antiinfective agent. these include a long half-life (~ days [ ]), good tissue penetration [ ] , and, depending on the isotype, the ability to either cross the placenta to provide antibody protection to fetuses and newborns [ ] or to be excluded from the placenta if fetal toxicity is a concern. heterologous mabs (i.e., murine mabs) have shorter half-lives in humans, but chimeric and humanized mabs have longer half-lives than their murine precursors. mabs with a longer half-life can, in principle, be engineered by altering the regions of the constant domain that regulate clearance. a disadvantage of antibody-based therapies is the need for systemic administration. oral administration is unlikely to be effective, with the possible exception of therapy for enteric pathogens [ , ] . the blood-brain barrier is a potential obstacle for antibody therapy for infections of the brain. however, in infections of the brain in which there is inflammation, there is increased antibody penetration, and intravenous administration of antibodies appears to have been successfully used for therapy of meningococcal meningitis [ ] . antibodybased therapies can also be administered directly into the subarachnoid space, as has been done for the treatment ofmeningococcal and h. influenzae meningitis [ , ] . enhanced penetration of the brain can be achieved by modifying the charge of the molecule [ ] or by linking it to carrier proteins that cross the blood-brain barrier [ ] . antibody therapy for brain infections may be feasible if the blood-brain barrier is leaky, if antibody is administered directly into the subarachnoid space, or if antibodies with greater brain penetration are used. additional research into the pharmacokinetics of antibodies in infection is likely to be required for optimal use of antibody-based therapies. studies of antibody binding to tumors have revealed complex pharmacokinetics [ ] . there may be problems with antibody penetration into abscesses as have been found with tumors. toxicity. immunoglobulins are generally safe drugs [ ] . nevertheless, serious side effects have been reported with highdose ( . - g/kg) antibody therapy, including rare cases of renal failure [ ] , aseptic meningitis [ ] , and thromboembolic events [ , ] . high-dose immunoglobulin therapy is unlikely to be required in antiinfective mab therapy. in the past, serum therapy was effective against various pathogens despite the fact that immune sera contained only small amounts of specific antibody. mabs have significantly higher specific activity than polyclonal preparations. for example, . mg of two anti-tetanus toxin human mabs provides the same activity as - mg of immune globulin [ ] . thus, the toxicities described for high-dose immunoglobulin therapy may not be relevant in antiinfective therapies with mabs. until fully human mabs are available, rodent, mousehuman chimeric, or humanized mabs are therapeutic options. for over a decade, murine mabs directed against t cells have been used to prevent organ graft rejection in humans [ ] . a murine igm to endotoxin (e ) was tested in patients with sepsis and appeared to be a safe treatment [ ] . although administration of murine mabs is generally well tolerated in humans, allergic reactions occur in %- % of patients [ ] and most patients develop antibody responses to the murine mabs that may interfere with their therapeutic function [ , ] . experience with human-mouse chimeric antibodies and humanized mabs is accumulating rapidly as clinical trials with several compounds progress. a chimeric anti-cd mab for the treatment of rheumatoid arthritis has been well tolerated [ ] , but approximately half the patients had infusion-related side effects (headaches, nausea, fever, and chills), which were diminished by slowing the infusion rate [ ] . mouse-human chimeric and humanized mabs are less immunogenic than their murine precursors [ , ] , but antiidiotypic responses occur after repeated treatments [ ] . overall, the experience with chimeric and humanized mabs suggests that they are relatively safe compounds [ , [ ] [ ] [ ] [ ] . antigenic variation and antibody resistance. the efficacy of antibody-based therapies may be diminished by antigenic changes in the pathogen. this could be minimized by antigenic surveillance systems, as is presently done for influenza virus. antibody-resistant mutants can be generated in the laboratory [ ] , and it is likely that the same can occur in patients. mechanisms of antibody resistance include mutations that change the antigenic site and protease production. antibody use may select for antigenically distinct variants. evidence for the horizontal transfer of iga protease genes exists in neisseria gonorrhoeae and it is conceivable that widespread antibody use would result in selection of protease-producing strains for many pathogens [ ] . thus, large-scale use of antibody-based therapies may result in rapid emergence of antibody resistance in a manner analogous to that of antibiotic resistance. however, one advantage of antibody-based therapies is the versatility of antibody compounds. for example, emergence of an antibodyresistant strain could be countered by a new antibody directed toward the mutated epitope or another antigenic target. emergence of protease-producing strains could be countered by designing antibodies without protease cleavage sites (although these may then be recognized as foreign and be immunogenic) or by addition of antiprotease mabs. selection of antibodyresistant organisms could be minimized by using cocktails of mabs directed at multiple antigenic targets or by using a combination of antibodies and antimicrobials. cost. cost effectiveness is a significant concern that is likely to be a major obstacle for the development of passive antibody therapies. antibody prophylaxis for cmv infections can cost $ , to $ , per patient [ ] . the cost effectiveness of antibody therapy for prevention of infection in leukemic patients has been questioned [ , ] . however, in selected populations such as premature infants, antibody therapy may be cost effective [ ] . furthermore, the cost of a drug when first introduced may not reflect its long-term costs given the potential for improvements in technology and production. mabs are presently made in tissue culture, so the cost of production is high. mabs can be made in bacteria or yeast, and less costly means of production might be developed [ ] . in the past, serum therapy was used despite its high cost because it was believed to be effective. new antibody-based therapies are likely to be used if they prove to be effective. for pathogens such as s. pneumoniae, n. meningitidis, and h infiuenzae, there is a large body of experimental and clinical evidence to support the development of passive antibody therapy. however, for many pathogens, antibody immunity has not been proven to be important. for example, cryptococcus neoformans is a fungus for which the importance of antibody immunity is uncertain. however, administration of preformed antibodies can modify the course of cryptococcal infection in various animal models [ ] [ ] [ ] [ ] [ ] , and the combination of antibody and amphotericin b is a more effective treatment than the use of either agent alone [ ] [ ] [ ] . studies with mabs have shown that there are protective, nonprotective, and deleterious antibodies to the c. neoformans capsule polysaccharide [ ] . heavy chain isotype [ , ] and epitope specificity [ ] are important determinants of protective efficacy. the existence of protective and nonprotective antibodies against c. neoformans, a fungus for which antibody immunity mayor may not be important, provides a paradigm that may be applicable to other pathogens. we propose the hypothesis that for every pathogen there exists an antibody that will modify the course of infection to the benefit of the host; such antibodies are candidates for development as antimicrobial drugs. our proposal includes the use of antibodies for intracellular pathogens; for example, anti-bodies may exist that could prevent cell entry of intracellular bacteria and/or shift the intracellular location of such bacteria by promoting internalization through fe receptors. an mab that inhibits intracellular toxoplasma gondii infection has recently been described [ ] . in addition, antibodies have been described that can neutralize viruses intracellularly [ ] or penetrate the nuclear membrane [ ] . our proposal is not limited to those pathogens for which antibody immunity has been demonstrated to be protective. conclusions on the importance of antibody immunity are usually based on observations made with polyclonal sera (i.e., passive protection or correlation of immunity with the presence of antibody). since polyclonal sera might contain protective antibodies, nonprotective antibodies, and disease-enhancing antibodies, the existence of protective antibodies cannot be ruled out on the basis of absence of protection in experiments involving passive transfer of polyclonal sera or immunizations. conversely, experiments that demonstrate that polyclonal sera can mediate protection indicate the existence of protective antibodies. for pathogens for which polyclonal antibody has shown no protection, the question of whether useful antibodies exist must be reevaluated with mabs since the presence of nonprotective or deleterious antibodies in polyclonal sera could create confounding variables. a corollary of our proposal is that antibody-based therapies may be developed against pathogens for which antibody immunity is not considered to be important. infections that are difficult to treat and that can be modified by antibody immunity provide a logical starting point for the development of antibody-based therapies. antibodies have historically been more effective in prophylaxis than in therapy. antibody-based therapies have traditionally been most effective in infections where viral and toxin neutralization modifies the course of the disease. however, most of the historical experience was gained with polyclonal preparations of uncertain composition and mayor may not be applicable to mab preparations with high activity. opportunistic infections. infections with low-virulence organisms in immunosuppressed individuals are often difficult to treat and are sometimes incurable. since the problem is deficient immunity, antibody therapy is an attractive option because antibodies can enhance immune function. table lists examples of opportunistic pathogens for which antibody can modify the course of infection. antibiotic resistance. drug-resistant organisms are targets for development of antibody-based therapies. the spread of penicillin-resistant s.pneumoniae, methicillin-resistant staphylococcus aureus, and multiply-drug-resistant e. faecium has limited the useful antibiotic arsenal against these pathogens [ , , ] . for pneumococcus, antibody therapy has been shown to be useful [ ] . antibody may be useful against staphylococcal infection [ , ] . for e.faecium, the role of antibody immu-ern ; (july) table . opportunistic pathogens for which experimental data suggest a potential role for antibody-based therapies. nity is uncertain, but some patients make opsonic antibodies, which might be protective [ ] . significant work has already been done in the development of pseudomonal antibodies for resistant gram-negative organisms (table ). the addition of antibody-based therapies to chemotherapy may slow the development of resistance and improve outcome. toxin neutralization. the ability to neutralize toxins is a unique characteristic ofantibody-based therapies. antitoxin antibodies are useful for the treatment of diphtheria, tetanus, botulism, snake bites, and black widow bites (see above). recent attempts to develop antibody therapy for gram-negative sepsis have focused on mabs to neutralize endotoxin [ , , ] and cytokines [ ] . pseudomonas aeruginosa sepsis, combination therapy with mabs to endotoxin and tumor necrosis factor was superior to therapy with a single mab [ ] . examples ofpotential targets for antibody-based therapy include the toxins and proteases associated with toxic shock syndromes [ , ] . combination therapy with antibodies to exotoxins could improve outcome, but cocktails of mabs may be necessary because of toxin heterogeneity [ ] . intravenous immunoglobulin administration has been associated with dramatic clinical improvement in a patient with streptococcus pyogenes toxic shock syndrome [ ] . antivirals. virus neutralization is an established property of antibodies. in the preantibiotic era, serum was used for prophylaxis ofmeasles, chickenpox, mumps, and poliomyelitis. recently, the potential of antibody therapy against many viruses, including hepatitis b [ ] , hiv [ ] , respiratory syncytial virus [ ] , cmv [ ] , and parvovirus [ ] , has received considerable attention. newly identified viral illnesses are targets for antibody drug research. combination ofantibody therapy and chemotherapy. antibody-based therapies are unlikely to be used alone unless they are the only available therapy or are being used for prophylaxis of infection. combinations of antibody therapy and chemotherapy offer theoretical advantages. antibodies promote microbial killing directly by a complement-mediated lytic process or indirectly by enhancing nonspecific immune mechanisms. table lists examples of bacterial, fungal, and viral pathogens for which combination therapy has shown promise. combination therapy could reduce the amount of either agent required to table . pathogens for which the combination of chemotherapy and antibody based therapy has shown some promise. pathogen chemotherapy antibody [ , ] neisseria meningitidis sulfanilamide horse immune sera [ ] streptococcus pneumoniae rabbit immune sera [ , ] haemophilus infiuenzae sulfonamide rabbit, horse immune sera [ ] staphylococcus aureus sparftoxacin human igg mab [ ] p. aeruginosa human igm mab [ ] p. aeruginosa murine e lps antibodies [ ] p. aeruginosa human gamma globulin [ ] escherichia coli murine e lps antibodies [ ] shistosoma mansoni praziquantel rabbit immune sera [ ] candida albicans amphotericin b human gamma globulin [ ] cryptococcus neoformans amphotericin b rabbit immune sera [ , ] c. neoformans murine igg mab [ ] c. neoformans murine igg mab [ ] lassa virus ribavirin monkey immune sera [ , ] cytomegalovirus ganciclovir murine immune sera [ ] herpes simplex virus adenine arabinoside rabbit and human sera [ ] herpes simplex virus acycloguanosine human immune globulin note. this is not a complete list. achieve a therapeutic effect. for example, some antibiotics are quite toxic, and the ability to reduce doses could lessen side effects. in a similar vein, the addition of chemotherapy to antibody therapy could reduce the amount of antibody necessary to achieve a therapeutic effect, which would lessen the cost of therapy. the availability of broad-spectrum antibiotics has diminished the need for making exact microbiological diagnoses. for example, gram-negative sepsis or presumed fungal infections can be treated with empirical therapy without identifying the pathogen. this situation is in contrast to the practice of infectious diseases in the s when identification of the pathogen (and its serotype) was necessary for choosing the correct antiserum. for antigenically diverse pathogens such as s. pneumoniae, rapid protocols were developed for typing strains. the present practice of infectious diseases is not unlike a gambling strategy where the probability of infection by a given microbe is matched to the likelihood of activity by the available antibiotics. this has resulted in great emphasis on broad-spectrum antibiotic "coverage" and less emphasis on making an exact microbiological diagnosis. although the relative merits of this practice are beyond the scope of this article, widespread antimicrobial resistance is decreasing the effectiveness of existing antibiotics, and increased caution is warranted when designing broad-spectrum combinations. a return to antibody therapy would force greater emphasis on precise microbiological diagnosis and would foster the development of more accurate and more rapid diagnostic strategies. the use of antibody-based therapies may also require antigenic screening in the clinical laboratory, a practice not unlike the present one of antimicrobial susceptibility testing. in cost-conscious 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phase i trial in bone marrow transplant recipients polyclonal and monoclonal antibody therapy for experimental pseudomonas aeruginosa pneumonia vitro and in vivo activity of polyclonal and monoclonal human immunoglobulins g, m, and a against pseudomonas aeruginosa lipopolysaccharide confronting drug-resistant pneumococci immunoglobulin g enhances c degradation on coagulase-negative staphylococci passive local immunotherapy of experimental staphylococcal pneumonia with human intravenous immunoglobulin roles of antibodies and complement in phagocytic killing of enterococci treatment of gram-negative bacteremia and shock with human antiserum to a mutant escherichia coli phase i study of a murine monoclonal anti-lipid a antibody in bacteremic and nonbacteremic patients therapy with antibody to tumor necrosis factor in sepsis the efficacy of combination immunotherapy in experimental pseudomonas sepsis microbiology of toxic shock syndrome: overview association of phenotypic and genotypic characteristics of invasive streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome intravenous immunoglobulin therapy for toxic shock syndrome human combinatorial antibody libraries to hepatitis b surface antigen passive immunization for the prevention and treatment ofhiv infection human monoclonal fab fragments derived from a combinatorial library bind to respiratory syncytial virus f glycoprotein and neutralize infectivity efficacy of human immunoglobulin and penicillin g in treatment of experimental group b streptococcal infection protection of mice against meningococcus infection by polyvalent antimeningococcic serum combined protective action of human gamma globulin and antibiotics when administered simultaneously in experimental staphylococcal infections synergism between human gamma globulin and chioroamphenicol in the treatment of experimental bacterial infections combined serum and sulphanilamide in the treatment of streptococcal infections in mice monoclonal antibodies for treatment of gram-negative infections efficacy of anti-endotoxin monoclonal antibody e alone or in combination with ciprofloxacin in neutropenic rats with pseudomonas sepsis praziquantelinduced exposure of schistosoma mansoni alkaline phosphatase: drugantibody synergy which acts preferentially against female worms. parasite immuno effects of immunoglobulin g and low-dose amphotericin b on candida albicans infections in burned mice combination of -flucytosine and capsule-binding monoclonal antibody in therapy of cryptococcus neoformans infection enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys - -dihydroxy- -propoxymethyl) guanine prevents death but not immunity in murine cytomegalovirus-infected normal and immunosuppressed balb/c mice combined effects of acycloguanosine and humoral antibodies in experimental encephalitis due to herpesvirus hominis synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to herpesvirus hominis unequalled but unobtainable key: cord- -nt jcjm authors: garwes, d.j.; lucas, m.h.; higgins, d.a.; pike, b.v.; cartwright, s.f. title: antigenicity of structural components from porcine transmissible gastroenteritis virus date: - - journal: vet microbiol doi: . / - ( ) - sha: doc_id: cord_uid: nt jcjm pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. similar results were obtained with serum from rabbits inoculated with whole virus and structural components. all three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus. the immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. in each case where the immunoglobulin class was determined, igg was found. one sow that received surface projections also had iga with neutralising activity in her colostrum. in contrast, infection of sows with live whole virus resulted in neutralising antibody of the igg, igm and iga classes. infection of pigs with the coronavirus causing transmissible gastroenteritis (tgev) results in disease that is most severe, frequently fatal, in young animals and of less severity in animals over weeks old. following recovery of the animals from infection, neutralising antibody can be detected in serum and secretions; transfer of this antibody, predominantly iga, in the colostrum and milk from a convalescent dam to her offspring confers reasonable protection against disease in her piglets (bohl et al., b; saif et al., ; stone et al., ) . parenteral administration of inactivated virus stimulates circulating antibody of the igg class saif et al., ; lucas et al., ) but, as little or no specific iga antibody is formed, the low level of igg in the milk affords little protection to newborn piglets during the period that they are most susceptible. work is in progress to investigate how an iga response can be stimulated by inactivated virus but more information is required about the nature of the viral antigen. we have previously demonstrated that inactivated purified tgev is capable of inducing a primary neutralising antibody response in pigs , suggesting that a virus structural element is the antigen responsible for stimulating the neutralising antibody. there is evidence, however, that at least one soluble component from tgev-infected swine testis cells can stimulate neutralising antibody production . following development of techniques to separate and purify the surface profections (sp) from the internal proteins of a subviral particle (svp) (garwes et al., ) it has been possible to determine which of these components are involved in the neutralising antibody response. this report describes the immune response in swine and rabbits inoculated with inactivated tgev and purified subviral components. antigen preparation the fs / isolate of tgev was used after six passes through gnotobiotic piglets aged -- days. a % (w/v) suspension of infected small intestine was clarified by centrifugation at × g for min and was then inoculated onto secondary pig thyroid (apt/ ) cells as monolayer cultures in medium containing galactose . virus was harvested following freezing and thawing the cultures at h after infection and was purified as previously described (garwes and pocock, b) . isolation of purified sp and svp antigen preparations was achieved by differential centrifugation after treatment of purified virus with % nonidet-p (garwes et al., ) . each antigen preparation (whole virus, sp and/or svp) was diluted : with growth medium, formaldehyde was added to . % and the preparations were held at °c for h. following this treatment, no infectivity could be detected in any of the samples. the preparations were stored at °c until used. each preparation of antigen for immunisation studies was derived from -- × pfu (sows) or × l s pfu (rabbits) of tgev. virus used for radio-immune assay was prepared from apt/ cells infected with tgev fs / passaged times through primary pig kidney cells. the medium contained h-uridine (specific activity ci/ mmole; the radiochemical centre, amersham) at pci/ml and the virus was harvested h after infection and purified as described (garwes and pocock, b) . infectivity was titrated by plaque assay. virus neutralisation test sera, colostra and milk samples were tested as previously described (cartwright, ) . purified tgev that had been grown in the presence of h-uridine was mixed with equal volumes of sera or % bovine serum albumin, and incubated at °c for i h. a sample was removed for titration of infectivity and the remainder was layered on to a linear -- % (w/w) sucrose gradient and centrifuged at × g for h. the gradient was fractionated by siphon into aliquots, material that had sedimented to the bottom of the tube was resuspended in distilled water and a sample was removed from each fraction for determination of radioactivity using a toluene-triton x based scintillant. measurement of virus agglutination by antibody was accomplished by incubating # samples of h-uridine labelled tgev with equal volumes of test sera diluted in isotonic saline at °c for h. to each reaction mixture was then added ul of rabbit anti-porcine igg serum (miles laboratories ltd., stoke poges) and incubation continued for h at °c followed by storage overnight at ° c. the samples were passed through cellulose acetate membrane filters of nm mean pore diameter (type eg, millipore (u.k.) ltd., london), the filters were washed with four changes of . ml distilled water and their radioactivity was determined by liquid scintillation. earlier attempts to use membrane filters composed of mixed esters of cellulose (mf filters, millipore (u.k.) ltd., london) were not successful as purified tgev is bound irreversibly to them (unpublished observations). this problem was overcome by using the cellulose acetate membranes to which the virus did not bind. solutions of serum igm and igg and colostral iga were obtained by gel filtration and ion-exchange chromatography (porter, ; vaerman and heremans, ) . antisera were raised in rabbits by injection of precipitin lines made by immunoelectrophoresis of the immunoglobulin solutions against rabbit antiserum to pig serum (goudie et al., ; higgins, ) . sera were made class specific by absorption with glutaraldehyde cross-linked immunoglobulin solutions (avrameas and ternynck, ) . specificity was assessed in immunoelectrophoresis and immunodiffusion tests against serum, colostrum and immunoglobulin solutions. sera and colostral wheys were examined by gel filtration in sephadex g- (pharmacia) in . m tris-hcl, ph . , containing . m naci and mm edta. wheys were also examined by ion-exchange chromatography on deae-cellulose (de- , whatman), using the six step buffer system described by stone et al. ( b) . the eluate obtained with each buffer was treated as a single fraction. presence of igm, igg and iga in the fractions was observed by microimmunodiffusion against specific antisera. fractions were concentrated about five-fold by dialysis against polyvinylpyrrolidone and examined for neutralising antibodies to tgev. the allocation of antibody activity to an immunoglobulin class was made by comparison of the distribution of antibody activity and immunoglobulins in the fractions. twelve pregnant sows were inoculated by the intramammary route: one with live, whole virus, three with formalin-inactivated virus, two with a mixture of purified sp and svp, four with sp alone and two with svp only. in addition one sow was given live virus orally and two sows were kept as uninoculated controls. for the intramammary inoculations, . ml of the antigen preparation was injected through the skin near the teat into three mammary glands of each sow three times. the same glands were inoculated at approximately , and week before farrowing. for the first inoculation, the antigen was emulsified in complete freund's adjuvant. colostrum samples were taken from inoculated and uninoculated glands separately at farrowing and milk samples were taken for several days after that. blood samples were removed before the first inoculation and at the intervals shown in table i . three rabbits were inoculated with tgev antigen: one with inactivated whole virus, one with sp and one with svp. each rabbit received two subcutaneous inoculations of antigen emulsified in complete freund's adjuvant at an interval of weeks, followed by three intramuscular inoculations of aqueous antigen at weekly intervals. one rabbit was kept as an uninoculated control. blood samples were taken from each animal before the first inoculation and at intervals up to months. p/o = orally; i/mam = by the intramammary route. --= not detected. those not tested are indicated by blank spaces. piglets born to the experimental sows were challenged at days of age by oral administration of ml of a suspension of tgev-infected intestine diluted to contain approximately x tcids per ml (equivalent to approximately x lds per ml in piglets reared away from the sows). the suspension was free, as far as it was possible to determine, from moulds, bacteria and other viruses. the results of neutralisation tests for serum and colostrum are shown in table i . after intramammary inoculation of sows, antibody was detected at the time of farrowing in serum and colostrum from all of three sows given whole killed virus, from out of given surface projections with subviral particles, from out of given surface projections only and from neither of the animals given subviral particles alone. neutralising antibody in colostrum only was detected in out of the given purified surface projections. there was little or no difference between antibody titres of colostrum from inoculated and uninoculated glands. the levels of neutralising antibody in milk from sows inoculated with inactivated whole virus or viral components fell to undetectable levels between to days after farrowing. neutralising antibody titres in sera of all sows, including the uninoculated controls, rose during the first weeks after farrowing. this reflects the immune response to infection of the dam by live virus administered to her piglets days after farrowing. the results are shown in table ii . the immune response to infection with live virus (sow no. ) resulted in neutralising antibodies in the three immunoglobulin classes reported previously (ristic and abou-youssef, ; bohl et al., b; salf et al., ; lucas et al., ) although in the example given igg antibody was detected in serum but not in colostrum. samples from sows that received intramammary inoculation contained neutralising antibodies of the igg class in all cases where the specific imunoglobulin could be characterised. no igm antibodies were detected in any sample and iga antibodies were demonstrated in the colostrum from only out of sows that received sp antigen. the only litter that survived challenge was that of sow no. which had been inoculated with sp antigen and had produced neutralising iga antibodies in the colostrum. the other litters experiences - % mortality and all piglets from the control uninoculated sows died. serum samples taken from the rabbit that had been inoculated with inactivated whole tgev showed neutralising antibodies rising to a maximum titre of l/ . neutralising antibodies were detected also in the serum of the rabbit that received sp antigen; the rate of antibody production was slower, however, and the peak titre reached was l/ . no neutralising antibody was found in serum from the rabbits inoculated with svp antigen and the uninoculated animal remained seronegative throughout the course of the experiment. the sedimentation profiles of h-uridine tgev after incubation with bovine serum albumin or with sera from sows that had been inoculated with viral antigen preparations are illustrated in fig. . virus incubated with either bovine albumin or normal pig serum sedimented as a sharp, homogenous band of radioactivity with very little material collecting at the bottom of the gradient. in contrast, however, virus incubated with sera from animals inoculated with viral antigen moved through the gradient as a broad band, suggesting heterogeneity of size and density, with a large proportion of the total radioactivity sedimented at the table i. bottom of the tube. there were no obvious differences between the profiles obtained after treatment of virus with sera that contained neutralising antibodies (sows no. , and ) or with serum from an animal that received svp antigen and which produced no neutralising antibodies (sow no. ). incubation of h-uridine tgev with sera from sows inoculated with viral antigen resulted in the formation of immune complexes that were retained by membrane filters (fig. ) . after incubation with serum from uninoculated animals approximately % of the virus was retained on the filter, probably due to aggregation that occurs in purified tgev preparations. sera from sows inoculated with whole virus, sp antigen or svp antigen preparations produced virus complexes that were retained most efficient samples of h-uridine labelled tgev containing ct/min were incubated for h at °c in dilutions of test sera followed by incubation with rabbit anti-porcine igg serum. after filtration through cellulose acetate membrane filters and repeated washing with isotonic saline, the retained radioactivity was determined by scintillation counting. test sera from: sow no. -" -', sow no. ~ e, sow no. = -', uninoculated sow d d. ly at a serum dilution of / . at higher serum dilutions there was reduced retention, implying that the level of antibodies was limiting. serum dilutions lower than / gave suboptimal retention due, probably, to an excess of antibodies and the formation of smaller complexes. omission of the rabbit anti-porcine igg serum from the test resulted in poorer retention of radioactive virus at all dilutions of experimental serum and a decline in the reproducibility of the test. discussion we report the immune response of sows and rabbits to tgev preparations containing whole virus, isolated surface projections or subviral particles produced by removal of the surface projections and envelope lipid from the virus by detergent treatment. sows receiving whole virus, isolated sp antigen and mixtures of sp and subviral particles produced antibody capable of neutralising virus infectivity; sows that received subviral particles alone, however, did not. similarly rabbits immunised with purified whole virus or surface projections produced neutralising antibody to tgev while administration of subviral particles produced no detectable neutralising activity. these data suggest that neutralising antibody raised during infection of swine with tgev is directed against a structural antigen of the virion and that this antigen is associated with the surface projection, a similar finding to that obtained with other lipid enveloped viruses (webster and laver, ; cartwright et al., ; jennings et al., ; hunsmann et al., ) . the fact that no neutralising antibody was produced in the pigs and rabbits that received subviral particles alone strongly suggests that the structural elements of these particles are not involved in virus neutralisation; conclusive proof of this, however, would necessitate the use of more animals than were available in the present study. the possibility existed that the tgev subviral particles were not antigenic in the animals used. this seemed unlikely as we had previously shown that these structures of nm diameter, readily visualised by electron microscopy, contained all the structural proteins except for the sulphated glycoprotein of the sp (garwes et al., ) . standard serological tests, such as complement fixation and radial immunodiffusion, are not sensitive enough to detect the levels of antibodies present in the experimental sera. the radioimmune assay techniques described above indicated, however, that antibody directed against the surface of the virus was produced in response to inoculation of sows with whole virus or either of the structural moieties. the sedimentation analyses showed that these sera caused an increase in the sedimentation coefficient of the virus, presumably by coating the particles with antibody molecules, and brought about sufficient aggregation to pellet radioactivity to the bottom of the gradient. application of this serum-induced clumping of radiolabelled virus to filtration studies demonstrated that sows receiving svp antigen were capable of producing antibodies to surface components at a similar level to those animals receiving whole virus or sp antigen. the use of this technique to quantitate agglutinating antibody is complicated by the need to incorporate rabbit anti-porcine igg serum into the test. as discussed in the text, purified tgev adsorbs irreversibly to membrane filters containing cellulose nitrate. this necessitated using cellulose acetate filters, for which the smallest pore diameter available is nm. assuming that virus aggregates are approximately spherical and composed of particles -- nm in diameter, the smallest aggregate that sould be retained reproducibly by such filters would need to comprise -- virions. the secondary antiserum to porcine immunoglobulin is required to achieve complexes of this size. the low level of retention of virus incubated with low dilutions of immune sera presumably reflects the solubility of antigen--antibody complexes in the presence of large excesses of either of the two reactants. analysis of the immunoglobulin classes responsible for neutralising activity after inoculation of sows by the intramammary route showed that igg was the predominant antibody species in both serum and colostrum, consistent with previous findings with inactivated tgev lucas et al., ) but differing from the stimulation of iga to ferritin reported by bourne et al. ( ) . whether this discrepancy is due to differences in the types of antigen used is not known. it is of interest that sow no. , which received sp antigen, produced neutralising antibody of the iga class in the colostrum and was the only animal capable of protecting her piglets against challenge with live virus. the possibility that this sow encountered live tgev prior to farrowing and that the experimental inoculation generated a secondary immune response cannot be totally ruled out, since infection with the live virus stimulates igm and iga antibody production (sow no. above; lucas et al., ; stone et al., ) and may result in limited protection of the litter. there was no other reason to believe that prior infection with tgev had occurred, however. all the control sows remained sero-negative throughout the experimental period. the apparent lack of neutralising antibody of the igm class in the pigs that received inactivated viral material may have resulted from the materials used and the route of inoculation; the times at which the serum samples were taken may have been too late, however, to have detected a transitory igm response. most of the sows showed rising neutralising antibody titres after challenge of the piglets, probably in response to cross-infection. those inoculated with sp antigen that had no detectable neutralising antibody at farrowing nevertheless developed titres by to weeks, comparable with those that were sero-positive at farrowing. the two sows inoculated with svp antigen alone, however, as well as having no detectable serum neutralising antibody at farrowing, developed lower titres that were more comparable with those achieved by the uninoculated controls. it has been reported that a soluble antigen from tgev-infected swine testis cells could induce neutralising antibody formation when injected into rabbits. from the data presented above we should anticipate that the soluble antigen concerned is likely to comprise viral sp either free in solution or bound to small fragments of membrane. since the viral sp is characterised mainly by the molecular weight of its component glycopolypeptide and has neither haemagglutinin nor enzyme activity associated with it, the task of recognising the presence of sp in crude tissue extracts is difficult. it should be possible, however, to develop a radioimmune assay using specific antibody to purified components. such a test would facilitate the preparation of potent antigen suspensions and work is in progress to this end. the cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents a. immunology of transmissible grastroenteritis antibody responses in serum, colostrum and milk in swine after infection or vaccination with tge virus the influence of route of vaccination on the systemic and local immune response in the pig surface structure of vesicular stomatitis virus a cytopathic virus causing a transmissible gastroenteritis in swine. ii. biological and serological studies effect of precipitation with methanol on antigenic potency of tge virus the polypeptide structure of transmissible gastroenteritis virus isolation of subviral components from transmissible gastroenteritis virus a simple method for producing antibody to a single selected diffusible antigen. the lancet fractionation of fowl immunoglobulins properties of mouse leukaemia viruses. ix. active and passive immunisation of mice against friend leukaemia with isolated viral gpt~ glycoprotein and its corresponding antiserum the immune response of hamsters to purified haemagglutinins and whole virus vaccines following live influenza virus infections attempts to vaccinate sows against tge the influence of ph on the growth and stability of transmissible gastroenteritis virus in vitro transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglets comments on the immunology of transmissible gastroenteritis isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies transmissible gastroenteritis in neonatal pigs: intestinal transfer of colostral immunoglobulins containing specific antibodies a. partial characterisation of the principal soluble antigens associated with the coronavirus of transmissible gastroenteritis by complement fixation and immunodiffusion chromatographic separation of gram quantities of immunoglobulins from porcine colostrum against transmissible gastroenteritis virus immunoglobulin a in the pig. i. preliminary characterisation of normal pig serum iga influenza virus subunit vaccines: immunogenicity and lack of toxicity for rabbits of ether-and detergent-disrupted virus key: cord- -uib h lb authors: mawle, alison c.; nisenbaum, rosane; dobbins, james g.; gary, howard e.; stewart, john a.; reyes, michele; steele, lea; schmid, d. scott; reeves, william c. title: seroepidemiology of chronic fatigue syndrome: a case-control study date: - - journal: clin infect dis doi: . /clinids/ . . sha: doc_id: cord_uid: uib h lb we performed serological testing for a large number of infectious agents in patients from atlanta who had chronic fatigue syndrome (cfs) and in controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. we performed serological testing for a large number of infectious agents in patients from atlanta who had chronic fatigue syndrome (cfs) and in controls matched by age, race, and sex. we did not find any agent associated with cfs. in addition, we did not find elevated levels of antibody to any of a wide range of agents examined. in particular, we did not find elevated titers of antibody to any herpesvirus, nor did we find evidence of enteroviral exposure in this group of patients. chronic fatigue syndrome (cfs) is an illness of unknown etiology, characterized by debilitating fatigue lasting longer than months and a variety ofnonspecific symptoms, including myalgia, arthralgia, lymphadenopathy, low-grade fever, sleep disorders, and inability to concentrate [ ] . an infectious etiology has been suggested for cfs, although the evidence is not compelling [ ] . many patients report the sudden onset of a flulike illness that presaged their fatiguing illness, and a number of infectious agents are known to cause postinfection fatigue. reports that viral antibody titers are elevated in cfs cases has led to the speculation that latent viruses may be reactivated in this illness as a result of an underlying perturbation of immune function, and that elevated titers of antibody to common agents may be a reflection of this disturbance. we conducted serological tests for a large number of infectious agents as part of a case-control study assessing risk factors for cfs. our goals were ( ) to determine whether we could detect a common exposure history among cfs patients who met a stringent research case definition and ( ) to determine whether levels of antibody to any of the agents examined were elevated in cfs cases. patients with cfs were recruited from the atlanta component of the centers for disease control and prevention's (cdc's) surveillance study ofcfs [ ] . cases ofcfs in atlanta received who currently met the cfs research case definition [ ] and who had been sick for~ years were eligible for the study; cases were recruited to participate in the study. we selected two controls for each case. they were matched for age (± years), race, and sex. controls were selected by random-digit dialing in the five-county area of atlanta covered by the surveillance system. all controls were screened to eliminate those with medical conditions that could bias the results (reyes et al., unpublished data). we collected blood and stool samples from each participant. serum and stool specimens were stored at - °c. each laboratory was requested to perform serological testing of samples as a single batch to minimize interassay variation. if that was not possible, samples from a case and two matched controls were tested in the same batch. all samples were coded so that the tests were performed in a blinded fashion. retroviruses. serological testing for hiv- and hiv- was performed with use of a u.s. food and drug administration (fda)-licensed elisa kit (genetic systems, redmond, wa). confirmatory testing was performed with an fda-licensed western blot test (cambridge biotech, rockville, md). antibodies against human t-lymphotrophic virus types i and ii were detected with an elisa, and confirmatory testing was performed by western blotting [ ] . to detect the presence of a retrovirus in peripheral blood lymphocytes (pbls) from cases, we cultured x pbls with allogeneic phytohemagglutinin-stimulated pbls for weeks. cultures were fed weekly and culture supernatants were screened weekly for reverse transcriptase activity [ ] . igm with use of a monoclonal antibody-capture elisa similar to that described previously [ ] . to detect active infection, we prepared rna from stool samples and performed a reverse transcriptase pcr to detect enteroviral sequences [ ] . for four cases and three controls, materials were insufficient for enteroviral rna testing. arboviruses. sera were screened with a hemagglutinationinhibition assay [ ] against a panel of arboviruses. antibodies against colorado tick fever virus and vesicular stomatitis virus were detected by means of a cf assay [ ] . titers of : were considered positive. herpesviruses. titers of antibody to cytomegalovirus, human herpesvirus , and varicella zoster virus were determined by means of an elisa against the whole virus. optical density readings were converted into antibody units with use of known standards [ ] . cytomegalovirus and varicella zoster virus values were considered positive at~ antibody units, and human herpesvirus values were considered positive at~ antibody units. epstein-barr virus (ebv) early antigen antibody titers were determined with a commercial elisa (gull laboratories, salt lake city), and a titer of~i: was considered positive. antibodies specific for ebv viral capsid antigen (vca) and nuclear antigen were titrated by indirect immunofluorescence assay (ifa) [ ] . a titer of~ : was considered positive. type-specific herpes simplex virus serology was performed with a glycoprotein g-based western blot assay [ ] . respiratory viruses. sera were tested for the presence of antibodies to adenovirus; parainfluenza viruses i, , and ; and respiratory syncytial virus. testing was performed by elisa with a : serum dilution [ ] . antibodies to coronavirus subtypes oc and sn e were detected with a microneutralization assay [ ] . hepatitis viruses. sera were screened for antibody to hepatitis b or hepatitis c by elisa, with use of commercially available kits (abbott laboratories, abbot park, il). elisapositive sera were subjected to confirmatory testing by western blotting (abbott laboratories). other viruses. antibody to measles nucleoprotein [ ] , rubella (rubestat g, bio whittaker, walkersville, md), and parvovirus b [ ] was detected by elisa. measles neutralization titers were also determined [ ] . rickettsiaceae. serological testing for antibodies to rickettsia typhi, rickettsia ricketts ii, coxiella burnetii, and ehrlichia chaffeensis was performed with use of ifa against fixed whole organisms [ ] . a titer of~ : was considered positive. bartonella (formerly rochalimaea species). sera were screened for antibodies to bartonella henselae, b. quintana, and b. elizabethae by means ofifa with fixed whole organisms [ ] . a titer of~ : was considered positive. borrelia burgdorferi. sera were screened by elisa for antibodies against flagellin. a positive result was confirmed by western blotting (robbins et ai., manuscript in preparation). candida albicans. precipitating antibodies were detected by both latex agglutination and immunodiffusion. the result was considered positive if either the immunodiffusion assay was positive or the latex agglutination titer was~ : . we also screened for the circulating candida antigens mannan a and mannan b [ ] . chlamydia. sera were screened by ifa at : dilution against chlamydia trachomatis, serotype l . this contains the lipopolysaccharide antigen specific for the genus chlamydia and thus detects antibodies to all chlamydia species [ ] . we analyzed our data using matched analysis procedures. we used a nonparametric test (cochran-mantel-haenzel statistic [ ] with modified ridit scores [ ] ) to assess differences in continuous variables between cases and controls. the logarithm in base of the titers of antibody to ebv-vca, ebvearly antigen, ebv -nuclear antigen, and coronavirus oc as well as the measles neutralization titer were used in the analysis, since they have a natural interpretation when titers are considered (each dilution represents unit in the log base scale). in all statistical testing we carried out, a p value of~. was considered significant. since the design of the study was exploratory and not hypothesis-testing, we did not correct for multiple comparisons [ ] . statistical analyses were performed with the statistical software sas (sas institute, cary, nc). we recruited patients with cfs ( female and male) and matched controls. two controls withdrew, leaving cases with only matched control. all participants were white (this finding is consistent with those of other studies). the median duration of illness for the cases was . years (range, . - . years), and the median age at onset of illness was . years (range, - years). occupation, income, and education were comparable between cases and controls. agents infrequently detected in or absent from cfs cases. reverse transcriptase pcr analysis of stool specimens revealed the presence of enteroviral rna in three cases ( . %) and four controls ( . %). there was no overlap between those who had enterovirus-specific igm and those who had detectable viral rna in their stool. there were no statistically significant differences between cases and controls for any of these agents. agents frequently detected in cfs cases. all other agents tested were detected in ;;::: % of cfs cases, and antibody levels were compared between cases and controls. these agents included the herpesviruses cytomegalovirus, varicella zoster virus, human herpesvirus , ebv (early antigen, nuclear antigen, and vca), and herpes simplex virus ; the respiratory viruses adenovirus, respiratory syncytial virus, coronavirus, and parainfluenza viruses , , and ; measles virus; parvovirus b ; and rubella (tables and ). the seropositivity rates in both cases and controls were ;;::: %. there were no differences between the two groups in terms of seropositivity rates or virusspecific antibody levels, as measured by titer, by antibody units, or by optical density in an elisa. we have screened a well-characterized group of patients with cfs for evidence of infection with a wide range of agents and have compared percentages of infection with age-, race-, and sex-matched controls. we found no evidence of a common infectious agent in the patients studied. there were no differ-ences in prevalence of current enteroviral infection between cases and controls, as determined by levels of circulating enterovirus-specific igm, nor in the frequency of detectable enterovirus in stool samples. thus, in the group of patients from atlanta, enteroviral infection was not more common in cfs cases. we were unable to detect evidence of infection with any known human retrovirus, and our inability to detect reverse transcriptase activity in cocultures suggests that there is no common, unknown retrovirus in these patients. this is consistent with other studies that have been unable to detect retroviruses in cfs [ , ] . we found no evidence of infection withb. burgdorferi, the causative agent oflyme disease. infection with b. burgdorferi can lead to a cfs-like illness, and in areas where lyme disease is endemic, it can contribute to cfsrelated estimates [ ] . in this group of patients from atlanta, there was no evidence that lyme disease is a factor in cfs. other agents that had < % seroprevalence in these patients included arboviruses, hepatitis b and c, rickettsiaceae, bartonella species, chlamydia species, and precipitating antibodies to c. albicans. we also looked for circulating candida antigens, since these are frequently found in severely immunodeficient patients. none of our patients with cfs had detectable mannan a or mannan b, which suggests that these patients were not immunodeficient. the lack of precipitating antibodies to c. albicans argues against the occurrence of a chronic candidal infection in patients with cfs. herpesviruses-in particular, ebv and human herpesvirus -have been associated with cfs. although elevated titers to ebv-vca and ebv early antigen were initially described as occurring in patients with cfs [ , ] , subsequent studies made it clear that these were not specific to cfs [ ] [ ] [ ] . in this study, we found no differences in titers to ebv-vca or ebv nuclear antigen between cases and controls. however, the titer of early antigen was slightly elevated in the cases compared with that in controls, although the difference was not statistically significant. in conclusion, we were unable to find evidence of a single infectious agent associated with cfs in this patient population, nor did we find evidence of elevated antibody titers consistent either with reactivation of a latent virus or with generalized immune activation. the chronic fatigue syndrome: a comprehensive approach to its definition and study is chronic fatigue syndrome an infectious disease? epidemiology of chronic fatigue syndrome: the centers for disease control study chronic fatigue syndrome: a working case definition simultaneous confirmation and differentiation of human t-lymphotropic virus types i and ii infection by modified western blot containing recombinant envelope glycoproteins isolation, characterization, and transmission of human t-lymphotrophic virus types i and ii in culture echovirus infection and aseptic meningitis in parents of children attending a child care center enteroviral rna sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses adaptation oflecf microtechnique in standardized diagnostic complement fixation method and adaptation to micro test prior herpes simplex type infection as a risk factor for hiv infection immunofluorescence in cells derived from burkitt's lymphoma evaluation of a test based on baculovirus-expressed glycoprotein g for detection of herpes simplex virus type-specific antibodies viral antibody screening system that uses a standardized single dilution immunoglobulin g enzyme immunoassay with multiple antigens antigen detection in human respiratory coronavirus infections by monoclonal time resolved fluoroimmunoassay baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays human parvovirus bi -specific igg, iga, and igm antibodies and dna in serum specimens from patients with erythema infectiosum evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus a comparison of the complement fixation, indirect immunofluorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases serological response to rochalimaea henselae antigen in suspected cat-scratch disease serodiagnosis of fungal diseases single antigen fluorescence test for chlamydia antibodies average partial association in threeway contingency tables: a review and discussion of alternative tests on the combination of independent two-sample tests of wilcoxon no adjustments are needed for multiple comparisons search for retrovirus in the chronic fatigue syndrome lack of evidence for infection with known human and animal retroviruses in patients with chronic fatigue syndrome jennekens fgl chronic muscle weakness caused by borrelia burgdorferi meningoradiculitis evidence for active epstein-barr virus infection in patients with persistent unexplained illness: elevated anti-early antigen antibodies persisting illness and fatigue in adults with evidence of epstein-barr virus infection frequency of chronic active epstein-barr virus infection a cluster of patients with a chronic mononucleosis-like syndrome: is epstein-barr virus the cause? antibodies to epstein-barr virus in patients with chronic fatigue the authors gratefully acknowledge drs. l. schonberger, w. gunn, a. khan, and g. holmes (cdc), whose initial work on cfs led to this study. they are indebted to drs. w. bellini, c. black, r. craven, d. erdman, t. folks, . hierholzer, n. karabatsos, r. lal, s. lambert, m. pallansch, r. regnery, e. reiss, and t. tzianabos (cdc), in whose laboratories many of the reported assays were performed, and to dr. . gow (institute of neurological science, scotland) for performing the enteroviral rtpcr. they also acknowledge d. connell, b. randall, and r. jacobs (abt associates) as well as e. parris (cdc), who were responsible for study logistics and data processing. key: cord- -kbwz xop authors: toubanaki, dimitra k.; margaroni, maritsa; prapas, athanasios; karagouni, evdokia title: development of a nanoparticle-based lateral flow strip biosensor for visual detection of whole nervous necrosis virus particles date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: kbwz xop effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. paper lateral flow biosensors (lfbs) have been established as attractive tools for such analytical applications. in the present study a prototype lfb was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. the lfb was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the lfb. formation of a red test line was indicative of nodavirus virions presence. nodavirus visual detection was completed in short time ( min). key factors of the lfb development influencing the assays’ detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. therefore, the proposed lfb assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. the proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. it is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures. instrumentation. the thin layer chromatography (tlc) applicator linomat and wincats software were from camag (muttenz, switzerland). the desktop scanner hp scanjet g was from hp (california, usa) . the nanodrop spectrophotometer was purchased from thermo fisher scientific (delaware, usa) and the ultraviolet-visible spectroscopy plate reader was the mrx model from dynatech laboratories (chantilly, va, usa). fish samples. european sea bass (dicentrarchus (d.) labrax) samples with or without vnn clinical signs were collected from sea-cage fish farms in cyprus, saronikos, korinthian and amvrakikos gulfs. dissected brain and retinas were isolated aseptically and stored in sterile polypropylene tubes at − °c until use . ethical statement. the collection of biological samples from fish farms was conducted by licensed personnel of the respective aquaculture facility. all qualified personnel were previously informed of the purpose of the study, the confidentiality of the data, and their voluntary participation. the present study was approved by the hellenic pasteur institute animal bioethics committee regulations according to greek (pd / ) and eu (directive / ) legislation for the protection, care and use of animals used for scientific purposes and all samples used were euthanized in the sites of fish farming according to the ethical principles and other requirements of the law . virus preparation and titration. nodavirus was isolated from naturally infected d. labrax fish. all infected samples were verified by rt-pcr . the virus was propagated in ssn- cells as follows: fish brains were homogenized either in emem or leibovitz l -medium containing % fbs. centrifugation ( , × g, min, °c) of the homogenates ( % w/v) resulted in supernatant, which was filtered through . μm filters and was inoculated on cell cultures. following inoculation, the ssn- cells were cultured at °c, in falcon primaria cell culture flasks containing leibovitz's l -medium, supplemented with μg/ml streptomycin, u/ml penicillin, mm glutamine and % fbs. virus titration was performed on monolayers of ssn- cells grown in a -well plate. viral suspensions were prepared with -fold serial dilutions in emem supplemented with % fbs. quadruplicates of µl of each dilution were added in a -well plate seeded with ssn- cells. cultures were incubated at °c for days. during this period, the cell monolayers were observed for the appearance of cytopathic effect and the final titer, expressed as tcid /ml, was estimated by the end-point titration method . rabbit anti-nodavirus polyclonal antibodies preparation. female new zealand rabbits were used for anti-nodavirus polyclonal antibodies production with prior approval by the animal bioethics committee of the hellenic pasteur institute (athens, greece) according to the ec directive / and the national law / . rabbits were reared in institutional facilities under specific pathogen-free conditions, receiving a diet of commercial food pellets and water ad libitum . for immunization, the rabbits were inoculated with culture supernatant of nodavirus infected ssn- cells. in immunization scheme , one subcutaneous injection containing μg of antigen emulsified with cfa was administered in final volume of μl. three weeks later, a subcutaneous booster [ μg of antigen emulsified with ifa] was injected. a final booster of μg antigen without any adjuvant was given days later. blood was collected from rabbit marginal ear vein days after the final booster, the serum was isolated by centrifugation at × g for min and kept in − °c. three weeks later, another boost ( μg of antigen) was injected subcutaneously to the same rabbit. fifteen days later, blood was collected from the marginal ear vein. centrifugation was performed as above for serum isolation. in scheme , two weeks interval between immunizations was applied instead of three weeks interval used in scheme . the anti-nodavirus igg was purified using the nab tm spin columns, following the manufacturer's instructions. briefly, the samples were diluted : in binding buffer ( . m phosphate buffer, . m nacl). the columns were centrifuged ( × g, min, room temperature (rt)), the flow through was discarded and were equilibrated with ml binding buffer, followed by centrifugation ( × g, min, rt). the procedure was repeated once. subsequently, the bottom ends of the columns were closed, the samples were added and the columns were stirred end-over-end ( min, rt). after centrifugation, the flow through containing non-bound serum components was collected and the columns were washed times with ml binding buffer. one ml of elution buffer (glycine . m, ph = - , × times) was added to the columns, the fractions were collected in tubes containing μl of neutralization buffer (tris m, ph = - ) and stored in − °c. finally, the columns were regenerated with ml of elution buffer (× times) and stored with ml of storage buffer ( . % nan in pbs). to avoid possible denaturation of purified antibodies due to sensitivity to elution buffer, purified antibodies were desalted using zeba tm spin desalting columns. briefly, the columns were centrifuged at xg for min, rt. ultrapure water was added in each column and centrifugation was repeated twice. samples were placed in each column and columns were centrifuged in order to collect the purified samples. the antibody concentrations were measured using the micro bca protocol. a specific elisa was utilized for anti-nodavirus polyclonal antibodies determination in rabbit serum. elisa plates ( -well flat bottom) were coated with μl of , % poly-l-lysine in bicarbonate/ carbonate buffer, ph . , (incubation h, rt). the plate was washed times with lswb and μl of nodavirus suspension were added, followed by incubation for h (rt). subsequently, μl of mm glutaraldehyde in pbs were added in each well and the incubation was continued for min (rt). the plate was washed with lswb ( times) and then blocked with μl % bsa in lswb, for h, at °c. following plate washing ( times with hswb), μl of serially diluted rabbit anti-nodavirus serum ( : , : , : and : in pbs/ ml/l tween- ), were added (incubation h, rt). the plate was washed with hswb and μl of anti-rabbit igg-hrp antibody was www.nature.com/scientificreports www.nature.com/scientificreports/ added; diluted in blocking buffer ( / , / , / and / ), and incubated for h at rt. the plate was washed with hswb and μl of chromogen (tmb) was added for min. the reaction was stopped using μl of . m h so . the optical density was measured at nm in spectrophotometer. nps. an aliquot of nm au nps solution (i.e. ml, . × particles/ml) was adjusted to ph by adding the appropriate amount of mm borax solution. in parallel, µg of polyclonal anti-nodavirus antibody ( . µg/µl) were diluted in µl of mm borax solution and added to the nanoparticles solution, gradually by stirring ( times × µl). the mixture was incubated for min at rt, and µl of % bsa in mm borax solution were added. the final solution was incubated for min at rt, and the excess of reagents were removed by centrifugation at , × g for min. the supernatant was discarded and the pellet was re-dispersed in washing solution ( ml % bsa in a mm borax solution), followed by centrifugation at , × g for min. the supernatant was discarded once again, and the red pellet was re-dispersed in µl of an aqueous solution containing . % bsa and . % nan in mm borax. all incubation steps were performed in darkness. finally, the functionalized antibody au nanoparticles were stored at °c . flocculation studies. gold colloid suspension ( . pmol/ ml) adjusted to ph . , was pipetted into a series of . ml siliconized tubes. colloidal gold and antibody ( - ng/µl) solutions were mixed and the au functionalization protocol was applied as described above. determination of the optimal antibody amount for au nanoparticles conjugation, was based on light absorption measurements. for that reason each tube received % nacl and was shaken for min. absorption of each tube at and nm was determined min later. preparation of the nodavirus lateral flow biosensors. the dry reagent lateral flow biosensors ( × mm) consisted of a cellulose immersion pad, a glass-fiber conjugate pad, a nitrocellulose diagnostic membrane, and a cellulose absorbent pad. the parts were assembled on a plastic adhesive backing as follows: the diagnostic membrane was placed on center of a laminated card by the manufacturer; the conjugate pad was placed below the membrane, the immersion pad was placed below the conjugate pad, and the absorbent pad was positioned above the membrane. each part overlapped mm to ensure that the solution could migrate through the biosensor. the tlc applicator was employed to construct the test zone (tz) and the control zone (cz) by loading polyclonal anti-nodavirus antibody (anti-pn) and anti-rabbit igg antibody (anti-r) on the membrane, respectively. the anti-pn and anti-r zones were constructed at distances of and mm from the edge of the membrane, respectively. in details, for the anti-pn zone, a solution consisting of mg/l purified polyclonal anti-nodavirus antibody, ml/l methanol, and g/l sucrose in freshly prepared mm nahco buffer (ph . ) was loaded at a density of ng per lfb, with dispensing velocity nl/s. for anti-r zone, a solution containing mg/l anti-rabbit igg antibody, ml/l methanol, and g/l sucrose in mm nahco buffer (ph . ) was loaded at a density of ng per -mm membrane, with dispensing velocity nl/s. the membrane was dried in an oven for h at °c, and the sensors were assembled as described before. lfbs with a -mm width were cut using a guillotine cutter and stored dry, at ambient temperature , . lateral flow biosensor detection assay of nodavirus virions. for detection of nodavirus virions, brain tissue samples were homogenized and centrifuged as described in section "virus preparation and titration". alternatively, aliquots of ssn- cell culture supernatant infected with nodavirus were used. the samples were directly applied to the conjugation pad next to . µl of polyclonal anti-nodavirus conjugated au nps. the immersion pad of the biosensor was then dipped into μl of the developing solution. the visual detection was completed within min. longer times did not affect the assay results . after completion of the assay, the lfbs were scanned with a desktop scanner and the band densities were quantified with imagej software . assay principle. the principle of viral particles detection based on lateral flow biosensor with functionalized nanoparticles is presented in fig. . nodavirus intact particles (virions), obtained either from ssn- cell culture supernatants or homogenized tissue samples, are directly applied on the lfb. the biosensor is dipped in a developing solution, which is transferred through the lfb by capillary action. the solution rehydrates the anti-nodavirus conjugated gold nanoparticles, and the virion -anti-nodavirus conjugated nanoparticles complex is captured from immobilized anti-nodavirus antibody at the lfb test zone. the resulted accumulation of au nps generates a characteristic red line. proper function of the biosensor is confirmed by immobilization of excessive au nps in captured anti-rabbit igg (control zone) forming a distinct red line. absence of virus particles in the sample results in the formation of the control zone only. the proposed lfb is based on the well-established immune reaction between antibody functionalized gold-nanoparticles and virions, which serve as antigens. since each virion surface i.e. the virus coat protein, contains multiple antigenic sites (e.g. b-cell epitopes) , a sandwich format was implemented for virion detection assay. it has been suggested that a sandwich elisa resulted in optimum quantitation results, with higher reproducibility and greater detectability range when it was compared with an antigen-immobilized elisa for nodavirus particles detection , therefore the sandwich format was chosen in the present project. moreover, the sandwich format has been successfully implemented in various other formats for low cost portable devices, e.g. nanoparticle-mediated photothermal assays with common thermometer as the signal reader , indicating its appropriateness for methodologies with on-site applications. therefore, polyclonal antibodies to nnv were immobilized on the test zone of the biosensor as well as on the gold nanoparticles. production and reactivity of rabbit anti-nodavirus polyclonal antibodies. the anti-rabbit igg concentration for the elisa assay was tested in the range of : , : , : and : dilution. the serum ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports www.nature.com/scientificreports/ dilution range was : , : , : and : . the results are presented in fig. s a . to optimize the polyclonal antibodies efficiency, the rabbit serum was purified with the appropriate columns and specificity of the purified and desalted antibodies was tested with elisa. optical densities (od ) for a representative fraction was . (elute ) and . (elute ) versus . of the unpurified serum. the use of : dilution of the anti-rabbit igg antibodies was selected since it results in similar signal intensity with higher amount of the antibody, for the same serum dilution. the immunization scheme consisted of two parts: times of antigen administration with weeks intervals between immunizations and a th immunization followed weeks later. antibody levels were quantified with a specific elisa, and the final immunization was performed to raise higher anti-nodavirus antibodies titer (od = . for immunizations vs od = . , for immunizations). in parallel, another female rabbit was immunized with scheme which allowed a weeks interval between the immunizations. as shown in fig. s b , scheme raised even higher titer of igg anti-nodavirus antibodies compared to scheme . therefore it was used for antibody production. colloidal gold nanoparticles were selected since they are the most common used nanosized labels for lateral flow biosensors, occupying more than % of the respective market in immunochromatographic assays, since they can be biolabeled with simple procedures, have excellent optoelectronic properties, have very low toxicity and are easily synthesized with low cost . conjugation of the anti-nodavirus antibody on the au nps was optimized in terms of: usage of a monoclonal versus the polyclonal anti-nodavirus antibody, the size of the used particles, the anti-nodavirus antibody amount on the nanoparticle conjugates and the amount of the conjugates that was applied on the lateral flow biosensors. effect of the use of polyclonal instead of a monoclonal antibody for au nps functionalization. the conjugation of a monoclonal anti-nodavirus antibody on the nanoparticles was tested in order to increase specificity. as shown in fig. a , the monoclonal antibody failed to provide functionalized au nps since not even the cz was visible. on the contrary, implication of a polyclonal anti-nodavirus antibody in the conjugation reaction resulted in a discrete signal in the test zone of the lfb and a strong signal in cz. effect of the gold nanoparticles size. the au nps size has important effects on the particles optical properties, the affinity of the conjugates with the used antibodies, their valency and the antigen binding kinetics . large sized particles develop steric hindrance in a high degree, affecting interactions of the target with a labelled antibody, which could impede the fluidity in the strip. on the other hand, small nanoparticles, are not optimal for visual signals formation . to determine the size where the signal would be optimal, commercially available nanoparticles were studied. gold nanoparticles stabilized either in citrate ( nm) or in pbs buffer ( , , , , nm) were conjugated with the polyclonal anti-nodavirus antibody. formation and intensity of tz and cz on the lfb was assessed for evaluation of the particles optimum size for the nodavirus lfb. figure b proves that all pbs stabilized particles were successfully conjugated with antibodies while no conjugation occurred on the citrate nm nanoparticles. therefore, au nps stabilized with pbs buffer facilitates proteins (antibodies) conjugation while the citrate buffer is more efficient on conjugation of sh-tailed oligonucleotides . use of and nm nps resulted in faint control zones. the and nm formulations formed strong control zones but the test zones were faint possibly due to difficulty in interaction with the larger nodavirus particle. the optimum signal was achieved with nm au nps, a size which is similar to the virus size. that size seems to be optimum for conjugation with different antibodies for different analytes intended for use on lfbs, e.g. anti-e.coli monoclonal antibody or anti-biotin antibody , indicating that nanoparticles in the range of - nm are better for antibody conjugation than smaller or bigger particles. effect of anti-nodavirus antibody amount on au nps conjugates. the antibody amount used for nanoparticles functionalization is a crucial factor for optimal lfb performance. variation in the antibody amount in the surface of the nanoparticles will affect its density on the conjugate, affecting the binding degree between the antigen and the conjugate. thus, the assays' detection limit can be changed based on the various binding efficiency. the minimum antibody concentration which inhibits nanoparticles aggregation in the presence of excess salt ( % nacl), ap: absorbent pad. a test zone (polyclonal anti-nodavirus antibody (tz) and a control zone (anti-rabbit igg antibody (cz)) have been immobilized on the biosensors' diagnostic membrane. the sample contains the nodavirus particles (virions) and is applied on the conjugation pad next to functionalized gold nanoparticles (au) with polyclonal anti-nodavirus antibody. the biosensor is dipped in the developing buffer, the sample and the nanoparticles are captured on the test zone and the positive signal is visualized, as a red zone. the excess nanoparticles bind to the control zone of the biosensor. the assay components are not in scale. is usually chosen as the optimum amount for au nps conjugation . several antibody amounts were studied and the optimal antibody concentration for the particle functionalization was determined by comparing the absorption between and nm (a -a ), in the presence of % nacl. the resulted conjugates are presented in fig. c , where all nanoparticles appear to be of the same color. the gold nanoparticles stability in the presence of concentrated nacl is indicated by the flocculation curve, which is an indirect measure of the immobilized protein surface coverage degree. a hydrated charged corona of proteins on the nanoparticles is formed by adsorption on their surface, which inhibits the particles aggregation, mostly due to steric hindrance and electrostatic repulsive forces. addition of a concentrated nacl solution inverts the au nps protection by uncovering their surface, leading to aggregation . the resulted flocculation curves indicated that ng/µl was the optimal antibody amount, which was therefore used throughout the project (fig. d) . application of that conjugate on the lfb resulted in a strong signal in the control zone, confirming the successful conjugation of anti-nodavirus antibody to au nanoparticles (fig. e) . the au nps amount on the conjugate pad was evaluated, in order to achieve satisfactory cz and tz intensity of the biosensor, combined with minimum background membrane coloration . the nanoparticles amount was optimized in the range of . - . fmol per lfb. the use of . fmol of au nanoparticles per lfb resulted in the optimum signal (fig. f) . less conjugate amount resulted in lower band intensities possibly because low conjugate content is inadequate for binding of specific epitopes amount on highly concentrated antigens. use of higher amounts of particles had a slight signal decrease, as expected since higher conjugate cause epitopes blocking on the antigen surface, and inhibits their binding in the lfb test and control zones . based on these observations, . fmol ( . μl of the prepared solution) of gold nanoparticles conjugates, corresponding to . μl of the prepared solution, were used in all subsequent experiments. important since a large number of parameters would affect the sensitivity and specificity of the assay. several factors were studied including the developing solution composition, the type of the used diagnostic membrane, the dispensing velocity, the use of a polyclonal instead of monoclonal antibody and the immobilized antibody amount for tz construction. effect of the composition of the developing solution. the developing solution composition has great impact on the antibody-antigen interaction on the biosensor. ideally, the developing solution facilitates the nanoparticles and sample mixture movement along the biosensor with flow rates that allows test completion in short time providing, in parallel, sufficient time for virion interaction with the modified nps. moreover, the developing solution should also prevent the non-specific nanoparticles absorption by the lfb membrane . glycerol increases the mixture viscosity causing a slower flow rate. addition of several surfactants (sds, tween- , sds, etc) or proteins (e.g. www.nature.com/scientificreports www.nature.com/scientificreports/ bsa) enables the particles movement from the conjugate pad. eleven different formulations were studied and their detailed composition is described in table , while the respective results are presented in fig. s . the use of high ph buffer or bsa did not result in any specific signal formation (fig. s a) . two formulations were optimal for non-specific signal elimination: i) g/ s/p, and ii) g/ s/ . t/p. the formulation that contained % glycerol resulted in prolonged running times (∼ min); therefore it was excluded from further studies. it was observed that tween- concentration seemed decisive for the biosensor specificity. for that reason, increasing tween- concentrations were tested in the range of . - % and the optimum results were obtained with . % tween- in developing solution (fig. s b) . concluding, the optimum developing solution for background elimination, rapid assay times, and specific bands was obtained using % glycerol, . % tween- and % sds in ×pbs, ph . . effect of the antibody dispensing velocity. the dispensing velocity for construction of the tz based on the polyclonal anti-nodavirus antibody was subsequently tested (fig. a) . the antibody dispensing velocity is an essential www.nature.com/scientificreports www.nature.com/scientificreports/ parameter for production of a uniform immobilized test zone. in general, high volatile solutions (e.g. acetone) are dispensed with high velocity (> nl/s) whereas low volatile solutions (water, ethanol) are sprayed with low velocity (< nl/s). based on previous studies for antibody immobilization on nitrocellulose membranes , a methanol containing solution ( ml/l) was used. methanol results in medium volatile solutions and the use of nl/s dispensing velocity was optimum for the uniform formation of the anti-rabbit igg control zone. however, the same dispensing velocity resulted in the formation of two 'pseudozones' when the anti-nodavirus antibody solution was sprayed on the membrane. it seems that high dispensing velocity creates a gap in the middle of the spraying zone, which doesn't allow proper absorption of the antibody solution from the membrane, perhaps due to non-uniform evaporation. the use of lower dispensing velocity resulted in a single uniform test zone and the velocity of nl/s was chosen since it resulted in the optimum signal. effect of the use of polyclonal instead of monoclonal anti-nodavirus antibody. in order to enhance the lfbs' specificity, a monoclonal anti-nodavirus antibody which was commercially available was used instead of the produced polyclonal antibody. it is obvious in fig. b , that the monoclonal antibody did not produce visual signal whereas use of the polyclonal antibodies led to high signal formation, since more antigenic epitopes interacted with higher antigen amounts and were subsequently immobilized. effect of the type of the diagnostic membrane. the porosity of nitrocellulose membranes plays a critical role for the specificity and the sensitivity of a lateral flow biosensor because it influences the capillary flow rate. two membranes, the hf and hf (millipore) were tested for the nodavirus lfb development, with corresponding migration times of and min, respectively. as shown in fig. c , the signals of the lfbs which were based on the hf membrane were more intense than the signals obtained with the hf membrane. thus, longer migration time is favorable for the sandwich-type complexes on the lfb, in accordance with independent observations . effect of the immobilized antibody amount. the anti-nodavirus antibody amount immobilized on the biosensor test zone was examined. ng of antibody per -mm lfb were used to obtain the optimum signal (fig. d) . the use of ng/µl for antibody immobilization resulted in slightly decreased signal, which was significantly lower when . ng/µl of the antibody were applied. interestingly, the signal decreased as the antibody concentration increased. the use of high amounts of antibodies on lateral flow biosensors could cause the so-called "hook effect". the prozone or hook effect is basically a false negative result of a particular analyte, when an antibody has very high concentrations. if too many antibodies that can bind to the antigen are present, then the antigenic sites are coated by these antibodies, and few or no conjugated au nanoparticles directed toward the analyte are able to bind more than one antigenic particle. since the antibodies do not bind to antigens, no signal is formed. therefore, the result is a false negative . lfb detectability was assessed by samples consisting of decreasing concentrations of nodavirus infected ssn- cell culture supernatants. all samples were studied by applying the optimized experimental conditions. in details, supernatant volumes ranging from . to . µl of supernatants were applied on lfbs. the respective lfbs are shown in fig. a ,b. the lowest detectable amount of nodavirus containing supernatant was . µl. to define a positive result and discriminate from any background signal, the signal/noise (s/n) ratio (s/n = ) was used. the signal definition refers to the tz optical intensity of each detected sample, whereas noise is the produced tz intensity when using culture medium alone. the lowest amount corresponded to . tcid (supernatant titer was . tcid /ml) which is the measure of infectious virus titer. high amounts of virus supernatant seem to result in stable densities. nodavirus lfb reproducibility. reproducibility is one of the most crucial parameters of the lfb evaluation procedure. for that reason, it was assessed with a constant amount of nodavirus containing ssn- cell culture supernatant ( µl). five biosensors were tested with the nodavirus supernatant for studying test and control zones. all lfbs were prepared in variable batches. the lfbs are shown in fig. s , and analysis by densitometry resulted in coefficient of variation (cv) of . % for the test zones and . % cv for the control zones. nodavirus lfb specificity. specificity of nodavirus biosensor was assessed by studying the assay cross-reactivity with non-infected ssn- cell culture supernatant and homogenized tissues of non-infected fish (fig. ) . only the samples which were characterized positive by cell culture resulted in detectable signal. developing solution-only negative controls were also tested in each run. concluding, the analytical performance of the proposed assay can be summarized as follows: × − tcid of cell culture supernatant was detectable, by using a standard centrifugation step and the proposed biosensor. the assay is specific for infected fish and highly reproducible. in table , the assay sensitivity of previously reported methods for nodavirus virion detection by sandwich/ capture elisa, as well as nodavirus detection with lateral flow assays in general, are summarized. even though comparison of these methods' sensitivity cannot be highly accurate, since the limit of detection is determined by slightly different way in each case, the present assay seems to have higher sensitivity for virion detection in comparison to elisa methodologies and the mab/ lfia www.nature.com/scientificreports www.nature.com/scientificreports/ brain homogenization and separation of the insoluble fraction by centrifugation or ssn- cell culture supernatant). the samples supernatants were applied directly to the nodavirus biosensors, which were run and scanned. eleven infected (positive; n = ) and non-infected (negative; n = ) d. labrax samples were applied on the lfb (fig. ) . all results were visualized within minutes. all tested samples were verified with ssn- cell culturing. five samples were originated from saronikos gulf area (s , , , , ), samples were from korinthian gulf region (s - ), sample was collected from amvrakikos gulf (s ) and samples were originated from aquacultures in cyprus (s , , ). a negative control, containing only developing solution, and a positive control, containing . µl of nodavirus containing cell culture supernatant (corresponding to the limit of detection), were included in each run. the use of the cell culture supernatant in an amount corresponding to the assays detection limit was intended to be used as a visual "yardstick", a comparison measure to indicate which signal could be positive. if the positive control was intended to be used just to confirm the lfbs' proper operation more supernatant could be applied to provide more intense signal. we observe that in the lfbs with positive signal the gold nanoparticle conjugates appear to bind non-specifically between the test and the control zones. in any case that non-specific binding is not crucial for the nodavirus presence assessment, since the test zone is clearly positive or negative. most of the non-infected samples (s - ) did not show any signal in the tz. the infected samples resulted in positive signals with varying intensities above the positive control signal, which correlates with different amounts of nodavirus in the sample. one negative and one positive sample (s and s , as characterized by ssn- cell culturing) resulted in similar faint zones. in order to decide the disease state of each sample, the test zones were quantified with the publicly available, easy to use imagej software, based on scanned lfb images. quantification confirmed that s was negative and s positive with very low virus load (fig. b) . in the case of such ambiguous results on the field, the lfbs images could be photographed and quantified with imagej with a smartphone. the scanned images of the lfbs were independently evaluated by a second person, and were compared to lfb images with known outcomes (positive and negative). cost-effective and labor-saving methods for fish infectious diseases identification have attracted an increasing interest to aquaculture industry in recent years, due to their high economic impact. the development of a highly promised robust and rapid lateral flow biosensor has been reported in the present study. the proposed lfb can be used for simple and accurate screening of nodavirus detection in various fish species. to enable on site analysis of nodavirus, we developed and optimized a paper lfb based on gold nanoparticles for easy, specific and sensitive visual detection of nodavirus viral particles (virions) in biological samples. the target virions can be simply detected by the red colored signal formation in the lfbs test zone. the lfb permits the visual detection of nodavirus particles within minutes without using any instrumentation. the total assay starting including tissue homogenization, centrifugation and virion detection with the biosensor was accomplished in less than min. the total cost of the assay is less than €. undergoing studies by our research group include complete validation of the nodavirus biosensor on fish culture facilities, and efforts to improve the biosensors' sensitivity of the lfb utilizing dual nanoparticles labels , and signal enhancement systems in general. moreover, the elimination of centrifugation from the sample preparation step is among our future goals to further simplify nodavirus detection on-site. concluding, the proposed biosensor could become a reliable tool for the rapid screening of questionable samples in aquaculture facilities. as a screening tool it can be used to decide whether a sample is positive or negative, and indicate which samples have ambiguous state of the disease and need further analysis by other methodologies in a laboratory setup. such a procedure could lower the cost and the analysis time to maintain healthy fishes in aquaculture facilities. fine mapping qtl for resistance to vnn disease using a high-density linkage map in asian seabass recombinant nodavirus vaccine produced in bacteria and administered without purification elicits humoral immunity and protects european sea bass against infection advances in aquaculture vaccines against fish pathogens: global status and current trends betanodavirus infections of teleost fish: a review understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy viral encephalopathy and retinopathy in aquaculture: a review cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, dicentrarchus labrax nanoparticle-based lateral flow biosensor for visual detection of fish nervous necrosis virus amplification products towards a dual lateral flow nanobiosensor for simultaneous detection of virus genotype-specific pcr products a nanostructured microfluidic immunoassay platform for highly sensitive infectious pathogen detection development of the sensitive lateral flow immunoassay with silver enhancement for the detection of ralstonia solanacearum in potato tubers paper-based surface-enhanced raman scattering lateral flow strip for detection of neuron-specific enolase in blood plasma flexible electronics based on micro/nanostructured paper a paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers biomarker detection for disease diagnosis using cost-effective microfluidic platforms lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey lateral flow assays: principles, designs and labels rapid immunochromatographic assay for ofloxacin in animal original foodstuffs using native antisera labeled by colloidal gold progress in rapid optical assays for heavy metal ions based on the use of nanoparticles and receptor molecules gold nanoparticle-based colorimetric elisa for quantification of ractopamine lateral and vertical flow assays for point-of-care diagnostics a rapid diagnostic assay for intact influenza virus using a high affinity hemagglutinin binding protein specific detection of avian influenza h n whole virus particles on lateral flow strips using a pair of sandwich-type aptamers factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus x enhancing the lateral-flow immunoassay for viral detection using an aqueous two-phase micellar system dual enhancement with a nanoparticle-based lateral flow biosensor for the determination of dna identification of immunoreactive leishmania infantum protein antigens to asymptomatic dog sera through combined immunoproteomics and bioinformatics a simple method for estimating fifty percent endpoints vaccination with leishmania histone h -pulsed dendritic cells confers protection in murine visceral leishmaniasis. vaccine image processing with imagej identification of b-cell epitopes on the betanodavirus capsid protein reproducibility of antigen-immobilized enzyme-linked immunosorbent assay (elisa) and sandwich elisa for quantitative detection of nnv particles nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer effect of different-sized spherical gold nanoparticles grown layer by layer on the sensitivity of an immunochromatographic assay a remarkable sensitivity enhancement in a gold nanoparticle-based lateral flow immunoassay for the detection of escherichia coli o :h . rsc adv oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for dna analysis by hybridization identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction: a dna biosensor for simultaneous visual detection of both alleles visual detection of microrna with lateral flow nucleic acid biosensor studies of the 'hook' effect in the one-step sandwich immunoassay development of a lateral flow immuno-chromatic strip assay for the detection of nervous necrosis virus (nnv, rgnnv genotype) double-enhanced lateral flow immunoassay for potato virus x based on a combination of magnetic and gold nanoparticles production of monoclonal antibodies against grouper nervous necrosis virus (gnnv) and development of an antigen capture elisa detection of betanodavirus in juvenile barramundi, lates calcarifer (bloch), by antigen capture elisa quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass dicentrarchus labrax rapid and sensitive detection of macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (cpa-lfd) for rapid and specific detection of red-spotted grouper nervous necrosis virus reagents. ssn- cells were purchased from the european collection of animal cell cultures (salisbury, uk). fetal bovine serum (fbs) and leibovitz l -medium were from biochrom (berlin, germany), and whatman filters (ptfe, . μm) were from ge healthcare (buckinghamshire, england). penicillin, streptomycin and l-glutamine were from gibco (paisley, uk). cell culture flasks falcon primaria were purchased from becton dickinson labware (franklin lakes, usa) and flat bottom -well elisa plates were obtained from greiner (kremsmunster, austria). micro bicinchoninic acid (bca) protocol for protein quantification, nab tm spin columns and zeba tm desalting columns were from pierce (thermo fisher scientific, delaware, usa). borax, dimethyl-sulfoxide, bovine serum albumin (bsa) and glutaraldehyde, were purchased from applichem (darmstadt, germany). eagle minimum essential medium (emem), complete and incomplete freund's adjuvant (cfa and ifa, respectively), anti-rabbit igg-horseradish peroxidase (hrp), poly-l-lysine, anti-rabbit igg (whole molecule) antibodies, , ′, , ′-tetramethylbenzidine (tmb) substrate, au nps stabilized in pbs ( , , , , nm), or citrate buffer ( nm), siliconized tubes and tween- were obtained from sigma-aldrich (steinhem, germany). monoclonal anti-nodavirus antibody was from aquatic diagnostics (stirling, uk). nitrocellulose membranes on laminated cards (hf mc and hf mc ), glass (gfcp ) and cellulose fiber pads (cfspoo ) were all from millipore (billerica, ma, usa). applichem or sigma provided all common reagents.low the research project was implemented within the framework of the action «supporting postdoctoral researchers» of the operational program "education and lifelong learning" (action's beneficiary: general secretariat for research and technology, ls _ ), and was co-financed by the european social fund (esf) and the greek state. d.k.t. conceived and designed the experiments; d.k.t. and m.m. performed the experiments; a.p. provided d. labrax samples; d.k.t. and e.k. analyzed the data; d.k.t. wrote the paper; a.p. and e.k. proof-read the manuscript; d.k.t. and e.k. supervised the work. all authors have approved the present article. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to d.k.t.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - wy rpqv authors: denman, a.m. title: viral etiology of polymyositis/dermatomyositis date: - - journal: polymyositis and dermatomyositis doi: . /b - - - - . - sha: doc_id: cord_uid: wy rpqv nan a.m. denman . . . for we all of us, grave or light, get our thoughts entangled in metaphors and act fatally on the strength of them. george eliot, middlemarch any attempt to ascribe polymyositis and dermatomyositis (pm/dm) to a viral etiology must take into account what is already known about the pathogenesis of this group of disorders. first, it is a rare disease, and there are few pointers to a conventional infec tious etiology. outbreaks of the disease have not been described, and the evidence for a seasonal onset is weak. thus, theories based on a viral etiology must invoke a highly un usual host response to ubiquitous agents, the existence of uncommon viruses with the abil ity to provoke the disorder, or the possibility that virus infections operate synergistically with other factors. second, the immunopathologic features of pm/dm strongly indicate the importance of immune mechanisms in the pathogenesis of most forms of the diseases [ ] . thus, advocates of a viral etiology are confronted with the same problems facing anyone seeking to implicate virus infections in autoimmune diseases in general. finally, pm/dm often accompanies a variety of other inflammatory connective tissue diseases, and these associations must also be satisfactorily explained by any theory based on viral infec tion. increasing understanding of the immunopathologic consequences of viral infections means that such points can be countered con- the author would like to thank mrs. kathy jameson for expert help in preparing the manuscript. attempted isolation electron-microscopic studies viral probes antiviral antibody titers establishing clones from infiltrating t lymphocytes and screening their antigen specificities exploring the antigen specificities of associated autoantibodies in vitro models of virus-infected muscle cells in vivo models of experimental polymyositis ceptually. however, one must concede that the evidence to date for a viral etiology is fragmentary. the available approaches for exploring this possibility are set out in table . . in recent years there have been several claims implicating specific virus infections in isolated patients with pm/dm. these reports have of ten been based on direct isolation of the agent or the ultrastructural appearances of the inflamed muscle. coxsackieviruses have at tracted particular attention because of the relative ease with which these agents induce inflammatory disease in cardiac and striated muscle of mice. in addition, coxsackieviruses have been linked with myocarditis in human infants on epidemiologic grounds [ ] . struc tures resembling picornaviruses were de tected by electron microscopy in muscle biopsy from two patients who died with subacute dermatomyositis [ ] . similar particles were seen in the diaphragmatic and intercos tal muscles of an -year-old girl who suc cumbed to a chronic myopathy [ ] . these authors also claimed that muscle suspensions induced cytopathic effects in culture of pri mary human amnion cells. furthermore, the isolate reacted with antibody to coxsackievirus a . other viruses have been the subject of similar reports. mckinlay and mitchell [ ] described eight children with transient myositis associated with elevated serum levels of creatine (ck) following acute virus infec tions. adenovirus type ii was isolated from one patient and a parainfluenza virus from a second patient. pm complicated the clinical course of a patient with undoubted hepatitis caused by the hepatitis b agent since the diag nosis was confirmed by muscle biopsy and marked elevations of the serum ck level [ ] . hepatitis b surface antigen (hb s ag) was de tected by immunofluorescence studies of the intranuclear and intracytoplasmic inclusions in the biopsy specimen of the affected muscle. mikol et al. [ ] have also described inclusion bodies in a muscle biopsy specimen obtained from a patient with long-standing pm; these workers also claimed to have cultured adeno virus type ii from the biopsy. in addition to the foregoing reports there have been several reports based almost exclu sively on ultrastructural appearances (sum marized by schiraldi and iandolo [ ] . very few of these reports have been confirmed by isolating the virus suspected on ultrastruc-tural grounds. nor indeed have there been many attempts to confirm the viral nature of the inclusion bodies by immuno-electron mi croscopic techniques or other specific stain ing methods. indeed, there are dangers in ascribing pm/dm to a viral etiology on the basis of ultrastructural changes in isolation. katsuragi et al. [ ] have emphasized this point by their description of histochemical studies of a mus cle biopsy specimen from an elderly man with pm. some unidentified objects in the biopsy specimens were initially considered to be in clusion bodies resembling picornaviruses but were later shown to have the chemical charac teristics of glycogen. the most striking im pression from these reports is their scattered nature, implicating several different agents. there is a dearth of reports showing that agents can be consistently isolated and pas saged in vitro from patients with pm/dm. nor have sequential studies of immune re sponses been appended indicating that pa tients with pm/dm attributed to a given agent mounted a cell-mediated or humoral immune response to that agent at the time of alleged infection. there are diagnostic difficulties over some reports. myalgia has often been reported in association with acute respiratory tract infec tions, and the symptoms may be accompa nied by electromyographic (emg) abnormal ities [ ] . an epidemic of acute myositis has also been described in children in association with influenza b virus infection [ ] . tran sient elevation of the ck level was noted in these children, and influenza b virus was iso lated from of the patients studied. how ever, the patients made a complete recovery in to days without specific treatment, and it is clear that postviral myositis of this nature is unrelated to persistent pm as generally en countered. interestingly, the symptoms were almost exclusively confined to the gastrocnemius and soleus muscles. acute myositis has also been described after rubella vaccination, although the accompanying clinical and patho logic features suggested that the muscles were involved as part of a more generalized arthus reaction to vaccination [ ] . the muscle dis orders most commonly provoked by viral in fections have usually taken the form of tran sient myalgia, in contrast to the more sustained character of idiopathic pm/dm. furthermore, the muscle disorders provoked by most spe cific infections have also been focal rather than generalized and, therefore, clearly re lated to localized infection [ ] . muscle symptoms after influenza-like illnesses have also been described in older patients [ ] , including objective muscle weakness and minimal emg changes. however, the viral etiology of the antecedent illness was not con firmed, biopsy proof was not obtained, and there was no consistent rise in the serum ck level or the levels of other muscle enzymes. muscle problems in patients with immu nodeficiency deserve separate comment. muscle inflammation attributable to echovirus infection presents a striking picture in patients with hypogammaglobulinemia [ ] . the muscle inflammation is often unilateral, usually localized, and rarely involves the clas sic sites of muscle involvement in idiopathic pm/dm. the virus has usually been isolated from the cerebrospinal fluid rather than from involved muscle [ ] . local abnormalities are revealed by muscle biopsies and consist of perivascular mononuclear infiltrates and muscle fiber atrophy, occasionally accompa nied by extensive interstitial fibrosis. how ever, the disease picture in these unusual cir cumstances has not been shown to have a wider relevance. there are additional reasons for doubting the relevance of most reports of this nature to the pathogenesis of pm/dm as commonly encountered. ultrastructural changes of a kind likely to be caused by virus infection have not been reported in any systematic study of biopsy specimens from patients with pm/dm diagnosed by accepted criteria. fur thermore, the epidemiologic evidence does not support the concept that exposure to common viruses is linked to the pathogenesis of pm/dm. thus, influenza virus causes tran sient myalgia and has also been isolated from the muscle of a patient with myoglobulinuric pm [ ] . yet massive immunization with swine influenza vaccine during a -week period in was not followed by any in crease in the incidence of pm/dm [ ] . most attempts to identify viruses in clini cal material obtained from patients with pm/ dm have depended on the presence of viruses that can either be recovered as fully infectious particles or recognized ultrastructurally. there is always the possibility that defective replication or latent infection might render the host cell the target for an immune re sponse and yet be undetectable by traditional methods. the powerful techniques now avail able for detecting viral nucleic acid sequences offer one obvious means of probing putatively infected muscle samples. this approach has been applied to the study of heart muscle from patients with myocarditis [ ] . coxsackievirus b has been implicated serologically in the etiology of myocarditis, but virus has not been convincingly detected in dis eased muscle by immunofluorescence or iso lated by conventional culture techniques. these authors used a complementary dna (cdna) copy of coxsackievirus b genomic rna as a hybridization probe for identifying virus-specific sequences in myocardial biop sies. they claim to have obtained positive hybridization signals in nine of samples from patients with myocarditis and related disorders. if confirmed and extended, these experiments will obviously be relevant to sim ilar attempts to identify viral sequences in biopsy specimens from patients with pm/dm. one can also speculate that latent infection might be more readily detectable if the in fected host cells could be cultured for periods of sufficient duration. improving techniques for culturing differentiated muscle cells and for inducing the growth and expression of latent viruses makes this an important area for future research. antibody screening has been frequently used as indirect means of obtaining evidence for a viral etiology in pm/dm. antibody titers to coxsackievirus b have received particular at tention because of the many experimental models in which this group of viruses induces myocarditis. this line of research has pro duced some suggestive but not conclusive evi dence for coxsackievirus infection in patients with cardiomyopathies. cambridge et al. [ ] encountered high neutralization titers to coxsackievirus b more commonly in patients with congestive cardiomyopathy than in con trols. moreover, this occurred more com monly in patients with a short history than in patients with a febrile illness at the onset of their symptoms. interestingly, endomyocardial biopsies did not produce evidence of direct viral infection. although myositis and myo carditis occur most commonly as clinically distinguishable entities, cardiac involvement is often an important feature of idiopathic pm/dm. thus, in one detailed study haupt and hutchins [ ] found evidence of active myocarditis in of patients with pm or related diseases. thus it is reasonable to sur vey antiviral titers to this group of viruses in patients with pm/dm in isolation. in a lim ited study of this problem travers et al. [ ] noted a marked rise of antibody titer to a specific serotype of coxsackievirus b in four patients with pm/dm without any rise in titers to other viruses. detailed surveys of this kind have not been reported in pm/dm. simi larly raised antibody titers to these viruses have been detected by complement fixation techniques in a preliminary study of children with juvenile pm/dm [ ] . the most informative studies of this kind have been those that have sought evidence for an igm response to defined coxsackievirus strains, using enzyme-linked immunosorbent assay (elisa) techniques, since this approach is more likely to detect serologic indication of recent infection. el-hagrassy et al. [ ] used elisa techniques to detect coxsackievirus b-specific igm responses in % of patients with acute myocarditis or pericarditis. in other areas of clinical investigation improved elisa assays have also proved more discrimi nating. king et al. [ ] detected igm responses to a single coxsackievirus b strain in % of children aged to years with insulin-dependent diabetes mellitus; their findings also emphasize the important point that it may be easier to identify recent infec tions by defined viral strains in children than in adults since the latter can be expected to have had cumulative exposure to the entire range of these ubiquitous viruses. surveys of antiviral antibody titers of pm/dm have not provided any other useful clues to agents that might be implicated in the pathogenesis of the disorder. occasionally, high antibody titers to specific agents have been described in isolated cases. thus, john and fink [ ] noted a sustained rise in anti body titers to echovirus type in one patient with pm. however, surveys of antiviral anti body titers to common viruses pose several problems in interpretation. first, antibodies if viruses induce pm/dm, the simplest mech anism by which they could induce this disease is by replicating in muscle cells, thereby serv ing as a direct target for an immune attack. furthermore, ephemeral or latent infection of muscle cells could initiate an immune response subsequently sustained by other mechanisms. in a search for myotropic vi ruses, klavinskis et al. [ ] found that two strains of influenza a virus lyrically infected human synctial myotubes. in addition, one strain was found to infect unicellular precur sor myoblasts. these authors showed that viral proteins were synthesized in the infected cells and they also identified viral antigens on the surface of the infected cells by immunofluorescence and immuno-electron microscopy ( fig. . ). there is little information about will be present in the majority of the popula tion. rising antibody titers or the detection of igm antibody against strains recently detected in the community might be helpful, but such surveys are difficult to organize, particularly given the rarity of the disease. positive finding in cases drawn from widely disparate geo graphical areas would need extensive control studies in those same areas. second, antibody titers to endemic viruses do not necessarily imply that those agents are pathogenetically related to the disease. this point is particu larly troublesome if the virus, such as mem bers of the herpes group, persists life-long in the human host; rising antibody titers may simply reflect nonspecific reactivation. finally, atypical infections with unusual clinical con sequences may be encountered in patients with generalized immunodeficiency or impaired immunity specifically to the infecting agent. this immunodeficiency could be reflected in inappropriately low antibody titers; the com plications of echovirus infections in hypogammaglobulinaemic patients vividly illustrate this problem. the extent to which other viruses implicated in the pathogenesis of pm/dm grow in mus cle cells. nor should interactions between viruses and muscle cells be analyzed exclu sively in terms of immune-mediated damage. there is also the possibility that pm/dm in duced by a putative infectious agent could result from a mixture of direct cytopathic changes and immune damage. for example, hemolytic paramyxoviruses induce perme ability changes in infected cells in addition to their ability to mediate cell fusion. patel and pasternak [ ] have described membrane permeability changes in cell lines infected by influenza virus at low ph which persisted when the ph was shifted to the physiologic range. persistent pm/dm is often character ized by degenerative changes in muscle fibers the suspected association between coxsackievirus infections and muscle disorders of man has prompted a particular interest in animal models of myocarditis and pm induced by this group of viruses. moreover, it has proved relatively easy to induce inflammatory disorders of muscle with these agents. coxsackievirus-induced myocarditis has been studied particularly intensively. the factors determining the outcome of such infections are set out in table . . to some extent the outcome of infections by coxsackievirus b is governed by the genes peculiar to the infecting strain. indeed, strains belonging to the same group differ in their ability to grow in host tissues and in their resulting virulence. virulent and avirulent strains may appear identical by criteria such as antigens detected by conventional polyclonal antisera, biophysi cal properties, and genomic structure. further more, even virulent coxsackievirus strains indistinguishable by conventional criteria induce a variety of clinical syndromes, including myocarditis. for example, adoles cent cd-i mice inoculated with one variant of coxsackievirus b developed readily observable myocarditis, whereas a second variant was virtually innocuous [ ] . these variants were indistinguishable in terms of their ability to grow in cell lines in vitro or heart muscle cells in vivo, to stimulate defective particles that might interfere with viral replication, or to stimulate host immune factors such as interferon. however, more stringent analysis may reveal basic differences between virulent and avirulent strains. some of the factors determining the outcome of coxsackievirus b infections are governed by the genes of the infecting strain. prabhakar et al. [ ] showed that monoclonal antibodies can detect differences between strains of virus considered antigenically homogeneous by conventional analysis. these authors used monoclonal antibodies to detect antigeni cally distinct variants of coxsackievirus b . they also found that the rate of antigenic mutation is extremely high in this strain, resembling that long recognized to occur in influenza viruses. further analysis of antigenic variants of coxsackievirus b shows that clinical isolates display a mixture of highly conserved, moderately conserved, and poorly conserved epitopes [ ] . their studies suggested that a high rate of error in rna replication is responsible for these frequent antigenic changes. moreover, this appeared to be independent of any selection pressure. receptor availability? interferon induction in interstitial cells? express la antigens? antibody-all relevant epitopes recognized? cell mediated immune responses: antiviral autoimmune access to myocardium interferon production see text for discussion and references. despite this marked antigenic diversity, all the isolates were neutralized by conventional polyclonal antiserum. although most of the antigenic changes solely detected by mono clonal antibodies may be innocuous, there is also the possibility that these changes deter mine viral tropism for different target cells in vivo and hence their subsequent virulence. however, while there is considerable antigen variation between different strains of coxsackievirus b, repeated passage in intermediate hosts seems to stabilize their properties since there is preferential survival of strains that are tropic for some target cells such as pan creatic islet cells and other endocrine tar get cells. thus, coxsackievirus b repeatedly passaged in mouse pancreata or beta cell cul tures acquired a specific tropism for those cells [ ] . the nature of the host immune response also influences the outcome of experimental coxsackievirus infection. this important point is borne out by all the earlier work on this model (reviewed by woodruff [ ] ). both humoral and cell-mediated immune responses contribute to host defense. in animal models, humoral mechanisms are decisive in limiting primary infection. cell-mediated immunity also contributes to host defense but some times at the price of initiating immunopathologic reactions against virus-infected myocar dial cells. thus cell-mediated responses are a mixed blessing for the infected host. there is good experimental evidence that cellmediated immunity may reduce the virulence of coxsackievirus infections. khatib et al. [ ] found that the virulence of one strain of coxsackievirus b was increased in a strain of swiss mice given antithymocyte serum at the time of infection. in addition, immunosuppressive treatment with cyclophosphamide converted infection by an avirulent strain of b coxsackievirus into one produc ing myocarditis, probably because the virus was thereby allowed to replicate to abnor mally high titers [ ] . however, the dual contribution of viral cytopathic effects and cell-mediated immunity is well illustrated by experiments in which athymic balbc-nu/nu mice were infected with coxsackievirus b [ ] . in the immunodeprived mice, the virus produced mild degenerative or necrotic changes only. however, intense inflammatory changes were induced after the adoptive transfer of spleen cells of immunocompetent nu/+ mice immunized with the same virus. in another athymic strain nu/nu mice could not eliminate infectious virus from their hearts, and this persistent infection was associated with myocardial abnormalities characteristic of cytopathology and also with increased mortality rates [ ] . euthymic nu/+ mice were infected for much shorter periods, yet developed persistent myocardial abnormali ties attributable to the cellular response. these observations emphasize that the myocardial changes are determined partly by viral factors and partly by the character of the t-cell response. the immunopathologic consequences of t-cell immunity often offset any advantage to the host. this outcome is suggested by the frequent presence of mononuclear cell infil trates at sites of coxsackievirus replication in myocytes indicative of myocarditis. in vitro studies of sensitized lymphocytes in coxsackie virus infections emphasize the complexities of cell-mediated immunity to these agents and point to some pathways that may lead to immunopathologic catastrophes. in general, the briskness of the cytotoxic t-cell reaction to infected myocardial cells correlates with the severity of the myocarditis, which sug gests that hypersensitivity to viral antigens contributes to the immunopathologic conse quences of infection. however, there are also important observations that sensitized lym phocytes in coxsackievirus infections are able to kill uninfected myocardial cells and fibroblasts (woodruff [ ] ), and there is now good evidence that this autoreactivity contributes to the myocarditis. in addition, paque et al. [ ] have described experiments in which peritoneal exudate cells from mice infected with coxsackievirus b were tested for their reactivity with cardiac antigens using the mi gration inhibition assay. they found that the migration of these cells was specifically inhib ited by cardiac antigens isolated from mice infected with coxsackievirus b but not by antigens prepared from uninfected hearts or from hearts infected with an unrelated virus. since the cardiac extracts failed to bind virusneutralizing antibodies, they concluded that virus-induced tissue antigens but not virusrelated antigens were responsible for this inhibition. paque et al. have extended these observations to baboons infected with this virus [ ] . in these experiments, sensitivity directed solely at antigens isolated from hearts infected with coxsackievirus was shown by a proliferative response of lympho cyte monitored by the incorporation of tritiated thymidine as well as by inhibition of migration. further evidence for autoimmunity to myocyte antigens is provided by experiments in which spleen cells of mice infected with coxsackievirus b were allowed to adsorb to uninfected or infected myocyte monolayers [ ] . one population of t lymphocytes adsorbed to and lysed uninfected myocytes and were assumed to be autoreactive. in contrast, lymphocytes lysing infected cells were considered to be virus specific. the autoreactive lymphocytes were injected into recipient mice infected with the same virus but deprived of t lymphocytes by prior ex perimental manipulation. the transferred cells induced myocarditis in mice not other wise able to mount a t-cell response to infected myocytes. these observations raise important questions about the nature of the autoantigens, the manner in which auto immune reactions are induced, and the fac tors that dictate their immunopathologic importance. so far, there is little information about the precise nature of the antigens in apparently uninfected heart cells which attract the t-cell response. these might be related to sites of viral transport across cell membranes during the initial infection or during the export of newly synthesized viral particles. nor is it clear whether the response is directed at viral antigens or, as seems more likely, normal host cells whose production or expression is al tered by viral infection. furthermore, while autoreactivity persists beyond the time in which viral persistence can be detected by conventional techniques, there is still the strong possibility that in reality it is sustained by viral persistence in latent or defective form. appropriate experiments with viral probes or with techniques for reactivating vi rus have not yet been reported. the sequence of events by which transient or persistent coxsackievirus infection induces an autoreactive t-cell response also remains largely unexplored. autoreactivity arises early in infection and is more vigorous in female than in male mice. the ability of t cells to gain access to infected myocardial fibers may also influence the outcome of the infection. huber and job [ ] in described two strains of coxsackievirus b type which are indistin guishable in terms of their antigenic proper ties and ability to grow to high titers in myo cardial cells. judged by in vitro techniques these strains are equally efficient in inducing cytotoxic t lymphocytes. however, it is likely that in mice infected by the virulent strain, these cells are able to reach and damage myo cardial cells, whereas lymphocytes in mice infected by the nonvirulent strain lack this ability. however, other crucial pieces of in formation are lacking, such as the expression of viral antigens in infected cells, local inter feron production, the expression of la anti gens by myocardial cells, and the kinetics of the infiltrating cell populations. it is also possible that the extent to which coxsackievirus infection interferes with the re gulation of the immune response also deter mines the severity of the immunopathologic damage. as with other models of experimental virus infection, coxsackieviruses induce sup-it has long been recognized that some strains of echo virus induce pm in mice [ ] . how ever, the pm results from acute necrosis of muscle fibers, and there is little evidence that the pm is mediated by immune mecha nisms. indeed, paralysis only occurs in mice in which viral replication has reached very high titers. thus, the disease is easily induced in newborn mice in which more than pressor t cells which affect immune responses in general and may also influence the character and efficiency of specific antiviral immune re sponses [ ] . deficient suppressor mechan isms could allow persistent autoreactive tcell responses that damage heart fibers even after infectious virus has been eliminated and viral antigens are no longer expressed. there is little evidence to date that coxsackieviruses interfere directly with lym phocyte subpopulations. however, it has been shown that these viruses readily establish per sistent infections in human lymphoid cell lines and particularly in b-cell lines [ ] . only a minority of the cells are infected, hinting that in vivo infection might be difficult to identify if only a minority of circulating lym phocytes are infected. these observations also suggest that the infection may be maintained by mutants of the original infecting virus. the relevance of this experimental model of lym phocyte infection to putative defects in immunoregulation has not yet been established. in addition to specific immune responses, other nonspecific factors influence the out come of experimental coxsackievirus infec tion. thus, reyes et al. [ ] have shown that myocardial damage is accentuated in mice infected with coxsackievirus b that were given forced exercise in the form of daily swimming. observations of this kind rein force the suspicion that excessive physical ac tivity may predispose to myocarditis and pm in clinical practice. % of skeletal muscle tissue is destroyed, older mice rapidly acquiring resistance to this process [ ] . similarly, acute myositis has long been recognized as a consequence of coxsackievirus b in neonatal mice, the dis order resulting from the cytopathic effects of viral growth [ ] . interestingly, even in this model, the myositis is selective for spe cific muscle groups, the hip extensors and hindquarter knee flexors being particularly susceptible. a more persuasive animal model of virusinduced pm is that described by strongwater et al. [ ] . in this model, the intraperitoneal inoculation of the tucson strain of coxsackievirus bl induced proximal hindquarter weak ness that persisted for more than weeks. moreover, the criteria for pm were satisfied by the characteristic emg and histologic changes. furthermore, these changes per sisted long after infectious virus could no the extraordinary diversity of clinical syn dromes associated with the acquired immu nodeficiency syndrome (aids) has alerted clinical investigators to the possibility that demonstrable or covert infection by retroviruses could be etiologically related to pm, or indeed any chronic disorder whose cause has continued to baffle them. however, pm has not emerged as a frequent complication of aids despite the close monitoring of such patients for neurologic disorders. thus, of patients with aids or generalized lymphadenopathy studied at one center between and , only five patients had muscle problems: two had persistent myalgias, two had myopathy, and only one had pm [ ] . more recently, however, two additional pa tients with pm associated with aids were reported [ a] , as discussed in chapter . yet in a primate model of acquired immunodefi ciency, % of monkeys infected with the d retrovirus called saids d showed muscle weakness and wasting, elevated serum con centrations of sarcoplasmic enzymes, and bi opsy features of pm [ ] . the etiologic agent could be transmitted experimentally to healthy rhesus monkeys by tissue homogenates, blood, saliva, and urine. reverse transcriptase assays revealed the presence of virus in muscle biopsies, but immunofluorescent studies with antibody to the agent longer be detected, suggesting that the disor der was mediated by immune mechanisms. infectious virus could only be recovered from the affected muscles for weeks after infec tion, during which period viral antigen could also be detected by immunofluorescence. no tably, electron microscopy did not reveal characteristic viral particle during this period of demonstrable infection. this model has many features in common with human pm and could also explain why it might be diffi cult to demonstrate virus infection in man. showed that the agent was localized to the infiltrating inflammatory cells and interstitial fibroblasts rather than to muscle fibers. inter estingly, the retrovirus replicated in myotubes and fibroblasts in culture but did not have any cytopathic effect on these cells. the predomi nantly immunopathologic features of this model make it an attractive analogue of the human disease. however, generalized immu nodeficiency is not a feature in the vast major ity of patients with pm/dm. nevertheless, these observations emphasize the protean na ture of retroviral infection and indicate that this etiologic possibility should not be lightly discarded. in the past, retroviral infection has been invoked rather simplistically in the pathogenesis of inflammatory connective tis sue diseases, such as pm/dm. it is worth noting that harbers et al. [ ] microinjected cloned retroviral dna into mouse zygotes and found that virus-specific rna was subse quently expressed at different concentrations in the organs of infected mice. in one mouse, retrovirus specific information was expressed at far higher concentrations in muscle than in other tissues. while such viruses can be ac quired by horizontal infection, it is also possi ble that any involvement of retroviruses in the pathogenesis of pm might be more complicated. the pathogenesis of autoimmune diseases in general continues to be keenly debated and frequently reviewed [ ] . viruses are com monly invoked in most schemata albeit in concert with genetic and immunologic factors [ ] . it is instructive to apply these general hypotheses to pm/dm in order to see how viruses could invoke this autoimmune disease and the extent to which there is supporting data ( table . ). it has long been a popular notion that virus infection might induce autoantigens and thereby make infected cells targets for auto immune attack. there is in fact little evidence that viruses induce novel antigens or alter the structure or expression of normal antigens. nevertheless, there is good evidence that vi rus infection affects the recognition of surface antigens by the immune system, thereby in creasing the possibility that these will induce an immune response. classically, lindenmann and klein [ ] in found that influenza virus infection of target cells increased the immunogenicity of host cell antigens expressed by the infected cells. the mechanisms of this phenomenon have been extensively studied. for example, it has clearly been shown that vaccinia virus infection of tumor cells increases cytotoxic t-cell and antibody responses against the in fected cells [ ] . there are also several models of experimental viral infection that provoke autoimmune responses against the infected organs, with pathologic consequences. mice infected with reovirus type i develop both transient diabetes and a runting syndrome [ ] . the infected mice produce serum autoantibodies reactive with cytoplasmic an tigens in beta cells of the pancreatic islets of langerhans, the anterior pituitary gland, and gastric mucosa. additional autoantibodies are generated against both insulin and growth hormone, and serum concentrations of these hormones are abnormally low. another perti nent example is provided by experimental in fection with the jhm strain of murine coronaviruses, which is neurotropic and which induces either acute or subacute encephalomyelitis, depending on viral and host genetic factors [ ] . subacute encephalomyelitis is determined not only by the antiviral immune response but also by an autoimmune reaction to brain antigens, including myelin. an important issue in such models is the role of viral replication in the target cells. in the reovirus model of experimental autoim mune endocrine disease it seems likely that virus in the target organs is mandatory since reovirus type , which does not infect the anterior pituitary gland, does not induce autoantibodies to growth hormone. how ever, the period of virus infection may be transitory since the autoimmune process per sists long after local infection can readily be detected. moreover, an immunopathologic basis for the syndrome seems certain, since it can be prevented by immunosuppressive treatment with antilymphocyte serum [ ] , the extent to which persistent infection of brain cells by jhm virus is essential for the subacute demyelinating disease in susceptible hosts is still debatable. one possible mechanism by which tran sient virus infection of target cells may induce a persistent t-cell response is the inappropri ate expression of la (class , or dr) antigens by the host cell. the expression of such anti gens has mainly been attributed to -interferon released by infiltrating t lymphocytes. in par ticular, this mechanism has been invoked to explain autoimmune disorders of the thyroid gland [ ] . however, more recently in the jhm-induced model of demyelinating encephalomyelitis, it has been shown that viral particles directly induce la antigen expres sion by astrocytes [ ] . moreover, the extent of this interferon production is likely to be genetically controlled. since both viral and host cell antigens are likely to be presented to the immune system in association with la an tigens, it is also evident that genetic factors control the generation of cytotoxic t cells directed at both sets of antigenic determinants. there is so far little evidence that similar mechanisms operate in pm. in particular, there is no information about the steps that initiate the infiltration of muscles by lympho cytes. analysis of the infiltrating cells by immunocytochemical techniques has shown that the majority of the infiltrating cells are t lymphocytes bearing antigen determinants characteristic of activated lymphocytes [ ] . furthermore, these cells predominate in un treated acute rather than chronic disease [ ] . interferons have also been detected in muscle biopsies [ ] . however, the antigens against which these cells react have not been charac-viral etiology of polymyositis terized. it is possible that after isolation and expansion by cloning techniques the infiltrat ing t cells may prove specifically reactive to muscle cells, using the kind of assays that cambridge and stern [ ] have shown are true of circulating blood lymphocytes in pm. cellular infiltrates resembling those en countered in pm/dm are also provoked by exercise, emphasizing that these do not pro vide specific clues to the nature of the local initiating event, infectious or otherwise [ a] . antimyosin antibodies have been detected by radioimmunoassay in % of sera from pa tients with pm [ ] , but the nature of the stimuli provoking these autoantibodies is un known. there have been claims that the stain ing pattern of autoantibodies in patients with acute myocarditis can distinguish between patients with preceding coxsackie b infection and those with other etiologies, but these an tibodies were present in low titer, and their reactivity showed considerable overlap with antigens in other tissues [ ] . it is purely conjectural that transient infec tion of muscle cells initiates the immunopathologic events of pm/dm. however, many vi ruses are able to replicate in vascular endothelial cells [ ] . these cells could serve as sites for the initial growth of viruses implicated in the pathogenesis of pm/dm. indeed, it has been shown that -interferon induces la ex pression on cultured human vascular endothelial cells and dermal fibroblasts [ ] , fur thermore, ashida, johnson, and lipsky [ ] have shown that these cells function as antigen-presenting cells in model systems. it is feasible, therefore, that muscle fibers become secondarily inflamed as the result of changes in the microenvironment of local blood ves sels and that persistent local immune responses are thereby established. indeed, fully developed lymphoid follicles have been noted as a histologic feature of pm/dm [ ] , it is customary to invoke genetic factors to account for the rarity of such events. how ever, in contrast to the strong hla associa tions with most organ-specific autoimmune diseases, there is only weak evidence for such associations in pm/dm [ ] . claimed associ ations with hla-b and hla-dr in both adult and juvenile dermatomyositis are not compelling and have been observed in rela tively small numbers of patients. the specificities of the autoantibodies characteristic of pm/dm have been invoked as evidence for a possible viral etiology for this group of diseases. several authors, nota bly plotz [ ] and bernstein et al. [ ] , have stressed that the autoantibodies associated with inflammatory connective tissue diseases, including pm/dm, are directed at a relatively small number of cellular components. these autoantigens have been fairly well character ized as proteins, often present in complexes with additional proteins or nucleic acid mole cules. the antinucleoprotein autoantibody peculiar to many connective tissue diseases reacts with small ribonucleoproteins (rnps) in which the antigens are commonly the small nuclear rna species, ul, u , u , u , or u [ ] . in addition, the antibodies characteristic of pm/dm commonly react with cytoplasmic antigens, notably aminoacyl-trna synthetases. these antigens are associated with mechanisms of protein synthesis [ ] . in the first stage of protein synthesis, rnapolymerases catalyze the formation of messenger rna complementary to the dna sequence. this messenger specifies the sequence in which amino acids are assembled in the na scent polypeptide chain. the amino acids are initially attached to trna molecules before their polymerization into polypeptides. a specific set of enzymes termed aminoacyl-trna synthetases couple each amino acid to its matching trna. anti-la autoantibody reacts with precursor forms of ribosomal rna and trnas for five different amino acids [ ] . the transcription of these rna sequences is controlled by rna polymerase iii. similarly, anti-ro autoantibodies precipi tate rnps containing rnas whose transcrip tion is also dependent on polymerase iii [ ] . the jo- antigen is a protein-rna complex incorporating the synthetase needed to com plex histidine with the corresponding trna [ ] , moreover, anti-jo autoantibody blocks the incorporation of histidine into nascent proteins, a reaction that depends upon this enzyme. since these protein synthetic steps are also utilized in the synthesis and assembly of some virus particles, it is conceivable that these autoantibodies might represent an im mune response by the host aimed at disrupt ing viral synthesis. anti-la antibody precipi tates not only cellular rnps but also rnp complexes containing rnas involved in the synthesis of adenovirus and epstein-barr vi rus (ebv) [ ] . many plant viral rnas and a few animal viral rnas contain a trna-like structure at their free end which may utilize trna synthetases to regulate viral assembly [ ] , equally, these autoantibodies might be directed at enzymes and proteins involved in protein synthesis which have been rendered autoantigenic by the transcription of viral se quences [ ] . this mechanism is analogous to that likely to explain lupus-like syndromes provoked by drugs such as hydralazine. however, it is only an assumption that the antigen specificities of the autoantibodies hint at a viral etiology. these autoantibodies may simply reflect an autoimmune response to a spectrum of antigens common to many tissues regardless of the provoking cause. similarly, there is an obvious analogy with experimental reovirus infection in that the autoantibodies might determine patterns of tissue damage but be of little help in determin ing the nature of the initial insult. a similar dilemma arises from the convinc ing observations that the sera of patients with autoimmune connective tissue diseases con tain antibodies to antigens coded by retroviruses. rucheton et al. [ ] have shown that sera from patients with anti-rnp and anti-la autoantibodies react with several viral polypep tides coded by the viral gag gene. how ever, as the authors emphasize, these antibod ies could be of secondary importance, simply reflecting the nonspecific activation of endo genous viral antigens rather than a response to exogenous retroviral infection. the advent of monoclonal antibodies has provided a powerful means of testing the long-held belief that autoimmune diseases may arise from cross-reactions between mi croorganisms and tissue antigens. molecular mimicry of this kind can now be sought by direct experiment and by computer tech niques comparing known microbial and tis sue antigenic sequences. these techniques have been used to test the idea that molecular mimicry between viruses and muscle antigens might account for the continued immune re sponses characteristic of pm/dm. srinivasappa et al. [ ] screened monoclonal antibodies to different viruses for their reactivity with different organs from normal mice. they found that . % of the antiviral antibodies reacted with specific cells in these organs and that several of the antibodies reacted with antigens in more than one organ. however, no cross reactions with muscle-specific anti gens were uncovered in this survey or referred to in the accompanying review of similar searches. immunofluorescence studies have revealed a monoclonal antibody to coxsackievirus b that reacts exclusively with a bands in the myofibrils of myocardial muscle, al though only one of antibodies tested showed this pattern of reactivity [ ] . walker and jeffrey [ ] adopted a different strategy by using a computer alignment procedure to seek microbial sequences comparable with amino acid sequences of histidyl-trna synthetase and alanyl-trna synthetase since these are known to be the target molecules for autoantibody responses in pm/dm. close matches were discovered between histidyl-trna synthetase and protein sequences of ebv. similarly, of the closest matches with alanyl-trna synthetase, more than half were with viral proteins, including those coded by ebv, some influenza virus hemagagglutinins, and a protein coded by adenovirus . these studies also uncovered sequence similarities with the myosin light chain tropomyosin and the skin component keratin. however, as em phasized by the authors, the trna synthetases are universally distributed, and it is diffi cult therefore to understand why skeletal muscle fibers should be a specific target organ in this disease. furthermore, sequence homologies may be entirely fortuitous or result when part of a cellular protein is incorporated into viral protein during the course of viral replication. furthermore, similarities in se quence do not take into account conformational differences which may in reality deter mine the ability of an antibody to fit a given epitope [ ] . moreover, regions of high mo bility in a protein also determine antibody binding so that comparison of sequences can never replace the need for experimentally studying the ability of an antibody which reacts with one structure to react with an other that appears to mimic it [ ] . it is also important to test experimentally the idea that any observed cross-reactivity accounts for persistent autoimmune reactions. thus fujinami and oldstone [ ] in reported sequence homology between amino acid se quences in myelin basic protein and hepatitis b virus polymerase; lymphocytes from rab bits immunized with the viral sequence pro duced a proliferative response in vitro to chal lenge with basic myelin protein and also developed histologic changes reminiscent of experimental allergic encephalomyelitis. it is now accepted that antibodies to novel sequences in the variable portion of the anti body molecule (idiotypes) contribute to the regulation of antibody production. further more, since there are sequence similarities be tween the provoking antigen, receptors for those antigens on cell surfaces, and the idiotype, anti-idiotype antibodies might be expected to recognize antigen receptors. this pathway for inducing autoantibodies reactive with cell membrane antigens has been recog nized as a possible source for autoimmune diseases and, indeed, anti-idiotype autoanti bodies have been shown to have immunopathologic consequences in some experimen tal models [ , ] . the complexities of possible derange ments of the idiotype network have been in creased by the recent demonstration that a human monoclonal antibody reactive with a membrane protein of multiple organs con tains an idiotype which in turn stimulates an anti-idiotype antibody, and that an antibody raised against this anti-idiotypic antibody it self recognizes the protein determinant that initiated the whole chain of events [ ] . ago nized readers are referred to ancient chain songs dealing with kids brought to market. hybridoma techniques have revealed monoclonal antibodies that react with epitopes shared by multiple organs and tis sues. one such epitope in sle is the phosphodiester bond [ ] , while monoclonal auto antibodies reactive with pituitary, thyroid, pancreas, and stomach have also been de scribed [ ] . antibodies to these idiotypes have been produced experimentally and used to screen the sera of patients with autoim mune diseases for antibodies bearing these idiotypes. if autoimmune diseases are pro voked by a persistent autoantibody response to idiotypes, one might predict that the sera of patients with autoimmune diseases would contain a higher concentration of antibodies with this idiotype than do sera of normal individuals. however, essani et al. [ ] found that idiotypic markers thought to be related to autoimmune diseases are expressed in normal individuals and that antibodies bearing these markers do not necessarily bind to autoantigens. similarly, the number of circulating b lymphocytes reactive with anti-idiotype antibody was identical in patients with autoimmune diseases and in controls. similar observations showing the public nature of autoantibody idiotypes have been made by other authors [ ] . thus far theories about the pathogenesis of autoim mune diseases based on generation of antiidiotype antibodies have not provided any etiologic clues in autoimmune diseases in gen eral. with respect to pm/dm, those autoreactive antibodies whose idiotypes have been analyzed do not react with muscle. however, the idiotypes of muscle-reactive antibodies in pm/dm have not been examined. there are also caveats concerning the idea that analyz ing autoantibody idiotypes might further im plicate viruses that are putatively involved in the etiology of pm. mcclintock et al. [ ] in reported that anti-idiotypic antibodies raised to mouse monoclonal antibodies reac tive with independent epitopes on coxsackievirus b did not block the binding of this virus to cells bearing appropriate receptors. the original studies of mice with an autoim mune polyendocrine disorder provoked by infection with reovirus type revealed that spleen cells fused with a myeloma cell line produced monoclonal antibodies reactive with cells in the anterior pituitary gland, pan creas, small intestine, and stomach [ ] . ad sorption studies on antigen affinity columns confirmed that the antibody was indeed re acting with antigens common to the different target organs. it subsequently became evident that cells producing monoclonal antibodies with the same repertoire of multiple organ reactivity can be isolated from the spleens of normal mice [ ] . similarly, dighiero et al. [ ] have established hybridomas with spleen cells of normal mice synthesizing monoclonal autoantibody reactive with myosin, doublestranded dna, actin, tubulin, and spectrin. in clinical studies, ebv transformed blood lymphocytes from both normal individuals and patients with autoimmune diseases pro duced monoclonal autoantibodies reactive with antigens in multiple organs [ ] . the target antigens were detected in thyroid, pan creas, stomach, stratified squamous epithe lium, and nerve axons. the reactions could only be detected by the highly sensitive avidin-biotin-immunoperoxidase assay. fur thermore, this group [ ] used the same tech nique of screening ebv-transformed b cells to reveal that b cells from patients with autoim mune diseases produce monoclonal antibod ies reactive with both pancreas and thyroid. evidently b cells in patients with autoimmune disease synthesizing serologically recogniz able autoantibodies may also synthesize autoantibodies reactive with different organs which are undetectable by standard tech niques. thus the association of pm/dm with other autoimmune diseases could result from the simultaneous production of muscle-re active autoantibodies that have not yet been characterized. it also follows that pm/dm, like other autoimmune diseases, could result from the inappropriate activation of b cells already programmed to react with muscle an tigens. the multiple ways in which this acti vation could be achieved include the bypass ing of suppressor t-cell mechanisms and the inappropriate presentation of muscle-specific antigens to b cells by t cells reacting with surface antigens expressed by muscle fibers [ ] . viral infections could provide the ini tiating signal; experimentally bromberg et al. [ ] have shown that herpes simplex, new castle disease, and vaccinia viruses can induce immune responses against membrane anti gens that are normally weakly immunogenic. there is only meager evidence to support the alternative possibility that viruses putatively inducing pm/dm latently infect suppressor cells, thereby leading to their functional abla tion. hollingworth et al. [ ] showed that herpes simplex virus failed to replicate in pha-stimulated blood lymphocytes from pa tients with pm/dm, suggesting that some cells might harbor virus particles that inter fered with the growth of the challenge virus. since viral infection of a minority of circulat ing lymphocytes has marked immunodepressive effects [ ] , this possibility can not be lightly discarded. it has recently been shown that measles virus has a strong tropism for lymphocytes and that continued infection of these cells may contribute to the pathogenesis of the persistent viral disease subacute sclerosing panencephalitis [ ] . pm/dm commonly occurs in association with other inflammatory connective tissue diseases in which autoimmune reactions are promi nent. thus, theories attributing autoimmune diseases to the uncontrolled proliferation of autoreactive lymphocyte clones are also ger mane to the pathogenesis of pm/dm. classic ally, the emergence of such clones has been considered to result from the failure of regula tory mechanisms that normally lead to their elimination. alternatively, mutations in the progenitor cells for autoimmune reactive lymphocytes might lead to their autonomous proliferation in a manner independent of con ventional antigenic stimulation. indeed, the proliferation of autoreactive cells has often been considered intermediate between a con-ventional immune response and overt malig nant proliferation. theoretically it is now possible to test the strength of such hypothe ses by comparing the characteristics of autoantibody secreting cells with frankly malig nant lymphoma cells (table . ). there is so far no evidence that autoreactive cells origi nate from a single progenitor cell in a manner analogous with the b cells of patients with chronic myeloid leukemia; the latter point has been clearly established in black female patients with the disease who are heterozy gous for the enzyme glucose- -phosphate hydrogenase [ ] . nor have gene rearrange ments of the kind observed in neoplastic lymphoid cells [ ] been detected in the genes coding for the antigen receptors of autoreac tive t cells or the immunoglobulin coding genes of autoreactive b cells. gene probes for t-cell receptors have been used to document the maturation of mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase (hprt) activity [ ] . furthermore, assays for rearrangements of genes coding for t-cell antigen receptors have provided insight into a clinically benign but biologically lymphoproliferative disorder, lymphomatoid papulosis [ ] . immunoglobulin synthesis involves a se ries of differentiation steps in which the prod- these mutations usually involve a sin gle base substitution, but it is not known whether these are entirely spontaneous or de pendent on antigenic stimulation. similarly, there is a high rate of mutation in the genes coding for the variable portions of the beta chain of the antigen receptor of t cell al though at a lower rate than that occurring in genes coding for the variable portion of the immunoglobulin molecule. thus, classic the ories postulating that autoimmune diseases arise from mutation in antibody synthesizing clones must be reconciled with observations showing that mutation is a physiologic step in the generation of antibody diversity. further more, such theories must explain the circum stances that confer continuous and appar ently autonomous proliferation on such clones. certainly a single somatic mutation can result in the production of an antibody with autoantibody specificity [ ] , but anal ogous mutations in human autoantibodyproducing cells have not yet been described. in malignant lymphomas, substantial evi dence has been adduced implicating the inap- propriate activation, mutation, or translocation of oncogenes in abnormal b-cell proliferation; moreover, many of these mod els postulate that these transforming events are initiated or enhanced by exogenous viral infection. there is as yet no evidence that similar events govern the proliferation of autoreactive b cells in connective tissue dis eases. the activation of the c-myc gene in sle, for example, seems simply to reflect the extent of b-cell proliferation rather than an unusual event initiating this proliferation [ ] . nevertheless, the wealth of interaction observed between exogenous infectious agents and the deregulation of myc-genes in b cell tumours [ ] means that such ideas should not be lightly discarded in other lymphoproliferative disorders. b-cell differentiation and maturation involves multiple steps, each of which is governed by interactions with ex ternal ligands, such as b cells and their prod ucts, activated components of the comple ment sequence, and other as yet unknown factors. it is noteworthy that the b-cell hyperplasia characteristic of mouse strains prone to spontaneous autoimmune disease is deregu- there is so far little evidence that pm/dm is caused directly or indirectly by viral infec tions. conventional methods have failed to isolate viruses in the vast majority of cases and serologic surveys have proved uncon vincing. the case for continuing to seek a viral etiology rests mainly on animal models. myocarditis and pm induced by coxsackievirus infection is mediated by immunopathologic mechanisms that may continue to dam age muscle fibers long after infectious virus and even viral antigens have been eliminated. clearly, if analogous mechanisms operate in human disease, it will prove extremely diffi cult to identify the provoking agent by the time the disease is clinically apparent. fur thermore, the combination of viral and host genetic factors necessary to induce muscle in flammation may be extremely rare and im-lated at one or more points in their matura tion [ ] . there is little information about the detailed regulation of human b cells syn thesizing conventional or autoantibodies. however, the introduction of improved tech niques for propagating human b cells in cul ture should remedy this situation. there is evidence, for example, that lupus b cells spon taneously synthesize b-cell growth factors and are refractory to exogenous growth fac tors [ ] . the failure of normal repair processes may increase the possibility for recombination events between immunoglobulin-coding genes and genes related to controlled proliferation. this mechanism has also been invoked as a factor in mouse strains susceptible to plasmacytoma induction [ ] , and it is note worthy that lymphocytes from patients with pm/dm are abnormally susceptible in vitro to irradiation, ultraviolet irradiation, and the mutagen methylnitrosourea [ ] . in general, the relevance of such concepts to the pathogenesis of pm/dm has not been experimen tally tested. possible to define by orthodox biologic and immunologic techniques. indeed, the succes sive steps leading to autoimmune inflamma tion of muscle in pm/dm or inflammation of organs in other autoimmune diseases are tortuous to unravel even when the disease is deliberately incited. the hope of progress lies in improved techniques for isolating, expand ing, and characterizing the effector cells from the blood and local lesions of patients with this group of disorders. the association of pm/dm with other auto immune diseases encourages the belief that genetically determined mutations in t or b lymphocytes may be responsible for the dis order. if pm/dm results from the quasi-malig nant proliferation of autoreactive lymphocyte clones, such hypotheses can only be tested by detailed analysis of the multiple steps leading to the emergence of such clones. such analo gies imply that viruses might not act by simple cause and effect. instead, it is possible that they might act at several stages in the emer gence of such clones. viruses could promote recombinational events between transform ing genes and genes coding for immunoglobu-lin or t-cell receptor sequences. alternative ly, viruses could act by overcoming restric tions to 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cells lymphocytes recognize human vascular endothelial and dermal fibroblast la anti gens induced by recombinant immune in terferon hu man endothelial cell-lymphocyte interac tion polymyositis with infiltration by lymphoid follicles autoimmune diseases of muscle hypothesis: viruses and autoantibodies. autoantibodies and anti-idiotype antibodies to anti-viral antibodies cellular protein and rna antigens in auto immune disease precursor molecules of both human s ribosomal rna and trans fer rnas bound by a cellular protein reac tive with anti-la lupus antibodies basic genetic mechanism genes for two small cytoplasmic ro rnas are adjacent and ap pear to be single-copy in the human ge nome myositis autoantibody inhibits histidyl-trna synthetase: a model for autoimmunity possible functional role of viral trna-like struc tures presence of circulating antibodies against gag-gene mulv proteins in patients with autoim mune connective tissue disorders molecular mimicry: frequency of reac tivity of monoclonal antiviral antibodies with normal tissues monoclonal antibody to coxsackievirus b reacts with myocardium polymyositis and molecular mimicry: a mechanism of auto immunity on the use of se quence homologies to predict protein structure: identical pentapeptides can have completely different conformations which structural features determine protein antigenicity? amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity idiotypes and autoimmunity multiple organ-reactive igg antibody induced by an antiidiotypic antibody to a human mono clonal igm autoantibody polyspecificity of monoclonal lupus auto antibodies produced by human-human hybridomas human multiple-organ reactive mono clonal autoantibody recognizes growth hormone and a , molecular weight protein anti-idiotypic antibodies against a human multiple organreactive autoantibody: detection of idiotopes in normal individuals and patients with autoimmune diseases anti-idiotypic antibodies to mono clonal antibodies that neutralize coxsackievirus b, do not recognize viral receptors multiple organ-reactive monoclonal autoantibodies lymphocytes capable of mak ing monoclonal autoantibodies that react with multiple organs are a common feature of the normal b cell repertoire murine hybridomas secreting natural monoclonal antibodies reacting with selfantigens epstein-barr virus-transformed lympho cytes produce monoclonal autoantibodies that react with antigens in multiple organs hu man monoclonal autoantibodies that react with both pancreatic islets and thyroid mecha nisms of autoimmunity: a role for crossreactive idiotypes viral antigen act as helper determinants for anti body responses to cell surface antigens effects of immunosuppression on herpes simplex virus growth in lymphocytes of patients with connective tissue diseases interactions of viruses with the immune system detection of measles virus rna in lym phocytes from peripheral-blood and brain perivascular infiltrates of patients with subacute sclerosing panencephalitis chronic myelocytic leuke mia origin of some lymphocytes from leukemic stem cells rearrangement of genes for the antigen receptor on t cells as mark ers of lineage and clonality in human lymphoid neoplasms use of t-cell receptor gene probes to quantify the in vivo hprt mutations of human t-lymphocytes clonal t-cell populations in lymphomatoid papulosis: evidence of a lympho-proliferative origin for a clinically benign disease somatic muta tion of the t- heavy chain gives rise to an antibody with autoantibody specificity induction of c-myc expression early in the course of b-cell activation: studies in normal humans and patients with systemic lupus erythematosus mechanisms in b-cell neoplasia spontaneous production of b cell growth factors by sle lymphocytes defective repair of o -methylguanine in autoimmune diseases key: cord- -xhzvp g authors: berencsi, györgy; szomor, katalin n. title: fetal and neonatal illnesses caused or influenced by maternal transplacental igg and/or therapeutic antibodies applied during pregnancy date: - - journal: maternal fetal transmission of human viruses and their influence on tumorigenesis doi: . / - - - - _ sha: doc_id: cord_uid: xhzvp g the human fetus is protected by the mother’s antibodies. at the end of the pregnancy, the concentration of maternal antibodies is higher in the cord blood, than in the maternal circulation. simultaneously, the immune system of the fetus begins to work and from the second trimester, fetal igm is produced by the fetal immune system specific to microorganisms and antigens passing the maternal-fetal barrier. the same time the fetal immune system has to cope and develop tolerance and t(reg) cells to the maternal microchimeric cells, latent virus-carrier maternal cells and microorganisms transported through the maternal-fetal barrier. the maternal phenotypic inheritance may hide risks for the newborn, too. antibody mediated enhancement results in dengue shock syndrome in the first month of age of the baby. a series of pathologic maternal antibodies may elicit neonatal illnesses upon birth usually recovering during the first months of the life of the offspring. certain antibodies, however, may impair the fetal or neonatal tissues or organs resulting prolonged recovery or initiating prolonged pathological processes of the children. the importance of maternal anti-idiotypic antibodies are believed to prime the fetal immune system with epitopes of etiologic agents infected the mother during her whole life before pregnancy and delivery. the chemotherapeutical and biological substances used for the therapy of the mother will be transcytosed into the fetal body during the last two trimesters of pregnancy. the long series of the therapeutic monoclonal antibodies and conjugates has not been tested systematically yet. the available data are summarised in this chapter. the innate immunity plays an important role in fetal defence. the concentration of interferon is relative high in the placenta. this is probably one reason, why the therapeutic interferon treatment of the mother does not impair the fetal development. at term, the amount of maternal igg antibody is higher in the neonate than in the mother. this pattern holds when the igg antibody is an anti-tnfa medication. a specific fc receptor neonate (fcrn) facilitates transfer of the igg antibodies across the syncytiotrophoblast into the fetal circulation (kane and acquah ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to the mother. by discontinuing this drug - weeks prior to delivery, the baby will likely be born with no or minimal serum levels, thus avoiding immunosuppression in a young infant. though there are some suggestive data that certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules (clowse ) . in early human placenta the exchange tissue area (villi) is formed around the entire surface of the conceptus. this placental shape is called diffuse placenta. by the third month of pregnancy, only the villi near the initial site of implantation have persisted, leading to the formation of the disc-shaped placenta. although chorioallantoic placenta in humans begins functioning already by the end of the fourth week of pregnancy, this process is completed with the formation of diskshaped placenta (sadler ) . during the first trimester, the human fetus is surrounded by two fluid cavities, i.e., the inner amniotic cavity and the outer extra-embryonic coelomic cavity. the chorioallantoic placenta is only formed of fetal vessels' endothelium and trophoblastic layer, bathed directly in the maternal blood (van der aa et al. ). for out of monoclonal therapeutic compounds, for which toxicity studies (in a broad sense) were performed, no significant maternal, fetal, or neonatal toxicity was observed. for the remaining seven products, the most common adverse effects on reproduction and development were reduced fetal weight, increased abortion rates, and reduction in fertility, indicating the general toxicity of these compounds (pentsuk and van der laan ). twenty-four ( %) of the children had one or more congenital anomalies that are part of vacterl association, but only one child (with maternal etanercept administration) was diagnosed with vacterl association i.e. v: vertebral defects, a: anal atresia or imperforate anus, c: cardiac abnormalities [atrial septal defect, ventricular septal defect, and tetralogy of fallot], t: tracheoesophageal fistula or tracheal atresia/stenosis, e: esophageal atresia, r: radial and or renal abnormalities, and pre-axial l: limb abnormalities (carter et al. (carter et al. , ). fc-fcgr interactions play a critical role in the biological function of antibody and are likely to be instrumental in preventing or modulating lentiviral infections. antibody responses that depend on fc-fcgr interactions may help widen the spectrum and increase the potency of vaccine-induced antibodies (forthal and moog ) . maternofetal transmission of non-neutralising dengue virus specific antibodies result in the development of hemorrhagic fever of the newborns even in the case of the first infection with a heterotypic virus. the mechanism is the formation of non-neutralised neonatal virus and heterotypic maternal igg complexes, which will be taken up by endothelial and hematopoetic cells by (virus-abfc)-fcgr pinocytosis. the risk disappears after the th month of age of the children, when the maternal igg disappeared from the circulation of the children (libraty et al. ). this phenomenon has been observed in the case of other flaviviruses and enteroviruses, too (ferenczi et al. ; wang et al. b ). dengue serotype cross-reactive cytotoxic lymphocytes (ctl) clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. high avidity cross-reactive memory ctl may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. these cells appear to be subsequently deleted leaving a more serotype-specific memory ctl pool. antibody-dependent enhancement ade is neither a sufficient nor an absolutely necessary precondition for the development of severe shock syndrome or hemorrhagic disease. ctl response to a viral infection can be modulated by the infection history of an individual in a manner likely to contribute to disease severity l€ uhn et al. ). respiratory syncytial virus (rsv) enhancer activity of transplacental maternal antibodies have been observed first by osiowy et al. ( ) . rsv infection is also enhanced by non-neutralising antibodies (johnson and graham ) . vaccination using inactivated virus particles were caused immune enhancement in the case of rsv in contrast to other, for example influenza viruses (openshaw et al. ; ye et al. ) . cytotoxic t-cells, cytokines and interferon induction by toll-like receptors may potentiate this enhancement (mobbs et al. ; boukhvalova et al. ; tregoning et al. ; lee et al. a, b; nguyen et al. ; ubol and halstead ) . tumour necrosis factor b block of cell-replication enhances rsv-replication (gibbs et al. ). the evidence of the enhancer effect of non-neutralizing antibodies to rsv can be prevented by the treatment of children at increased risk with neutralizing monoclonal antibodies directed against the fusion (f) protein of rsv (motavizumab and/or palivizumab). this monoclonal antibodies were not found to cause harmful effects in connection with the preventive treatment of neonates and infants at risk (martin-mateos ; nieri et al. ; weisman ; groothuis et al. ). anti-rsv-f protein specific antibodies can prevent also the rsv-s. pneumoniae enhancement of respiratory infections (hament et al. ) . the blocking of influenza specific anti-neuraminidase antibodies using antiidiotypes indirectly enhanced the hemagglutination-inhibition titers of antisera (dowdle et al. ) . influenza h specific antisera were found to enhance virus replication in a macrophage-like cell line p d , when p d cells, previously had been treated with neuraminidase to remove the viral receptors (ochiai et al. ). maternal antibodies systemic lupus erythemadosus (sle), is characterised by antibodies towards dsdna and ro (e ligase regulating tlr signalling) which, are present several years before the onset of disease and the neonatal disease is caused by some of these transplacental antibodies (watson et al. ) . systemic lupus erythematosus (sle) is the most common autoimmune disease affecting women of reproductive age and is associated with poor maternal and fetal outcomes. cd (+)cd (+) t reg cells are a subset of t lymphocytes with potent immunosuppressive activity that play crucial roles in controlling immunological self tolerance. evidence suggests that they are augmented in pregnancy, especially in the first trimester, suggesting an important role in early placental development. the literature describing t reg cells in sle is conflicting, but sle is associated with reduced numbers and functionally defective t reg cells, which may predispose pregnant women with the disease to pregnancy complications. this article discusses the role of t reg cells in sle and pregnancy, and how these cells may contribute to poor pregnancy outcome in sleaffected women (blois et al. ; clark et al. ) . sle was induced by interferon a administration, and by a specific stimuli i.e. sunshine exposure and smoking. the hla-dr haplotype was also found to be a risk factor (klareskog et al. ) . alcohol consumption was shown to be protective in these illnesses. vaccinations in adult age was found to be innocuous concerning the risk of ra according to the results of a case-control study when common vaccinations years before onset of ra had been followed up. the effects of environment, however, are unknown for the neonatal heart block in p and ro positive women, but the active immunisation does not increase the risk of it (bengtsson et al. a, b) . in sle and sj€ ogren's disease pregnants with high antibody titers against the p epitope of ro are those who almost exclusively carry the risk that their fetuses will develop neonatal heart block between gestational weeks - . monitoring during these period the anti-ro antibodies, steroid treatment or preventive in utero pacemaker treatment may reduce the risk (wahren-herlenius ). foetal genes, maternal age and infectious agents may contribute to the risk of congenital heart block. especially inherited high level interferon production was also shown to be a risk factor for sle (niewold et al. ) . heparin treatment and intravenous gamma globulin (ivig) treatment and specific anti-idiotypes reduce the risk of the disease (clark et al. ) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. the id:anti-id ratio was significantly higher in mothers whose offspring developed neonatal lupus compared to mothers who gave birth to a healthy child (p < . ). removal of anti-id antibodies substantially increased the reactivity against la( - ) in sera from five of seven mothers tested. ivig from batches administered to mothers who gave birth to a healthy child had an id:anti-id activity ratio of < , in contrast to that given to mothers who gave birth to a child with neonatal lupus (brucato et al. (brucato et al. , routsias et al. ) . the main pathomechanism of the development of neonatal lupus erythematosus is the transcytosis of maternal antibodies, and probably microchimeric cells. the pathogenesis of the maternal disease is extremely complex as summarised recently by perl ( ) and perl et al. ( ) . mitochondrial hyperpolarization underlies mitochondrial dysfunction, depletion of atp, oxidative stress, abnormal activation, and death signal processing in lupus t cells. nitric oxide production, expression of endogenous retroviral and repetitive elements such as hres- , (the long interspersed nuclear elements ), trex , interferon alpha (ifn-alpha), toll-like receptors and (tlr- / ), high-mobility group b protein, extracellular signal-regulated kinase, dna methyl transferase , histone deacetylase, spleen tyrosine kinase, proteasome function, lysosome function, endosome recycling, actin cytoskeleton formation, the nuclear factor kappa b pathway, and activation of cytotoxic t cells were shown to be components of the pathogenesis (varghese et al. ) . the hres- human endogenous retrovirus (erv) encodes a k nuclear autoantigen and a -kd small gtpase, termed hres- /rab . hres- /p is a target of cross-reactive antiviral antibodies, whereas hres- /rab regulates the surface expression of cd via endosome recycling. hres- /rab is overexpressed in lupus t cells where it correlates with increased recycling of cd and cd and contributes to downregulation of cd /tcrs via lysosomal degradation. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulates interferon and anti-dna antibody production. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulate interferon and anti-dna antibody production in sle (perl ; perl et al. ) . these complex of pathogenetic factors many of them influenced by pregnancy results the clinical disease of the newborns (ruiz-irastorza and khamashta ). neonatal lupus erythematosus (nle) is an inflammatory disorder of neonates characterized by transient cutaneous lesions and/or congenital heart block. the cutaneous lesions usually heal with minimal scarring within - months, but may be delayed for many months in occasional cases. the maternal antibodies disappear from the circulation of the newborns within - months, the long-lasting clinical symptoms indicate, that irreversible events, or impairment of fetal cells occur during the fetal life. photosensitivity is recognized as a component of this syndrome. u ribonucleoprotein (u -rnp) specific antibodies can be detected in the circulation of the newborns. skin and cardiac manifestations coexist in only % of patients. hepatic, hematological and, less commonly, pulmonary, neurological and gastrointestinal abnormalities may also be present (watson et al. ; perez et al. ) . neonatal lupus is a model of passively acquired autoimmunity in which a mother-, who may have systemic lupus erythematosus (sle) or sj€ ogren's syndrome (ss) or may be entirely asymptomatic-synthesizes antibodies to ssa/ro and/or ssb/ la ribonucleoproteins that enter the fetal circulation via trophoblast fcrn receptors and presumably cause tissue injury (lee ) as mentioned above. congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with ro/ssa autoantibodies. during pregnancy, the antibodies are transported across the placenta and affect the fetus. it has been previously demonstrated that antibodies directed to the - amino acid (aa) stretch of the ro component of the ro/ssa antigen correlate with the development of congenital heart block. the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the - aa stretch (ottosson et al. ). smith (sm) antigen, which is highly specific for sle is composed of at least nine different polypeptides with molecular weights ranging from to . kda, b (b , kda), b (b , kda), n (b , . kda), d ( kda), d ( . kda), d ( kda), e ( kda), f ( kda), and g ( kda). sle patients develop antibodies against the sm complexes of the small nuclear rnas u to u in the mother and recently these were also detected in neonatal le patient (ortiz-santamaria et al. ) . in neonatal lupus, mothers with high anti-idiotypic antibody activity against anti-la autoantibodies are at lower risk of giving birth to an unhealthy child, as compared with mothers without anti-idiotypic antibodies. usually anti-idiotypic antibodies may confer protection from the harmful effect of autoantibodies in certain autoimmune diseases (tzioufas and routsias ) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. a high id:anti-id ratio in both the ivig preparation and the maternal serum may explain the absence of an effect of ivig in preventing recurrent neonatal lupus in some cases (routsias et al. ) . the children of the mothers suffering from sle and sj€ ogren's syndromes are at risk also for other rheumatic/autoimmune diseases without carrying antibodies reactive with ssa/ro or ssb/la antigens. among the siblings without neonatal lupus developed later juvenile rheumatoid arthritis, hashimoto thyroiditis, psoriasis and iritis, diabetes mellitus, congenital hypothyroidism and nephrotic syndrome (martin et al. ; winter and schatz ) . experimental systemic lupus erythemetosus could be induced in mice using immunisation with anti-idiotype antibodies (ab ) specific to the anti-dna-specific monoclonal antibodies (mendlovic et al. ) . identical twins of mothers suffering from sle had also transient neonatal bullous skin disease (nakajima et al. ). autoimmunity has been defined as a normal physiological state with control mechanisms that prevent autoimmunity from progressing to overt pathology. the onset of a subset of rheumatoid arthritis (ra) begins with appearance of citrullinated protein antigen (cpa) positivity when tolerance to certain citrullinated proteins/peptides is broken supported by certain hla-dr haplotypes (hla-dr and hla-drb in smokers). local expression of peptidylarginine deiminases and occasional elevation of inflammatory cytokines can be measured (klareskog et al. ) . methotrexate was significantly more effective than placebo in preventing progression to this disease state in anti-cpa-positive patients with undifferentiated arthritis (ua) whereas no difference between methotrexate and placebo was seen in the acpa-negative group of patients with undifferentiated arthritis (ua; arthritis without arthralgia). the risk of ra was lower in this group without treatment (ehrenstein et al. ; van dongen et al. ). the treatments using therapiesinfliximab (a tumour necrosis factor (tnf-a) blocker) and abatacept (t cell costimulation inhibitor; anti-cd and anti-cd ) did not result in significant improvement of the acpa patients with ua (saleem et al. ; emery et al. ) . the analysis of myeloid-related proteins in serum is an excellent tool for the diagnosis of systemic onset of juvenile idiopathic arthritis (jia), allowing early differentiation between patients with systemic-onset of jia and those with other inflammatory diseases. mrp- /mrp- and il- b represent a novel positive feedback mechanism activating phagocytes via two major signaling pathways of innate immunity (tlr ) during the pathogenesis of systemic-onset of jia (frosch et al. ). the rheumatoid arthritis was found to be improved during pregnancy probably due to galactosylation of igg molecules (f€ orger and Østensen ). women with ra can acquire the susceptibility allele through microchimeric cells. very high amounts ( . % of total pbmcs) of drb * microchimerism in patients with ra were observed. similarly, drb * microchimeric dna was observed in significantly higher quantities in women with ra compared with healthy control subjects. in contrast, there was no difference between women with ra and control subjects when microchimerism for non-ra-associated alleles (hla-dqb * and drb * / ) was analyzed (rak et al. ). by analogy to graft-versus-host disease (gvhd), pioneer studies on microchimerism in women with scleroderma proposed direct and indirect recognition mechanisms as underlying reactions of microchimeric cells (nelson ) . in gvhd, donor cells invade the recipient, which is not what is observed with microchimerism in patients with ra. however, the presence of drb * microchimerism ( . %) and drb * microchimerism ( . %) is not negligible, with frequencies among total host pbmcs similar to the frequencies of antigen-specific cd + t cells, which thus are sufficient to impact the host immune reactions. the haplotypes of the patients influence the progression of the rheumatoid arthritis, too (liu et al. ). autoimmune neonatal bullous skin disease caused by placental transfer of maternal igg autoantibodies is rare. it has been reported in neonates born to mothers with pemphigus vulgaris, pemphigus foliaceus, and gestational pemphigoid. vertically acquired congenital autoimmune blistering disorders appear to be self-limited and resolve with supportive therapy, concomitant with the presumed clearance of maternal autoantibodies from the neonate's circulation (abrams et al. ). antiphospholipid antibody syndrome (apas) is regarded as the most frequently acquired risk factor for thrombophilia. thrombophilia is the tendency to thrombosis. the antiphospholipid antibody syndrome (apas) is a disorder of recurrent vascular thrombosis, pregnancy loss and thrombocytopenia, associated with persistently raised levels of anti-phospholipid antibodies (apa). the apa are phospholipids (part of a cell's membrane) recognized by the body as foreign and antibodies are produced against them. maternal autoimmune diseases significantly reduce the pregnancy outcome of the women. the most frequent illnesses were antiphospholipid syndrome, antiphospholipid syndrome associated with a rheumatic disease (aps/rd), other rd patients, isolated autoantibodies (autoabs) in the absence of a definite autoimmune disease (aabs) and reactive arthritis or spondyloarthropathies. of these patients, . % had previous pregnancy complications with an anamnestic livebirth rate of . %. in these patients, . % of pregnancies resulted in preterm delivery and . % newborns had low weight at delivery. aps/rd patients had the worse outcome: . % resulted in miscarriage, . % resulted in growth restriction and % resulted in preterm delivery. this result was mainly due to patients with aps/systemic lupus erythematosus (sle) that had the lowest gestational age at delivery ( . ae . weeks) and the lowest newborn weight (canti et al. ) . antiphospholipid syndrome was suggested to be the result of antibodies, directed against cardiolipin as a result of antigenic mimicry. the suspected antigen is beta- -glycoprotein-i (b gpi) (sherer et al. ) . clinically significant are lupus anticoagulant, anticardiolipin antibodies and anti-b glycoprotein-i (anti-b gp-i) antibody. neonates born to mothers with primary aps are at risk of prematurity, being small for gestational age, and having thrombocytopenia (chou et al. ). antiphospholipid antibodies (apl) can impair the physiologic development of a fetus during pregnancy not only by causing thrombosis of the placental vessels, but also by directly binding throphoblast cells and modifying their functions (tincani et al. ). transplacentally transferred antiphospholipid antibodies act as a risk factor, but are not usually a sufficient condition for thrombosis and other thrombophilic risk factors should be systematically evaluated. long-term studies of children born to antiphospholipid-antibody-positive mothers provided the evidence of possible neurodevelopmental changes in these children and regular neuropsychological assessments are recommended. antiphospholipid-antibody-related thromboses in children are frequently associated with multiple antiphospholipid antibody positivity and concomitant presence of inherited prothrombotic disorders can be also detected in addition to nonthrombotic manifestations, particularly hematological, skin and neurological manifestations (avcin ) . treatment of pregnant women with apas results in marked improvement in the live birth rate ( . - . %). however, complications like preeclampsia and intrauterine growth restriction (iugr) occur even after treatment, requiring strict monitoring and timely delivery. aberrant concentrations of fetuin a and heat shock protein might have also role in the preeclamptic inflammation (molvarec et al. a, b; dadhwal et al. ). foetal/neonatal disease is due to transplacental thyrotrophin receptor stimulating antibodies (trab). it's extremely important recognising and treating graves' disease in mothers as soon as possible, because a thyrotoxic state may have adverse effects on the outcome of pregnancy and both on the foetus and newborn. neonatal grave's disease tends to resolve spontaneously within - weeks as maternal thyroid stimulating immunoglobulins are cleared from the circulation but subsequent development may be impaired by perceptual motor difficulties. hashimoto's thyroiditis is a very common autoimmune thyroid disease. in presence of maternal hashimoto's thyroiditis, there are usually no consequences on foetal thyroid, even if antitpo and antitg antibodies can be found in the newborn due to transplacental passage. however there are some reports describing foetal and neonatal hyperthyroidism in the affected mothers' offspring (radetti et al. ; hemminki et al. ) . a number of autoimmune diseases; especially autoimmune thyroid diseases, erythema nodosum and sarcoidosis parity might somehow be involved in maternal disease development (jørgensen et al. ) . maternal thyroid status assessment and treatment improves fetal outcomes and neuropsychological developmental of the newborn (staii et al. ). juvenile myasthenia gravis is associated with antibodies to the acetylcholine receptor (achr) in most patients. thymoma is rare, but often malignant in children. the frequency of juvenile myasthenia gravis with antibodies to the musclespecific kinase (musk) varies markedly in different countries. neonatal myasthenia gravis associated with musk antibodies is often a severe and protracted albeit transient disease (béhin et al. ; evoli ) . transient neonatal myasthenia gravis (mg) is a human model of passively transferring the disease. although all newborn babies of myasthenic mothers have anti-achr antibodies at birth (morel et al. ; tzartos et al. ) , only a small percentage of them ( - %) express the myasthenic syndrome (namba et al. ) . the myasthenic symptoms usually appear a few hours after birth and their average duration is about weeks. neonatal myasthenia gravis is transiently transferred from the mothers to the newborn. nicotinic acetylcholine receptor (achr) antibodies result in loss of achrs and also directly block the function of the remaining achr molecules, thereby causing a defect in neuromuscular transmission. the majority, though not all, of both myasthenic and non-myasthenic infants were found to have a repertoire of anti-achr specificities very similar to their mothers. no significant differences were observed between sera from the two groups of mothers. adequate treatment in mothers can reduce both frequency and severity of neonatal disease. neonatal disease will recover following ivig treatment (béhin et al. ; o'carroll et al. ). the absence of neonatal myasthenia gravis might be caused by the antigenic differences between the fetal and adult enzymes similar to those detected in rats (hall et al. ; hesselmans et al. ). the human fetal acetylcholine receptor (achr) is present until weeks gestation, when the fetal (g) subunit is replaced by the adult (e) subunit. the term "fetal acetylcholine receptor inactivation syndrome" has been proposed for the illness, when other developmental disorders were also caused by the maternal antibodies (oskoui et al. ). neonatal guillain-barré syndrome (gbs) was observed to occur - days postpartum in children born to mothers with gbs. serum from mother and infant depressed quantal content by approximately % and reduced the amplitude of postsynaptic currents by - % in mouse, newborn and juvenile rats. the antibody nature of the blockade could be confirmed by showing that monovalent fab fragments were similarly effective as purified immunoglobulin (ig) g. both cellular and humoral immune mechanisms are operative in guillain-barré syndrome (gbs). transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction (luijckx et al. ; buchwald et al. buchwald et al. , . guillain-barré syndrome and sydenheim's chorea are diseases, which were shown to be associated with the immune response after microbial infections. the occurrence of guillain-barré syndrome noted in infants whose mother had harmless autoimmune antibodies during pregnancy (buchwald et al. ; sladky ) . the syndrome's occurrence within families is also of interest. the mmp c(- )t and tnfa c(- )a snp were associated with severe weakness and poor outcome, indicating that these snps may be one of the factors predisposing to a severe form of gbs (geleijns et al. ). the associated features of er / ek carriers consist of favorable metabolic and body compositional conditions. in contrast, the n s polymorphism was reported to be associated with an enhanced sensitivity to glycocorticoids. haplotypes carrying the minor allele of the bcli polymorphism of the glycocorticoid receptor gene was related to the phenotype and outcome of gbs explaining the family dependence of the syndrome and other neonatal disorders (dekker et al. ). mutations in about a dozen of genes have been linked to the development of permanent neonatal diabetes mellitus (pndm). the most frequent causes of pndm are heterozygous mutations in the kcnj , ins and abcc genes. although pndm is a rare phenomenon (one case in about , live births), this discovery has had a large impact on clinical practice as most carriers of kcnj and abcc gene mutations have been switched from insulin to oral sulphonylureas with an improvement in glycemic control (aguilar-bryan and bryan ; rubio-cabezas et al. ). the majority of transient neonatal diabetes mellitus (tndm) cases have an abnormality in chromosome q . half of the ndm cases are transient (tndm) and the other most frequent causes of ndm are missense mutations in the pancreatic b-cell k atp channel genes kcnj , ins and abcc (chr ), and in the preproinsulin gene, ndm has been linked to numerous other genetic causes including point mutations in gck (chr ), glis (chr ), eif ak (chr ), pdx (chr ), ptf a (chr ), slc a (chr ), hnf b(chr ) or foxp (chrx) (aguilar-bryan and bryan ; bonnefond et al. ) . genetic mutations were identified in~ % of non-consanguinous probands with pndm/mdi, using sequential screening of kcnj , ins and abcc genes in infants diagnosed within the first months of age. this percentage decreased to % in those with diabetes diagnosed between and months. patients belonging to the latter group may either carry mutations in genes different from those commonly found in pndm/mdi or have developed an early-onset form of autoimmune diabetes. islet-cell antibodies (ica), glutamic acid decarboxylase autoantibodies (gada), tyrosine phosphatase-related proteins-islet antigen autoantibodies (ia- a), insulin autoantibodies (iaa), zinc transporter autoantibodies (znt a) were found in the sera of the children, suggesting autoimmune origin of their disease (russo et al. ). biliary atresia (ba) is a devastating disease of infants, invariably leading to cirrhosis, end-stage liver disease, and death if untreated. it has been shown using microarray technique, that the t-cell regulatory gene rras seems to be a key factor in the development of the disease (zhao et al. ) . a recent review reported that ba may involve a primary perinatal hepatobiliary reoviral or rotaviral infection and a secondary autoimmune-mediated bile duct injury (mack ). the maternal virus infections followed by maternofoetal microchimerism seems to be a very impressive explanation of the etiology (muraji et al. ) . in a mouse model, oral vaccination before mating with rotateq and rotarix prevented most rhesus rotavirus-induced ba (turowski et al. ). biliary atresia is probably the endresult of different aetiological factors, among which viruses and other agents may cross the placenta. otherwise it cannot be understand, why only one of the twins obtain the disease (morris et al. ) . the anti-idiotypes transcytosed by different efficiency into the circulation of the twins, might be an additional explanation for the asymmetric disease. maternal symptomless paraproteinemia was also found to be transmitted to the fetus. the paraprotein was detected after birth for months in the serum of the child and caused prolonged immunosuppression without later consequences (littlewood et al. ; littlewood and payne ) . transient neonatal acquired von willebrand syndrome (avws) has been observed peripartum. its clinical management is analogous to monoclonal gammopathy of undetermined significance (mgus) of the mother since it is the consequence of transplacental transfer of maternal igg antibodies (simone et al. ; nageswara rao et al. ). immune trombocytopenic purpura associated with pregnancy. fetal and neonatal alloimmune thromocytopenia (fmait) results from transplacental transfer of maternal antibodies that develop in response to alloimmunization against paternal human platelet antigens (hpas) expressed on fetal platelets. this thrombocyte loss could be prevented using modified igg molecules (ghevaert et al. ) . at present seven biallelic human platelet antigen (hpa) systems have been determined and can be typed using genomic dna. platelet genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patients' sera to complement the clinical history in the diagnosis of alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait). prenatal platelet typing of the fetus in suspected cases of fnait became also available (curtis ) . half of the infants were borne with low platelet counts and of required replacement therapy upon birth (ozkan et al. ; gasim ) . the maternal, transplacental igg binding to the fetal platelets was suggested to prevent their recirculation by fcgr binding to and phagocytosis by macrophages. the pathogenesis of immune trompcytopenic purpura and that associated to myelodysplasia and leukemia were shown to be different (psaila and bussel ; psaila et al. ). neonatal endarteritis has been diagnosed in the newborns of mothers suffering from endarteritis nodosa. fatal myocardial infarction in a neonate due to coronary arteries is compared with two lethal cases of mucocutaneous lymph node syndrome and/or infantile periarteritis nodosa (mlns/ipn). cutaneous polyarteritis was transmitted to the newborn, too (kitzmiller ; krapf et al. ; stone et al. ) . during the last decade no publication could be found on neonatal endarteritis. probably the pathogenesis of this disease has been reevaluated in the light of the molecular diagnostic findings. hereditary autoinflammatory syndromes are caused by monogenic defects of innate immunity and are classified as primary immunodeficiencies. these syndromes are characterized by recurrent or persistent systemic inflammatory symptoms and must be distinguished from infectious diseases, autoimmune diseases, and other primary immunodeficiencies. the sited review describes the epidemiological, clinical and laboratory features, prognosis, and treatment of the main autoinflammatory syndromes, namely: familial mediterranean fever; tnf receptor associated periodic syndrome; the cryopyrinopathies; mevalonate kinase deficiency; pediatric granulomatous arthritis; pyogenic arthritis, pyoderma gangrenosum and acne syndrome; majeed syndrome; and deficiency of interleukin receptor antagonist. the cryopyrinopathies discussed include neonatal-onset multisystem inflammatory disease (also known as chronic infantile neurologic, cutaneous and articular syndrome), muckle-wells syndrome, and familial cold autoinflammatory syndrome (jesus et al. ) . primary immunodeficiencies (pids) were analyzed to gain insight into the physiopathology of sle. some pids have been consistently associated with sle or lupus-like manifestations: (a) homozygous deficiencies of the early components of the classical complement pathway in the following decreasing order: in c q, % of affected patients developed sle; in c , %; inc r/s, %; and in c , up to %; (b) female carriers of x-linked chronic granulomatous disease allele; and (c) iga deficiency, present in around % of juvenile sle. mutations of the complement system (c -inhibitor; c q, c r, c s, c , c , c , c , c , c , c and c deficiencies were shown to facilitate the development of sle in addition to the facilitation of bacterial infections (c and c , encapsulated bacteria; c to c neisseria). other autoimmune diseases, i.e. polyendocrinopathy candidiasis ectodermal dystrophy (apeced), immune dysregulation polyendocrinopathy enteropathy x-linked (ipex), and autoimmune lymphoproliferative syndrome (alps), suggesting that mechanisms considered as critical players for induction and maintenance of tolerance to autoantigens, such as ( ) aire-mediated thymic negative selection of lymphocytes, ( ) foxp + regulatory t cell mediated peripheral tolerance, and ( ) deletion of auto-reactive lymphocytes by fas-mediated apoptosis, were not found to be associated with sle physiopathology (blois et al. ; carneiro-sampaio et al. ). behçet syndrome (bs) is a multisystem chronic inflammatory disorder, which is characterized by relapsing oral and genital ulceration and iridocyclitis. while being of unknown etiology, vasculitic changes of possible autoimmune origin are common to all involved organs, and thrombotic complications, which may adversely affect gestation, are frequently seen was shown to possess genetic etiology on the basis of the comparison of its incidence among monozygotic and dizygotic twins (masatlioglu et al. ). pregnancy does not have a deleterious effect on the course of bd and may possibly ameliorate its course. however, it seems that bd may adversely affect pregnancy. the miscarriage rate was higher, and the pregnancy complications and cesarean section rates were significantly elevated (jadaon et al. ) . overall, parity was associated with an % increased risk of female predominant autoimmune diseases. pregnancies resulting in liveborn children therefore seem to contribute only little to the general female predominance in autoimmune diseases. crohn's disease and ulcerative colitis; in idiopathic inflammatory bowel diseases (ibd), including crohn's disease and ulcerative colitis, there is pathogenic build-up of cd + t cells at sites of inflammation, mediated in part by il- , which provides (as described earlier) an anti-apoptotic signal to t cells through the induction of bcl- and bclxl and promotes th lineage differentiation. il- is upregulated in patients with both types of ibd. il- levels are elevated markedly in the serum of patients with ibd, decrease with treatment of inflammation and are predictive of ibd relapse. microbial pattern-recognition receptors, such as the tlrs and nod (nucleotide oligomerization domain ), which activate nf-kb, have been implicated in these conditions. neutralisation with an anti-il- r antibody largely prevented the colitis in mice (arad et al. ) . most women and men with ulcerative colitis (uc) and crohn's disease (cd) can expect a healthy child with neither preterm birth nor low birthweight. no neonatal forms have been described (ludvigsson and ludvigsson ; van assche et al. ) . later crohn's disease was shown to impair the fetal outcome in the case of the affected mothers (naganuma et al. ). colitis-associated-cancer (cac); tgf-b suppresses the formation of cancer by inhibiting il- trans-signaling and human samples of colon cancer have low-levels of il- r, as do samples of inflamed colon. adenomatous polyposis coli (apc) gene of mice with apc mutation develop cancer, genetic deletion of the tlr-adaptor protein myd decreased the number of cancers markedly. because myd activation in turn activates nf-kb, it is not surprising that il- production was decreased greatly in mice deficient in myd and supports prior data showing that il- is one of the effector signals in the tlr-nf-kb activation pathway (becker et al. ) . tgf-b was found to suppresses tumor progression in colon cancer by inhibition of il- trans-signaling. huntington disease was found to be of congenital origin, and the diagnosis can be obtained before implantation (hdcrg ; peciña et al. ) . the causal mutation is the expansion of a cag trinucleotide repeat tract in exon of a large gene on chromosome that results in the extension of a polyglutamine tract at the n-terminus of the encoded, ubiquitously expressed protein called huntingtin. from the maternal blood the dna from the fetal-maternal transport can be also applied for the prenatal diagnosis (bustamante-aragones et al. ). hypoparathyroidism with autoimmunity due to the q . deletion syndrome. the majority of patients acquired autoimmune antibodies, but without antiparathyroid antibodies (lima et al. ). wegener granulomatosis (wg) is systemic disease of unknown etiology characterized by necrotizing granulomatous inflammation, tissue necrosis, and variable degrees of vasculitis in small and medium-sized blood vessels. the classic clinical pattern is a triad involving the upper airways, lungs and kidneys. ninety percent of patients present with symptoms involving the upper and/or lower airways, and % will eventually develop renal disease (mubashir et al. ) . pregnancy in patients with wg requires preconceptional planning, careful clinical management, and vigorous treatment of active disease. the management is individualized and the pregnancy outcome is variable. antenatal management and therapeutic options are important (koukoura et al. ) . no neonatal disease have been described, and ivig treatment could be successfully applied in the steroid resistant patients (bellisai et al. ). kawasaki disease (kd) is an illness mostly of infants and young children, the majority being less than years old. the peak age of onset of illness is - months. it is unusual in very young infants. from to , only six instances of kd occurred in infants days of age or younger in japan, and infants days of age or younger accounted for only . % of all patients. the youngest infant of kd to date (a neonate week old) was reported by stanley and grimwood. the youngest reported from india is a -day-old infant (thapa et al. ). therapeutic monoclonal antibodies (mabs) are most commonly of the igg subclass, which is transported most efficiently to the fetus. in all animal species used for testing developmental toxicity, fetal exposure to igg is very low during organogenesis, but this increases during the latter half of gestation such that the neonate is born with an igg concentration similar to the mother. the therapeutic monoclonal antibodies might cause developmental and reproductive toxicity (dart) requiring testing of antibody-based therapeutics (pentsuk and van der laan ). guillain-barré syndrome during pregnancy can be also treated with ivig and plasmapheresis. (niklasson et al. ; goyal et al. ; bahadur et al. ; modi et al. ; ohlsson and lacy ) . polyclonal antisera, administered for passive immunization, are mixtures of different proteins, sharing binding activity against the antigen (ag) determinants of the same immunogen preparation. immunoadhesins (or immunoglobulin fusion proteins) are antibody-like molecules resulting from the fusion of a constant region (e.g., fc portion) of an immunoglobulin and the ligand-binding region of a receptor or an adhesive molecule. antibody fragments such as fab, scfv, diabodies, and minibodies are molecules devoid of part or whole of the fc portion. therefore, they have faster clearance and better tissue/tumor penetration than whole immunoglobulins and they will perform better than whole iggs in conditions where a short half-life is desirable, such as in radio-imaging and/or radio-therapy. they have no adcc and cdc triggering activity. the smallest proteins retaining antigen binding are a single variable domain antibody (nieri et al. ). ivig prevention improves the risk of heart block of sle patients (friedman et al. ) . several members of the idiotype-anti-idiotype network and antibody dimers including antigen-antibody complexes were found in the ivig preparations (luijten et al. ; osterhaus et al. ; clark et al. ). ad anti-idiotype monoclonal antibody can mimic the cd antigen. the molecular basis of ad mimicry has been identified with three cdr regions of ad showing similarity to three regions of cd . these regions have been analysed for potential t-cell epitopes, and sequences that are predicted to bind to hla/a , , and to hla/dr , , have been identified within the cdrh region of ad . ad can stimulate cd and cd responses in colorectal cancer patients with the appropriate haplotype. only a few patients produce a sustained memory response (durrant et al. ; reinartz et al. ) . most of these vaccines for the lymphoma therapy use the tumor b cell idiotype (the unique variable region of the surface immunoglobulin) as a tumor-specific antigen. in spite of several problems anti-idiotype vaccines prospect towards integration of this strategy in the therapeutic armamentarium for lymphoma (houot and levy ) . clinical studies have been published using different preparations and summarised at the end of table . . abagovomab (aca ) acts as an antigen mimic of the carbohydrate ovarian cancer antigen (wagner et al. ) . the frequency of peripheral t reg s was increased during abagovomab therapy in a high percentage of patients. despite higher t reg counts compared with baseline levels, the suppressive capacity of t reg s was reduced in a subset of patients. the data further indicate that the ability of t cells to proliferate in response to ca- in vitro could be associated with diminished t reg activity. importantly, ca- -specific immunity could not be enhanced by in vitro t reg depletion, as ca- induced cd + foxp + t reg s with suppressive capacity. abatacept (orencia) human igg fc domain ctla- ; anti-cd and anti-cd . abatacept (abt, orencia, bristol-myers squibb ltd) rheumatoid arthritis tumor necrosis factor inhibitor in phase clinical trial, but it was found effective only in very early phase of the disease (malottki et al. ; emery et al. ) . prophylactic withdrawal of drugs before pregnancy is mandatory (Østensen et al. ) . at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (Østensen and f€ orger ) . abciximab (reopro) igg k-fab, chimera -integrina b (platelet-gp)haemostasis/trombosis (nieri et al. ). using immunohistochemistry, reopro was only detected attached to maternal and fetal platelets, and to the trophoblastic surface of the placental villi (miller et al. a, b) . the effect of abciximab to icam- was excluded, but an effect to monocytes could not be excluded (voisard et al. ) . adalimumab (ada, humira; abbott) human recombinant, igg k, anti-tnfa, as, psa, cd, pp, jia (van schouwenburg et al. ) tnf-alpha blocker therapy (adalimumab). the therapy of pregnants showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (Østensen et al. ) . expression of hla-g on pmbcs is up-regulated in ankylosing spondylitis (as), correlates with acute phase reactants and decreases after tnf-alpha blocker therapy (chen et al. ) . anti-tnfa is used for the treatment of infectious bowel diseases even during pregnancy, which might influence the development of the fetal immune system (arsenescu et al. ) . anti adalimumab anti-idiotype antibodies (bartelds et al. malottki et al. ; nieri et al. ). ( ) ? teplizumab anti-cd t. diabetes herold et al. ( herold et al. ( , ? integrin a b direct exposure to anti-tnf treatment during pregnancy was not related to a higher incidence of adverse pregnancy outcomes than infectious bowell diseases overall (schnitzler et al. ). the anti-tnf agent was started prior to conception and continued until there was evidence of fetal cardiac activity. in women treated with a combination of anti-tnf therapy, anticoagulation therapy, and ivig, the live birth rates ( %) were greater than those in women treated with anticoagulation therapy alone ( %) or in those receiving a combination of anticoagulation therapy and ivig ( %). fetal outcomes, including gestational age and birth weight, were similar across the groups, and no congenital anomalies were reported after anti-tnf agent exposure (winger and reed ; vinet et al. ). rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, crohn's disease (Østensen and f€ orger ) . all users requested at least one time repeatedly the treatment in norway (mahic et al. ) . long term treatment did not resulted in reactivation of chronic hepatitis b virus infection (mori ) . the treatment caused the improvement in two bone formation markers -b-alkaline phosphatase and osteocalcin (veerappan et al. ) . alemtuzumab (mabcampath, genzyme) humanized, igg k, anti-cd anti-ca immunoglobulin directed against cd antigen expressed on t-and b lymphocytes, monocytes, macrophages, nk cells, and a subpopulation of granulocytes, but not on hematologic precursors. induction of cdc or adcc on an fcreceptor g-binding mechanism. pancreas transplant recipients on alemtuzumab maintenance therapy suffered frequently from red cell aplasia, and autoimmune hemolytic anaemia. (elimelakh et al. ) cord-blood-hematopoetic-stem-cell expansion and increase the availability of cord-blood units for transplantation (lim et al. ) . in contrast to ivig and rituximab the compound may be an effective therapy for complex immunohematologic disorders complicating hematopoietic stem cell transplantation. the paper emphasizes the importance of t-cells in transplant associated immune cytopenias (chao et al. ). b-ccl (nieri et al. ). alicaforsen is a human monoclonal antibody a b -integrin, also known as leukocyte function antigen (lfa)- , and its ligand, intercellular adhesion molecule- (icam- ), is important for the recruitment of leukocytes to inflammatory sites (bosani et al. ). anti-cd and anti cd intratumoral microvessel density (imvd) monoclonal antibodies. anti-cd was more effective against non-small cell lung cancer than anti-cd (tanaka et al. ) . anti-cd mab (bms- , bristol-myers squibb) - bb (cdw ), a member of tumor necrosis factor receptor (tnfr) superfamily stimulating t-cells, nk-cells and dcs . anti-cd mab enhances rituximabdependent cytotoxicity against the lymphoma cells (lee et al. ; kohrt et al. ) . anti-leu- b or anti-leu- c, igg a, marker of t-cell subpopulation (clement et al. ; champlin et al. ) . apomab (genentech) igg , against extracellular domain dr /tumor necrosis factor related (trail) receptor apoptosis inducing ligand (nieri et al. ). basiliximab (simulect) chimeric, igg k, anti-cd , il r antagonist (aktas et al. ) % reduced fetal body weight (pentsuk and van der laan ) . bavituximab (peregrine pharmaceuticals, inc., tustin, ca), anti-phosphatidylserine bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma (he et al. ). hcv therapy (immunostimulant) (dammacco et al. ; quer et al. ) . belatacept (ctla -ig) is a new recombinant molecule that interferes with the signal of t lymphocyte activation and prevents acute rejection after renal transplantation. hla-g acts as a naturally tolerogenic molecule in humans. patients treated with ctla -ig displayed significantly higher soluble hla-g (shla-g) plasma concentrations than patients treated with calcineurin inhibitors or healthy donors (bahri et al. ). ctla -ig-treated dc acted as tolerogenic apc through shla-g secretion as they suppressed t cell alloproliferation, which could be restored by using a neutralizing anti-hla-g ab (bahri et al. ). the use of anti tumour necrosis factor mabs are not recommended (partlett and roussou ) . cytotoxic t-lymphocyte-associated protein (ctla- ) is a negative regulator of t cell activation and may modulate peripheral self-tolerance (kaufman et al. ) . bevacizumab (avastin) humanized, igg k, anti-vascular endothelial growth factor-a (vegf-a) used for the treatment of non-small cell lung cancer, (nsclc) colorectal cancer (cc); (nieri et al. ). efd rabbit: dose-dependant decrease in maternal bodyweight, increase in fetal malformations and late resorptions; (pentsuk and van der laan ). intravitreal bevacizumab therapy during pregnancy for off-label ocular indications can result in significant visual improvement without adverse fetal events related to treatment (tarantola et al. ) . intravitreal . mg bevacizumab may reach the systemic circulation in plasma concentrations of ng/ml (csáky and do ) . two pregnants have lost their babies within days following intravitreal injections of bevacizumab (petrou et al. ). placenta gf levels are elevated in the plasma of colorectal and rectal carcinoma patients receiving bevacizumab (xu and jain ) . prevention of angiogenesis by msc in pancreatic cancer (beckermann et al. ) . blinatumomab (epcam, antigen -epithelial cell-adhesion molecule, -present on % of cancer cells) and mt- and mt present on all non-hodgin lymphoma cells (armstrong and eck ; nieri et al. ). cd /cd bispecific, single chain recombinant antibody topp et al. ) . catomaxomab (removab) bifunctional recombinant anti epidermal cell adhesion molecule epcam and anti-cd . it is inducing adcc in ovarian and stomach cancer. epcam is the ligand for human leukocyte immune-globulin like receptor (lair- ) (armstrong and eck ; nieri et al. ; bokemeyer ; r€ ussel et al. ) . ceavac (mimicking carcinoembryonic antigen) colon cc (foon et al. ). certolizumab (cimzia®; ucb) pegylated humanized antibody fab' fragment of tnf-a monoclonal antibody; ra, cd; (Østensen and f€ orger ) . certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules, thus reducing the harmful consequences to the fetus (clowse ) . cetuximab (erbitux) chimeric, igg k, anti-epidermal growth factor receptor- (her- ), apoptosis, cdcc colorectal cancer (cc); squamous cell cancer of head and neck (scchn) weight loss and reduced food consumption in high-dose group dose-dependent increase in abortion rates (not known, if could be associated with treatment) weight loss and reduced food consumption in high-dose group (powell et al. ; pentsuk and van der laan ; rech and vonderheide ) . chaglycd (cd -specific) a humanized antibody, an aglycosylated human igg antibody directed against cd was shown to reduce the insulin-requirement of the patients. residual beta-cell function was better maintained with chaglycd than with placebo (keymeulen et al. ) . cixutumumab (img- or cix) targeting insulin-like growth factor receptor (igf-ir) treatment for multiple cancers. human igg , blocks interaction between igf-ir and its ligands, igf-i and -ii, and induces internalization and degradation of igf-ir. its combination of cetuximab (mab against egfr) inhibited the growth of pancreatic cancer and promoted its regression. an antiangiogenic mechanism was associated with cix treatment. reviewed recently (quatrale et al. ) . hyperglycemia is a regular side effect, but the fetal consequences during pregnancy have not been evaluated yet (mckian and haluska ) . eyelash trichomegaly in adults (bouché et al. ; garrido et al. ) . cnto chimeric monoclonal antibody with high affinity for human il- myeloma multiplex sensitivity to glycocorticoid (voorhees et al. ). ct- (curetech) humanized anti-pd- igg mab that binds to mouse and human pd- , programmed death receptor . pd- but not ctla- blockage abrogates the protective effect of regulatory t cells in a pregnancy murine model. mkrtichyan et al. ; stagg et al. ; wafula et al. ). dacetuzumab (sgn- ; seattle genetics) humanized; and hcd fully human (novartis/xoma); cd , tumour necrosis factor receptor; b-cells, dcs, macrophages lymphomas . daclizumab (zenapax) humanized, igg k, anti-a-chain of cd , il- r antagonists (elimelakh et al. ; aktas et al. ) transplant rejection. eculizumab (soliris) humanized, igg / j, anti-human complement c ; paroxismal nocturnal haemoglobinuria (pnh); (thomas et al. ; nieri et al. ; danilov et al. ). there was no evidence of complement blockade from cord blood samples taken at delivery. eculizumab appears safe to use in this setting and is likely to prevent many of the complications usually observed (kelly et al. ) . edrecolomab (panorex) igg a, epcam antigen cdc-adcc cancer (nieri et al. ). efalizumab (raptiva) humanized, igg k, anti-integrin-cd a -psoriasis (nieri et al. ). progressive multifocal leukoencephalopthy was found to be a rare, but lethal disease associated with long term efalizumab therapy (kothary et al. ). epratuzumab, a humanized igg unconjugated anti-cd antibody effective against non-hodgkin lymphoma and follicular lymphoma (leonard et al. ; watanabe ) . consequences of the application during pregnancy has not been reported. etanercept (etn, enbrel, wyeth pharmaceuticals) human igg fc domain tnfr /p ; anti-tnfa as, psa, pp, jia; ng/ml cord blood. ng/ml week postpartum ng/ml; weeks postpartum; undetectable weeks postpartum (clowse ) . reduction of post-partum microchimerism (rak et al. ). complications of the therapy may be acute anterior uveitis (etanercept), psoriasis (infliximab > etanercept) and infectious bowel disease (ibd) (etanercept > infliximab) which were observed in association with the treatment using tnfantagonists. the paradoxical consequences, however, affected less than % of the treated patients (fouache et al. ). eternacept, which is a recombinant human p soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued. the recombinant receptor proved to be useful for the treatment of rheumatoid arthritis (malottki et al. ), but its use in pregnants has not been approved (Østensen et al. ) . etanercept treatment ( mg  /week) has been stopped weeks before pregnancy. the treatment had to be reinitiated from the th week of pregnancy and no fetal complication was observed (umeda et al. ). the etanercept treatment was initiated weeks before pregnancy ( mg/sq-m  weekly). the cord blood contained ng/ml etanercept in contrast to the maternal serum ( , - , ng/ml). no detectable etanercept was found in the newborn's blood th week after delivery, although the breast milk contained . ng/ml etanercept (murashima et al. ). all patients requested at least one times the repetition of the treatment according to a publication from norway (mahic et al. ) . fontolizumab used for the preventive treatment of newborns at risk for respiratory syncytial virus infection (rsv) resulted a significant decrease in c-reactive protein levels suggested a beneficial biological effect. (nieri et al. ; reinisch et al. ) . galiximab, a human-primate chimeric anti-cd antibody: galiximab is a human-primate chimeric anti-cd antibody with excellent tolerability and singleagent effectiveness for recurrent follicular lymphoma (fl), resistant to other therapeutical means (watanabe ) . gemtuzumab (mylotarg k ) igg k; humanised; cd -monocyte, myeloid cell drug targeting (nieri et al. ). golimumab (simponi®; centocor ortho biotech) human monoclonal igg antibody ra, as, psa (Østensen and f€ orger ) . hcd (novartis/xoma) cd -specific fully human igg mab with antagonistic activity that mediates adcc and blocks cd l-induced survival and proliferation of normal and malignant b cells (chatenoud and bluestone ) . ibritumomab tiuxetan (zevalin) murine, igg k, anti-cd ; radiol (yttrium ) imc-c (nieri et al. ). infliximab (ifx, remicade, schering-plough ltd) chimeric, igg k, anti-tumor necrosis factor alpha (tnfa) rheumatoid arthritis (ra) and crohn's disease (cd) (saleem et al. ; van schouwenburg et al. ) . fatal case of disseminated mycobacterial infection has been reported in an infant who received bcg vaccine at months of age. the mother had been treated with infliximab throughout her pregnancy. vaccination with live bacteria and viruses should be postponed in infants exposed to infliximab in utero, until serum levels are undetectable which may require more than months (djokanovic et al. ) . the fetal concentration of infliximab was found to be higher than that of the mother. this might be a risk for the postnatal development of the immune system (zelinkova et al. ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to their mothers (clowse ) . all reported pregnancy outcomes under treatment with infliximab showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (Østensen et al. ) . only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide. the potency of this agent in moderate-to-severe ulcerative colitis (uc) and cd has been one of the most important advances in the care of inflammatory bowel disease (ibd) in the past decade (d'haens and daperno ) . anti-infliximab anti-idiotypes. no association was found between the patients' allotypes and the presence or concentration of anti-infliximab antibodies ) reduction in fertility (not known, whether related with male or female animals) (pentsuk and van der laan ). vacterl association? acute graft versus host disease (couriel et al. ) were described, but it proved to be useful for the treatment of rheumatoid arthritis (malottki et al. ) . inotuzumab ozogamicin (cmc- ), the calicheamicin-conjugated anti-cd monoclonal antibody and rituximab combination were used for the treatment of ankylosing spondilitis, psoriatic arthritis and ulcerative colitis; (nieri et al. ; smith et al. ) the concentration in the blood of the newborn was . mg/ml week post-partum and slowly declined over months (clowse ) . ipilimumab (fcgriib binding) overcoming tcla- -mediated immunosuppression, increasing anticancer immune-response (melanoma malignum; mdx- ; bristol-myers squibb/medarex); ctla- (cd ) t cells; t reg cells; colon and prostatic cancer; (nieri et al. ; houot et al. ) . iratumumab (sgn- and mdx- ) cd -specific igg used for the treatment of hodgkin's lymphoma. myelosuppression, fatigue, elevated liver enzymes were documented during therapy (klimm et al. ) . lfb-r , a fully human anti-rhesus d (rhd) antibody, for the prevention of feto-maternal allo-immunization in rhd-women, as a substitute for human polyclonal anti-rhd immunoglobulins (urbain et al. ). lfb-r , a monoclonal antibody directed against cd , for the treatment of b cell malignancies. antibody-dependent cellular cytotoxicity (adcc) activity and enhanced affinity to fcgriii (cd ), both correlated to a glycosylation pattern characterized by a low fucose content (urbain et al. ). mapatumumab; trail receptor activation (death receptor ) mediator of apoptosis in cancer cells (nieri et al. ). matuzumab (humanised anti-egfr monoclonal antibody; reviewed by seiden et al. ; and recently by quatrale et al. ) . melimmune: anti-idiotype antibody that mimic the high molecular weight chondroitin sulfate proteoglycan antigen of melanoma cells (pride et al. ; murray et al. ; ward et al. ) . mitumomab (bec , imclone systems) bec- anti-idiotype (giaccone et al. ; bottomley et al. ) bec is an anti-idiotypic antibody that mimics gd , a ganglioside that is expressed on the surface of tumor cells and is of neuroectodermal origin. ganglioside gd can be used as a vaccine against small cell lung cancer (sclc) (nieri et al. ). mln- anti-a b integrin antibody of igg type, humanised (reviewed by bosani et al. ) approved for the treatment of crohn's disease. motavizumab (humanised mouse monoclonal antibody). motavizumab targets a highly conserved epitope in the a antigenic site of the rsv fusion (f) protein, which is important in the invasion of rsv from cell to cell. motavizumab, which differs from palivizumab by just amino acids, has exhibited a -fold enhancement in binding to the rsv f protein compared with the first-generation mab, with an -fold faster association rate and sixfold slower disassociation rate (nieri et al. ; weisman ). muromonab, igg a, murine, t-cell cd blocade. cd -specific monoclonal antibodies can re-establish immune homeostasis in treated individuals. this occurs through modulation of the t-cell receptor (tcr)-cd complex (also termed antigenic modulation) and/or induction of apoptosis of activated autoreactive t cells, which leaves behind 'space' for homeostatic reconstitution that favours selective induction, survival and expansion of adaptive regulatory t cells establishing long-term tolerance. it is used for early treatment of diabetes type (chatenoud and bluestone ; nieri et al. ). natalizumab (tysabri) humanized, igg k, anti-a -integrin (vla- ), in the treatment of sclerosis multiplex (van schouwenburg et al. ) . natalizumab blocks both alpha- b integrin (vcam ) and alpha- b integrin (madcam ) interactions (rutgeerts et al. ). therapy of sclerosis multiplex will be more efficient in combination with interferon (miller et al. a, b; nieri et al. ). in animal experiments efd g. pig: reduced pregnancy rates in high-dose group; ppnd cyn: increased abortion and stillbirth rates (pentsuk and van der laan ). in cynomolgus monkeys, however, the abortion rate had not been increased, but hematopoetic changes were observed. natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy (wehner et al. a, b) . % of natalizumab therapy has been stopped because of pregnancy. three of patients treated at least for months developed progressive multifocal encephalopathy (pml; piehl et al. necitumumab (imc- f ) anti-egfr human monoclonal antibody (kuenen et al. ) . nimotuzumab (theracim) humanised, anti-egfr- /her- ; apoptosis, adcc; head and neck cancers (hncc), (spicer ; nieri et al. ; reviewed recently by quatrale et al. ) . omalizumab (xolair) humanized, igg k, anti-ige -asthma. causing marked reduction in serum levels of free ige and down-regulation of ige receptors on circulating basophils. effective in monozygotic twins (holgate et al. ; just et al. ; nieri et al. ) tolerability (corren et al. ). onercept, is a recombinant human p soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued (bosani et al. ). palivizumab (synagis) humanized, igg k, anti-respiratory syncytial virus "a" epitope of fusion protein. it is used for the prevention of respiratory syncytial virus infection of newborns with different risks for respiratory infections (martin-mateos ; nieri et al. ; weisman ) . panitumumab (vectibix) human, igg k, anti-human epidermal growth factor receptor binding the catalytic kinase domain of the receptor of colorectal cancer (cc; nieri et al. ). increased frequency of abortion/fetal death rates were observed in high-dose group (reviewed by pentsuk and van der laan ; nieri et al. ). eyelash trichomegaly in adults were seen (zhang et al. ; morris et al. ) . racotumomab( e ), an anti-idiotypic vaccine mimicking the n-glycolyl-gm ganglioside (guthmann et al. ; hernández et al. ) effective against breast and lung cancers. ngcgm is practically undetectable in healthy human tissues as a result of an alu-mediated inactivation of the gene, the ganglioside is highly expressed in several human cancer cells presumably due to incorporation of dietary ngc . ramucirumab (dc- ) (an antibody to the vegf receptor- ) (tonra et al. ; krupitskaya and wakelee ) . ranibizumab (lucentis, genentech) humanized, igg -fab, anti-human vasc. endothel. growth factor-a (vegf-a); for the treatment of choroidal neovascular (wet) age-related macular degeneration (armd) reviewed recently (ferrara et al. (ferrara et al. , ; neovascular acute myeloid leukemia (neovascular-aml); (csáky and do ; nieri et al. ). retuximab (epstein-barr virus) anti-cd- (sodani et al. ) . rituximab (rtx, mabthera k , roche) chimeric, igg k, anti-cd (sulesomab, leukoscan). murine fab, binds to surface granulocyte non-specific crossreacting antigen present on neutrophils. rhinitis, fever, chills and toxic laboratory findings occurred during the treatment (klimm et al. ) . hcv cryoglobulinaemia could be also treated (dammacco et al. ) . the treatment of pregnants because of bukitt's lymphoma resulted high rituximab concentrations and a transient complete b-cell depletion in the cord blood. b-cell recovery was fast, showing a regular immunophenotype without loss of cd antigen, no functional deficits and adequate vaccination igg titers (friedrichs et al. ) . administration in third trimester of pregnancy suppresses neonatal b-cell development, but without later neonatal consequences (klink et al. ) , in spite of these the prophylactic withdrawal has been recommended before pregnancy (Østensen et al. ) . human fetal b-cell depletion and lymphocytopenia in cynomolgus, were observed, too (vaidyanathan et al. ) . reduction of post-partum microchimerism was documented (rak et al. ). useful in rheumatoid arthritis therapy (malottki et al. ) in combination with chemotherapy depending on human concentrative nucleotide transporter (hcnt ) gene expression rate (rabascio et al. ) . non-hodgkin lymphoma, rheumatoid arthritis were the indications (nieri et al. ). cd is a costimulatory molecule expressed on a variety of immune cells after activation, including nk cells. cd stimulation by specific igg enhances the antilymphoma activity of anti-cd antibodies by enhancing adcc . of pregnancies with known outcomes, resulted in live births. twenty-two infants were born prematurely; with one neonatal death at weeks. eleven neonates had hematologic abnormalities; none had corresponding infections. four neonatal infections were reported (fever, bronchiolitis, cytomegalovirus hepatitis, and chorioamnionitis). two congenital malformations were identified: clubfoot in one twin, and cardiac malformation in a singleton birth. one maternal death from pre-existing autoimmune thrombocytopenia occurred. women should continue to be counseled to avoid pregnancy for months after rituximab exposure; however, inadvertent pregnancy does occasionally occur. practitioners are encouraged to report complete information to regulatory authorities for all pregnancies with suspected or known exposure to rituximab (chakravarty et al. ) . due to ongoing bleeding, rituximab was given in the th week of pregnancy. the platelet count rose to over  ( )/l after weeks. the neonatal b-lymphocyte count normalized at months after delivery. there were no neonatal complications of rituximab therapy (gall et al. ) . passenger lymphocyte syndrome has been described by lee et al. ( a, b) . siplizumab (cd or medi- ) is a humanised igglk monoclonal antibody that binds to human cd antigen. preclinical studies demonstrated that siplizumab kills target cells by adcc (fanale and younes ; watanabe ) . teplizumab (cd -specific, hokt g -ala-ala), a humanized fc mutated anti-cd monoclonal antibody induced tolerance, on the progression of type diabetes in patients with recent-onset disease even years after the first diagnosis (herold et al. (herold et al. , . tocilizumab (toc, roactemra, roche) against receptor of il- (mouse anti-human il- r antibody into human igg -k chain to create a human antibody with a human il- r binding site il- r a-chain or cd ; b-chain or cd ) at a low concentration of microg/ml, tocilizumab (anti-human il- receptor monoclonal antibody) inhibited the il- -induced matrix-metallo-proteinase (mmp) secretion which was shown to be stimulated in preterm premature rupture of membranes (pprm) (sato et al. ; mano et al. ; malottki et al. ; pham et al. ) . clinical phase trial for the treatment of rheumatoid arthritis has been approved. inherited autoinflammatory syndrome can be sometimes treated with anakinra and tocilizumab (goldfinger ). at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (Østensen and f€ orger ) . normal pregnancy is characterised by elevated th activity and anti-inflammatory cytokines during the first trimester, followed by increased th activity and proinflammatory factors near term (challis et al. ). in contrast, preeclampsia (pe) is marked by an increase in proinflammatory tumor necrosis factor-a (tnf-a) and interleukin (il- ) cytokines as well as a decrease in the anti-inflammatory cytokines il- and il- . in cases of restricted fetal growth, tnf-a is also elevated when compared with normal pregnancy (dávila et al. ) . women living at , versus , m in colorado had higher proinflammatory (il- , tnf-a) relative to anti-inflammatory (il- ) cytokines during the second and third trimesters (coussons-read et al. ) . multigenerational andean versus shorter duration european high-altitude residents were found to be protected from altitudeassociated fetal growth restriction. higher il- b might play a role in protection from altitude-associated reductions in fetal growth (coussons-read et al. ; dávila et al. ) . tocilizumab treatment increased serum levels of il- and soluble il- r (sil- r; nishimoto et al. ). in combination with other drugs adult onset of still's disease can be improved using monoclonal antibodies (efthimiou and georgy ) . tositumomab anti-cd igg, b-cell lymphoma (armstrong and eck ; nieri et al. ). trastuzumab (herceptin) humanized, igg k, erbb , anti-her . induction of cdc or adcc on an fcreceptor g-binding mechanism. human anhydramnion and oligohydramnion will develop because of the caused fetal kidney insufficiency (watson ; robinson et al. ; katsumi et al. ; matsumoto et al. ). this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich ) . transfer using aav-recombinant in mice does not induce anti-idiotypes (wang et al. a, b) . the mechanism of toxicity to the fetal kidneys is proposed to be associated with the different structure of egfr in the fetal renal-tubule epithelial cells (heterodimer of egfr and erbb in fetus vs. homodimer of egfr in adults). thus, trastuzumab will have a damaging effect on the fetal renal function, but it does not affect the kidneys of the adult (robinson et al. ) . anti-trastuzumab (ladjemi et al. ): anti-trastuzumab anti-id scfv , used as a therapeutic or prophylactic vaccine, protects mice from developing her -positive mammary tumors by inducing both anti-her ab antibody production and an anti-her th -dependent immune response. these results suggest that scfv could be used as an anti-id-based vaccine for adjuvant therapy of patients with her -positive tumors to reverse immunological tolerance to her . calmodulin inhibitors rescue trastuzumab sensitivity of breast tumours (kulkarni et al. ). the majority of these patients were able to tolerate therapy; however, oligohydramnios or anhydramnios occurred in out of the patients. this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich ) . tremelimumab (cp- , ; pfizer); ctla- (cd ) t cells; t reg cells; colon and prostatic cancer; . triab d (triab) breast cancer (reece et al. (reece et al. , (reece et al. , . trigerm (disialoganglioside gd ) melanoma . tositumomab (iodine labelled), murine cd , cdc, adcc, radio-cytotoxicity non-hodgkin-lymphoma (nieri et al. ) veltuzumab is a humanized anti-cd antibody with structure-function differences from chimeric rituximab (watanabe ) . visilizumab (cd -specific) for the management of both crohn's disease (cd) and ulcerative colitis (uc). biologics under evaluation or approved for uc that are discussed include monoclonal antibodies to tumor necrosis factor ([tnf] infliximab), inhibitors of adhesion molecules (mln and alicaforsen), anti-cd antibodies (visilizumab), and anti-interleukin (il)- receptor antibodies (daclizumab). biologics under evaluation or approved for cd that are reviewed include three monoclonal antibodies to tnf (infliximab, adalimumab, and certolizumab pegol), monoclonal antibodies against il- , interferon-g, and il- receptors, inhibitors of adhesion molecules (natalizumab, alicaforsen), and growth factors. only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide (d'haens and daperno ) . yttrium-ibritumomab tiuxetan and iodine-rituximab are anti-cd monoclonal antibodies combined with radioactive materials for diagnostic and/or therapeutic applications (watanabe ) . zalutumumab anti-egfr mab able to facilitate complement lysis of cancer cells (klausz et al. ) . reviewed recently (quatrale et al. ). expressed by antigen presenting myeloid cells (apc) (magnani et al. ). cd t-cell receptor (tcr)-cd complex resulting in the cells becoming 'blind' to antigen, a process that is also known as antigenic modulation (chatenoud and bluestone ) . cd over % (sato et al. ). cd ctla- negative regulator of t cell activation (kaufman et al. ). trail dr /tnf related receptor (nieri et al. interleukin (naugler et al. ; naugler and karin ; voorhees et al. ; reinartz et al. ). pd- programmed death protein ). complement factor thomas et al. ( ) breast cancer protein reece et al. ( reece et al. ( , reece et al. ( , ige immunoglobulin e, hypersensitivity (corren et al. ). anti-idiotypes carrier of the mimicry of epitopes (ab ) of antigens (k€ ohler ) . respiratory syncytial virus (nieri et al. ). cytotoxic t lymphocyte antigen- (ctla- ) and programmed cell death (pd- ) are members of the known t reg -associated molecules. blocking pd- abrogate the protective effect of t reg , resulting in a higher median abortion rate in comparison with the t reg / isotype-treated control while ctla- blockage did not interfere with the protective effect of t reg . pd- as an important mediator in t reg -induced fetal protection in the cba/ j · dba/ j murine model (wafula et al. ). ctla- was shown to interact with cd and cd resulting in termination of immune response (alegre et al. ) . mice genetically deficient in ctla- expression develop a lymphoproliferative disease which terminates in death by - weeks of age (tivol et al. ; waterhouse et al. ) . the cd possesses also role in the regulation of t-cells (sansom and walker ) . blockade of the interactions between cd and their ligands, cd and cd , has been shown to induce antigen-specific peripheral tolerance in organ transplantation. this knowledge has been successfully used in animal models to prevent allograft rejection by blocking cd and/or cd , thereby leading to long-term graft survival. cytokines favoring the maintenance of fetal survival mainly belong to the th -type (e.g. il- , il- , tgf-b), whereas pregnancy failure is associated with the th -type cytokines (e.g. ifn-g, tnf-a) at the materno-fetal interface and/or the absence of th -type cytokines. the combined use of anti-cd and anti-cd mabs in mice was effective in inducing maternal tolerance to the allogeneic fetus. blockade in vivo of cd and cd costimulation could prevent abortions by shifting cytokines from th predominance to th bias and expanding peripheral cd + cd + regulatory t cells (jin et al. ) . breakdown of immunologic self tolerance maintained by activated t cells expressing il- receptors (cd- ) results in the development of autoimmune diseases (sakaguchi et al. ; sakaguchi ) , which can be mitigated using anti-cd monoclonals. suppressive cd + cd t reg cells are elevated also during pregnancy (somerset et al. ) . intergins: progressive multifocal leukoencephalopathy was observed (pml) probably of polyomavirus etiology after natalizumab (anti-integrin-a ) therapy of crohn's disease (edula and picco ). tnfalpha blockers were shown to induce autoimmunity on ana and anti-dsdna antibodies in ra and spa patients. autoimmunity was induced more frequently with infliximab than etanercept and to a lesser degree to adalimumab therapy but, more importantly, this emergent autoimmunity was exceptionally associated to clinical manifestations of lupus (bacquet-deschryver et al. ) . the effect of infliximab, etanercept or adalimumab on spermatogenesis has been studied in patients with spondylarthritis (villiger et al. ) . sperm abnormalities were found in healthy controls. patients on anti-tnf therapy showed significantly better sperm motility and vitality than untreated patients (Østensen and f€ orger ). antibody products licensed for prevention or treatment of viral diseases include non-immune human immunoglobulin for use against hepatitis a and measles, virusspecific polyclonal human immunoglobulin against cytomegalovirus, hepatitis b, rabies, respiratory syncytial virus (rsv), vaccinia, and varicella-zoster, and the humanized monoclonal antibody palivizumab, fonolizumab and motavizumab (groothuis et al. ) . polyclonal immunoglobulin has also been used with various success for diseases caused by other human viruses including parvovirus b (pv b ), lassa virus, west nile virus, some enteroviruses, herpes simplex virus, crimean-congo haemorrhagic fever virus (cchfv), junin virus, severe acute respiratory syndrome-associated coronavirus (sars cov) and human immunodeficiency virus (hiv). serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot to lot variation and uncertain dosing have limited their use (casadevall (casadevall , . the use of rabies and tick-borne encephalitis virus-specific hyperimmune gamma globulins are used in several countries immediately following virus exposure (animal injuries or tick bites). cytomegalovirus-specific hyperimmune gamma globulin is used in the transplantation surgery (schmitz and essuman ) before the era of gancyclovir preventive therapy. sars cov surface glycoprotein, also called spike glycoprotein, (s protein or s glycoprotein) mediates viral entry into the host cell and has two functional domains s and s . the s domain is involved in the binding of the cellular receptor ace whereas the s domain facilitates the fusion between viral and host cell membranes. infections by many viruses, including coronaviruses, elicit potent neutralizing antibodies (nabs) that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. an improved method for epstein-barr virus (ebv) transformation of human b cells has been developed based on cpg oligonucleotides that increases the b cell immortalization efficiency from - % to - %, and this method was used for selection of human abs specific for sars cov proteins. one of the selected antibodies, which was specific for the s glycoprotein on the viral spikes, was about -fold more efficient in neutralization than convalescent serum. nipah virus (niv) and hendra virus (hev) are closely related emerging paramyxoviruses that comprise the henipavirus genus. they are biological safety level- (bsl- ) pathogens, and are on the niaid biodefense research agenda as zoonotic emerging category c priority pathogens that could be used as bioterror agents (zhu et al. ). monoclonal antibodies of enzyme activity have been developed. these can be used in cancer therapy, but the application for the treatment of pregnant women is at present not yet approved (kulkarni et al. ; quatrale et al. ). immunotherapy offers a range of potential treatment options: drug treatment, as well as the treatment of overdose, prevention of brain or cardiac toxicity and fetal protection in pregnant drug abusers. clinical trials, cocaine and nicotine vaccines have been shown to induce antibody titers while producing few side effects (haney and kosten ) . plasmin may serve as a major driving autoantigen for some anticardiolipin (acl) in anti-phospholipid syndrome (aps) patients who are positive for igg anti-plasmin ab. one mab displayed the anti-cardiolipin (acl) and the lupus anti-coagulant (lac) activities and induced fetal loss when injected into pregnant mice ). molecular mimicry has been suggested to play a role in the pathogenesis of many autoimmune diseases, such as allergic encephalomyelitis, experimental myocarditis, and experimental autoimmune keratitis and uveitis antigenic molecular mimicry is characterising anti-dna antibodies. these are reacting with different proteins i.e. enzymes (blank and shoenfeld ) . in case of schizophrenia, the overall finding has been that, when a monozygotic twin has this serious neuromental disorder (nmd), the other identical twin has a % risk; whereas among dizygotic twins, the risk -when one is afflicted -is only %. neuromental disorders (nmd) might be caused indirectly by maternal transplacentally-acquired antibodies, to agents with epitope molecular mimicry with the developing nervous system, and cause alterations which will clinically manifest years later (nahmias et al. ) . serological evidence of previous exposure to ebv in children with ms supports a role for ebv infection early in ms pathogenesis, as already indicated by prospective studies in adults. higher antibody titers and t-cell responses to ebv in patients compared to healthy ebv carriers indicate possible continuous viral reactivation. ms patients have increased cd + and cd + t-cell responses to ebv antigens, particularly ebna . there is some evidence that ebv could break immune tolerance to myelin antigens through molecular mimicry. detection of ebv-infected b-cells in patients' brain raises the possibility that intrathecal b-cell abnormalities and t-cell-mediated immunopathology in ms are the consequence of a persistently dysregulated ebv infection. accordingly, targeting t-cells and/or b-cells with monoclonal antibody therapies ameliorates ms. whether ebv has a causative or pathogenic role in ms can now be addressed in relation to genetic, hormonal and other environmental influences that may affect ebv-host interactions (salvetti et al. ). functional suppression by cd + cd + regulatory t cells was also found to be impaired in ms patients (viglietta et al. ). newborns of pregnants suffering from multiple sclerosis (ms) were impaired by the disease. in case the father of the newborn was suffering from ms, no negative consequences could be documentet i.e. safe paternity characterises ms-patients. the results of mothers does not seem to have an impact on birth weight, however, ms may contribute to a reduced birth weight (hellwig et al. ) . the mothers suffering from ms are usually treated with long-term interferon (ifn) beta-therapy in spite of the pregnancy. the foetal exposure to subcutaneous interferon beta- a therapy before treatment discontinuation was at least days; most pregnancies ( / ; . %) were exposed for days. the rates of spontaneous abortion and major congenital anomalies in live births were in line with those observed in the general population (amato et al. ; sandberg-wollheim et al. ) . the in vitro susceptibility of bewo cells was increased for toxoplasma gondii following treatment with interferon gamma, interleukin and transforming growth factor -beta (barbosa et al. ), but similar consequences were not observed during pregnancy in vivo. congenital epidermolysis bullosa acquisita: 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-mediated gene transfer enterovirus infection of monocytes with antibody-dependent enhancement a glycoengineered anti-cd antibody with potent antibodydependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo treatment strategies for nodal and gastrointestinal follicular lymphoma: current status and future development lymphoproliferative disorders with early lethality in mice deficient in ctla- herceptin (trastuzumab) therapy during pregnancy: association with reversible anhydramnios neonatal lupus erythematosus. a clinical, serological and immunogenetic study with review of the literature postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab, an alpha integrin inhibitor embryo/fetal development in cynomolgus monkeys exposed to natalizumab, an alpha integrin inhibitor motavizumab, a second-generation humanized mab for the prevention of respiratory syncytial virus infection in high-risk populations treatment with tumor necrosis factor inhibitors and intravenous immunoglobulin improves live birth rates in women with recurrent spontaneous abortion prevention strategies for type diabetes mellitus: current status and future directions down-regulation of placenta growth factor by promoter hypermethylation in human lung and colon carcinoma mhc class ii tetramers containing influenza hemagglutinin and ebv ebna epitopes detect reliably specific cd + t cells in healthy volunteers high intra-uterine exposure to infliximab following maternal anti-tnf treatment during pregnancy acquired trichomegaly and symptomatic external ocular changes in patients receiving epidermal growth factor receptor inhibitors rras: a key regulator and an important prognostic biomarker in biliary atresia exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody key: cord- -fp hljbq authors: rather, shabeer ahmad; sharma, sukesh chander; mahmood, akhtar title: antibodies generated against dextransucrase exhibit potential anticariostatic properties in streptococcus mutans date: - - journal: appl microbiol biotechnol doi: . /s - - -x sha: doc_id: cord_uid: fp hljbq streptococcus mutans is a common principal causative agent of dental caries. in this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of s. mutans. the purified enzyme showed -fold enrichment, . % yield and a specific activity of . units/mg protein. purified igg fraction of the antibody showed significant affinity with the antigenic protein. immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. the growth of s. mutans was also inhibited by % in the presence of μg of igg fraction of the antibody. antibodies also impaired glucosyltransferase activity ( . %) and biofilm formation by . % in s. mutans. western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. dot blot analysis showed little reactivity with lactobacillus acidophilus and staphylococcus aureus and there was no reactivity with other bacterial strains like enterococcus faecalis, escherichia coli and salmonella typhimurium. these findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of s. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent. dental caries is globally the most prevalent disease of mankind causing demineralisation of tooth enamel by acids produced by the oral bacteria (yang et al. ; talbman and smith ) . it is a multifactorial biofilm-mediated disorder, which is initiated by dysbiosis in the biofilm complex, where certain bacteria take the dominance over others in the oral flora. it has been shown that many bacteria are involved in the genesis of caries formation at different stages. among these streptococcus mutans is the primary causative agent of dental caries (alam et al. ). s. mutans is an acidogenic and aciduric microorganism, well characterised to produce dental caries (loesche ) . it has the ability to generate large quantities of extracellular polysaccharides (dextrans) from sucrose under the action of dextransucrase (ec. . . . )/glucosyltransferases (gtfs) accompanied with its adhesion and acid-producing activities (lynch et al. ). these dextrans help in the attachment of microbe to tooth surface leading to infection (kuramitsu ) . a number of compounds such as penicillin, cationic agents (chlorhexidine), plant products (polyphenols, flavonoids, anionic agents (sodium dodecyl sulphate) and non-ionic agents (triclosan) have been used for the prevention of dental caries by inhibiting growth and adherence of these cariogenic bacteria to the tooth surface (jarvinen et al. ; chen and wang ) . but these organisms are either resistant to them (alam et al. ; bhattacharya et al. ) or the drugs exhibit side effects (craig ) . studies on the prevention of cariogenicity have also focussed on antibody production and hence vaccine development from adaptive immunity. for vaccine development, attention was paid on the purified antigens involved in the pathogenesis of dental caries for the development of potentially safer vaccines, which may reduce the viability of bacteria in the saliva, impairing the surface adhesion and inhibiting the metabolically active enzymes involved in caries formation (chen and wang ) . many surface molecules of s. mutans such as lipoteichoic acid, glucosyltransferases (gtfs), antigen a (a -kda protein antigen), antigen c (a -kda protein antigen), antigen d (a -kda protein antigen), agi/ii (a -kda protein), agiii ( -kda protein), gbp (glucan-binding protein) (kruger ) , gtfb (kim et al. ) and dna-based active vaccines, synthetic peptides and mucosal adjuvants (heat-labile enterotoxins (hlt) from vibrio cholera (lt-i) or escherichia coli (lt-ii), bupivacaine, chitosan) have attracted great attention for passive immunisation in the prevention of the dental caries (yan ; chen and wang ; fan et al. ; xu et al. ; alam et al. ) . fusion vaccines (pgja-p/vax and pgjg/gac/vax) encoding pac and glu of s. mutans were also tested in gnobiotic animals (kt et al. ) and flagellin-pac fusion protein (kf-rpac) was also tested in rats for anticaries vaccine (bao et al. ) . antibodies raised against recombinant form of substrate binding component of the phosphate uptake system (rpsts) of s. mutans have shown protective response against caries formation (ferreira et al. ) . cao et al. ( ) found no significant effect of specific s-iga antibody on caries formation. yang et al. ( ) developed the intranasal cold-adapted influenza vaccine, which was limited by the large size of the vector than s. mutans epitope, this resulted in memory immune response thus reducing the duration and intensity of exogenous antigens. among the various proteins of s. mutans, dextransucrase has an essential role in the synthesis of glucan from sucrose, thus play a crucial role in the pathogenesis of the caries (talbman and smith ) . strategies of using adaptive immunity for the generation of antibodies against various purified molecules of s. mutans have shown encouraging results related to dental caries protection, but were limited by the cross-reactive epitopes against human heart and skeleton muscle tissues as detected by indirect immunofluorescence and crossed immunoelectrophoresis (kt et al. ) . hajishengallis and michalek ( ) however reported that glucosyltransferase when tested for cross reactivity with human heart tissue showed negative results. in the present study, we have tried to evaluate the effect of anti-dextransucrase antibodies on caries formation by using purified dextransucrase as the antigen from s. mutans. the evaluation of anti-dextransucrase antibodies demonstrated that they inhibited several of the cariogenic characteristics of s. mutans, thus have the potential for development of anticaries agent. some of these results are described in this communication. the study was approved by central animal ethics committee panjab university chandigarh (iaec no. pu/iaec/s/ / ). s. mutans strains mtcc- were grown in brain heart infusion (bhi) broth, supplemented with % dextrose, % peptone, . % glucose, . % sodium hydrogen phosphate and . % nacl (ph . ) to late-exponential phase at °c. s. oralis was grown in tryptic soy agar (tsa) (himedia, mumbai, india). e. coli, s. aureus and s. typhimurium were grown in nutrient agar at °c and l. acidophilus was grown in mrs media (sisco research laboratories pvt. ltd., new mumbai, india) . all studies relating to dextransucrase were carried out using mtcc- strain of s. mutans. antigenic protein dextransucrase was purified from culture supernatant of s. mutans mtcc- by ammonium sulphate precipitation followed by sephadex g- column chromatography. the pooled fractions from column chromatography were treated with peg- . after centrifugation at , g to separate the dextransucrase fraction, the pellet was dissolved in mm sodium maleate buffer (ph . ) dialysed overnight using dialysis membrane- (himedia, mumbai, india) (liu et al. ) . the dialysate obtained served as the antigen. except otherwise stated all procedures were carried out at °c (goyal ) . concentrations of the purified proteins were detected by bradford protein assay (campion et al. ) . the purity of dextransucrase protein was assessed by sds-page (laemmli ). dextransucrase activity was assayed by using standard reaction mixture containing . m sodium maleate buffer (ph . ), . m sucrose, in total volume of . ml. after incubation for min at °c, the samples were assayed for glucose using the glucostat kit (reckon diagnostic p. ltd.). the results were expressed in enzyme units per milligram of protein. one unit of dextransucrase activity was defined as the amount of enzyme required to release μmol of glucose per min under standard assay conditions. new zealand white rabbits - months old were purchased from animal facility of indian institute of microbial technology (imtech), chandigarh, india. animals were kept in the animal facility of the department of biochemistry panjab university chandigarh under specific-pathogen-free (spf) conditions. they were housed in a metallic cage and were provided with food and water ad libitum. animal studies were performed in compliance with the guidelines of committee for the purpose of control and supervision of experiments on animals (cpcsea). six-month old new zealand white rabbits were immunised subcutaneously with purified dextransucrase protein ( mg/ml) emulsified in freund's complete adjuvant (f , sigma, usa). the injection sites were clipped and disinfected with % alcohol before injections were made. the injections of around μl each were given at four different sites in the limbs. blood sample of ml was taken before injection. serum was collected from blood by centrifugation for analysis. booster injections were given post immunisation after weeks and repeated again after weeks and weeks using the antigen emulsified with freund's incomplete adjuvant. test bleeds were taken after days of each booster and checked for serum antibody using dot blot assay. sera was collected weeks after injecting the booster immunisation dose for the analysis of dextransucrase antibody. presence of serum antibody raised against dextransucrase antibody was checked by dot blot analysis (vera-cabrera et al. ). dot blot assay was performed by spotting μl of samples slowly to minimise area onto the nitrocellulose membrane at the centre of the grid drawn by a pencil. the membrane was allowed to dry at room temperature and non-specific sites were blocked by soaking in % skim milk in phosphate buffered saline tween- (pbst) for h at °c. the membrane was washed three times in phosphate buffered saline (pbs) (nacl mm, kcl . mm, na hpo mm, kh po . mm) ph . and overnight incubated at °c in : dilution of rabbit serum in % skimmed milk. after washing three times with pbs, it was incubated with hrp-conjugated goat anti-rabbit secondary antibody diluted : for h at °c. excess secondary antibody was washed three times min each with pbs developed by enhanced chemiluminescence system (ecl) and observed under gel-doc uvtech for fluorescence. samples positive for serum showed black dots and negative showed clear dots. to localise the target protein in the cells of s. mutans, immunocytochemistry was performed by culturing bacterial cells on glass coverslips in -well culture plates (greiner bio-one india pvt. ltd.) at °c for h. after -h of growth, the media were aspirated and cells were washed gently with pbs. cells were fixed in fixation buffer ( % formaldehyde and . % glutaraldehyde) for h. after fixation, cells were rinsed twice gently with pbs and blocking solution % bsa (bovine serum albumin) was added and incubated for min. blocking solution was aspirated and cells rinsed with pbs. primary antibody raised against the target protein diluted in blocking buffer was added and incubated overnight at °c. primary antibody was aspirated from wells and washed times with pbs and fitc (fluorescein isothiocyanate)-tagged secondary antibody (sigma-aldrich, usa) diluted in blocking solution was added and incubated for h at room temperature in the dark. after incubation with secondary antibody, the cells were washed with pbs, counterstained with dapi ( , -diamidino- -phenylindole) sigma-aldrich, usa, diluted : in pbs for min and washed with distilled water. the glass coverslips were mounted using mounting medium cells-side down onto microscope slides. the coverslips were observed under confocal microscope. the experiment were repeated in triplicates. serum collected from immunised rabbits was analysed for dextransucrase-specific antibody titre by elisa. flat-bottom -well elisa plates (nest biotech co. ltd., china) were coated with μg of antigenic protein in . m bicarbonate buffer (ph ) and incubated for overnight at °c. wells were washed three times with pbs- . % tween- to remove unbound proteins and blocked in % blocking buffer for h. after washing wells with pbs- . % tween- , rabbit serum samples were serially double diluted, loaded in the wells (first well with : dilution of antibody) and incubated for h at room temperature. subsequently, the wells were washed three times with pbs- . % tween which was followed by addition of hrp (horseradish peroxidase)-conjugated goat anti-rabbit igg (dilution : ) antibody (genei, bangalore, india) and incubated for h at °c. the liquid was aspirated and wells washed three times with pbs- . % tween . tetramethyl benzidine (tmb) solution μl/well was added for - min followed by addition of tmb stop solution (conc. h so ) μl to each well. absorbance was read at nm using microplate reader (synergy/hi biotek, india). the experiment was repeated three times. values of the dilution giving an o.d reading that was two times of background were determined as the titre. igg fraction from rabbit serum was purified by affinity chromatography by using ready to use prepacked protein a-sepharose column (bio vision, usa) with a binding capacity of ≥ mg rabbit igg/ml protein a-sepharose with a flow rate of . ml/min. the column was equilibrated with pbs as binding buffer (nacl mm, in mm na-k phosphate buffer ph . ). the serum sample was diluted with binding buffer in the ratio of : volumes, mixed well and applied to the column. the flow through was collected and reapplied times to the column. the column was washed - times with the × volume of binding buffer and the antibodies were eluted with the elution buffer ( . m citric acid, ph . ). the fractions were collected and neutralised immediately by adding μl of m tris ph . per ml of eluate. protein concentration was assayed by measuring the absorbance at nm. the minimum inhibitory concentrations (mic) of serum antibody against s. mutans was determined by microdilution method as described by hasan et al. ( ) . s. mutans was grown in basal bhi medium with increasing concentrations of serum antibody ranging from to μg/ml at °c for h under aseptic conditions. the mic was the lowest concentration that inhibited the visible growth of the bacteria. however, the bacterial colonies could not be counted as the growth was essentially negligible. all the determinations were performed in triplicates. immunotritation of dextransucrase was performed using standard reaction mixture containing . m sodium maleate buffer (ph . ), . m sucrose, in total volume of . ml in presence of increasing concentrations of igg ( - μg). the reaction mixture was incubated at °c for min and assayed for glucose using the glucostat kit (reckon diagnostic p. ltd.). the results were expressed in enzyme units/mg protein. the tissue samples were placed in ice cold lysis buffer (ripa buffer sigma-aldrich, usa) (product code r ) which contained mm tris-hcl, ph . , with mm sodium chloride, . % igepal ca- (np- ), . % sodium deoxycholate, and . % sodium dodecyl sulphate and phosphatase inhibitor cocktails (sigma-aldrich, usa). the tissues were lysed by homogeniser and incubated at °c overnight and centrifuged at , g at °c for min. the supernatant was stored and quantified for protein concentration. the protein lysate was run on % sds-page and transferred on to nitrocellulose membrane. western blot analysis was done following the method described by mahmood et al. ( ) . primary antibody was diluted in the blocking buffer in the ratio of : (v/v). hrp-conjugated goat anti-rabbit igg (dilution : v/v) was used as the secondary antibody. the membrane was developed by enhanced chemiluminescence (ecl) and the bands were analysed by imagej software for densitometry. to examine the inhibitory effect of serum antibody on biofilm formation by s. mutans, biofilm microplate assay was performed by standard protocol (merritt et al. ) . s. mutans were grown in bhi (brain heart infusion) supplemented with % sucrose with a concentration of - × cfu/ml or culture optical density adjusted to . at nm (od ) in -well microtitre plates precoated with saliva to mimic the conditions of oral cavity. sub mic of serum antibody were added and wells without serum igg served as control. after incubation at °c for h, the planktonic cells were decanted and adherent cells were stained with . % crystal violet solution for min. the wells were washed with pbs twice, dried and the bound dye was extracted by adding % acetic acid to completely destain the wells. biofilm formation was measured by measuring the absorbance of resuspended solution at nm on a biotek microplate reader. cross reactivity of dextransucrase antibody with mammalian tissues was evaluated by western blot analysis. proteins from different tissues such as the liver, lungs, spleen, heart, kidney and gall bladder of rat, mice, rabbit and human were resolved on % sds-page. samples containing μg of protein mixed with loading dye and heated for min were loaded in each well and the molecular weight marker was run in a separate lane so as to identify the band of interest during western blotting. resolved unstained proteins were transferred onto nitrocellulose membrane and detected as described above. cross reactivity of anti-dextransuccrase antibody was evaluated by using dot blot method. the cell extracts of various bacterial strains were spotted on the nitrocellulose membrane, allowed to dry and placed in % skimmed milk for h to block non-specific sites. after blocking, the membrane was incubated in serum antibody (dilution : ) raised against dextransucrase from s. mutans for h at °c followed by incubation in hrp-conjugated goat anti-rabbit secondary antibody diluted : for h at °c. the affinity of serum antibody was observed by developing the membrane by chemiluminescence using gel-doc uvtech. transferase activity of dextransucrase enzyme was assayed by modified method of mukasa et al. ( ) . an adequate preparation of enzyme solution containing . m phosphate buffer (ph . ), . mm sucrose, . μm dextran and . % sodium azide with and without purified anti-dextransucrase antibody was incubated for h at °c. the mixture was centrifuged at , g for min to collect water-insoluble glucan formed. the water soluble glucan was precipitated with % ethanol and collected by centrifugation at , g. the glucans formation was determined by phenol sulphuric acid method (dubois et al. ) in which glucose is dehydrated to hydroxyl methyl furfural which forms a yellow brown coloured product with phenol with an absorption maxima at nm. enzyme activity was expressed in units/mg protein. one unit of glucosyltransferase activity was defined as the amount of the enzyme catalysing the transfer of μmole of the glucose to glucan per min. purification of the antigenic protein dextransucrase from s. mutans the culture supernatant of s. mutans grown at °c for h was used as protein source. after % ammonium sulphate precipitation, the dextransuccrase activity was enriched by -fold with % recovery. enzyme preparation after fractionation on sephadrexg- chromatography followed by treatment with peg- the dextransucrase activity was purified -fold with recovery of . %. the analysis of the enzyme purification on sds-page using % gel showed that the purified enzyme after peg- fractionation gave the single protein band corresponding to kda as shown in fig. . antibodies were raised against the purified dextransucrase by injecting to rabbits subcutaneously as mentioned in material and methods. the serum collected from rabbit after booster immunisation was screened for the presence of the dextransucrase antibody by dot blot analysis and confocal microscopy. three spots of purified protein, culture supernatant of s. mutans and bsa respectively were made on the nitrocellulose membrane and incubated with the dextransucrase antibody. the antigen antibody complex was detected using hrp-conjugated secondary goat anti-rabbit antibody. as shown in fig. a , dextransucrase antibody showed strong affinity with the purified protein, culture supernatant of s. mutans demonstrating presence of antibody generated against dextransucrase. however, no reactivity was seen with the bsa which served as the control. to further validate the generation of antibody against dextransucrase, immunofluorescence by confocal microscopy of s. mutans cells for the affinity of the target protein was carried out. as shown in fig. b , green fluorescence of s. mutans cells depicted significant immunoreactivity of the antibody against dextransucrase thus confirming the generation of specific antibody against antigenic protein dextransucrase. the antibody titre of serum from immunised rabbit with dextransucrase from s. mutans was determined using elisa. antigenic protein dextransucrase of μg coated to wells of -well plate and reacted with different dilutions of antidextransucrase serum. wells with no coating of antigen served as blank, wells treated only with primary antibody but no secondary antibody, served as primary control and wells treated with secondary antibody only served as secondary control. the linear regression was used to calculate r that is linear correlation between serum dilution and od. the results of elisa as shown in fig. c demonstrated that the affinity decreased with dilution. the affinity of antisera was detected at a dilution of : and the non-coated wells showed estimate of similar to cut-off i.e. negative results. purification and characterisation of igg serum igg fraction was purified from immunised rabbit serum using affinity protein a-sepharose column (bio vision milpitas, usa). igg was detected only in first four fractions collected in affinity column chromatography by measuring the absorbance at nm using uvspectrophotometer which had concentrations of . mg/ml, . mg/ml, . mg/ml and . mg/ml respectively. the fractions containing igg were analysed on % sds-page which showed two clear bands of igg having molecular weight of kda and kda against a prestained molecular weight marker of kda (pink plus gene direx) fig. a . the fractions containing purified igg were pooled and tested by immunoblots using the samples of purified dextransucrase, culture supernatant and bsa as control. the antigen antibody complex was detected by using hrp-conjugated secondary goat anti-rabbit antibody ( : ) dilution by enhanced chemiluminescence (ecl) system. the results are shown in fig. b . the effect of dextransucrase antibody on the growth of s. mutans was studied under in vitro conditions. s. mutans cells were grown in presence of different amounts of antibody ranging from to μg/ml for h at °c. as shown in fig. , μg/ml concentration of igg was found to be the minimum inhibitory concentration (mic) which inhibited the growth of s. mutans to nearly % of the control indicating significant antimicrobial activity. to further study the inhibitory effect of purified antibody on the enzyme activity of dextransucrase, immunotritation was evaluated. increasing concentrations of the igg from to μg/ml to enzyme protein were added and enzyme activity was assayed after min at °c. as shown in table , there was a progressive increase in the percentage inhibition from . to . as compared with the control. thus, antibody against dextransucrase exhibited inhibitory effect on the activity of dextransucrase isolated from s. mutans. cross reactivity of the antibody against various mammalian tissues was studied by western blot analysis. protein samples of the liver, heart, spleen, kidney of mice, rat and liver, kidney of rabbit were analysed by western blot analysis to evaluate the cross reactivity of purified antibody with mammalian tissues fig. a . evaluation of western blot analysis showed that the anti-dextransucrase antibody in the dilution of : did not cross react with any of the mammalian tissues tested; however, there was a strong reactivity of the antidextransucrase antibody with the s. mutans-derived dextransucrase used as control. western blot analysis of the human heart, liver and gall bladder was also tested for cross reactivity with anti-dextransucrase antibody. as shown in fig fig. growth inhibition of s. mutans by purified igg under in vitro conditions. the cell culture was treated with different amounts of igg ranging from ( - μg protein) per culture tube. bacterial growth was determined by measuring o.d at nm after -h incubation at °c. the values are mean ± sd, n = b, there was no cross reactivity with these organs but showed a strong reactivity with the s. mutans-derived dextransucrase which served as the positive control. immunoblot analysis of cell extracts from gram-positive and gram-negative bacterial strains such as s. oralis, l. acidophilus, s. aureus, e. faecalis, e. coli and s. typhimurium respectively was also studied to check the reactivity of these strains with dextransucrase antibody. as shown in fig. , there was little reactivity of antibody raised against dextransucrase with l. acidophillus and s. aureus and no reactivity with other bacterial strains confirming the specificity of the antibody against s. mutans. the anti-biofilm formation tendency of antibody raised against dextransucrase from s. mutans was investigated by crystal violet assay. the s. mutans cells were grown under in vitro conditions treated with μg purified igg. the assay was carried out in triplicates in three sets of samples as blank, treated and control in triplicates in -well plate and incubated for h at °c. as shown in fig. , the value of crystal violet assay of antibody treated sample was . as compared with control . as quantified by photometric estimation at o.d . biofilm formation in antibody treated sample was reduced by . % as compared with the control and statistically significant reduction in biofilm formation was observed at μg/ml of serum igg. dextransucrase antibody showed significant inhibition of glucosyltransferase activity in s. mutans dextransucrase have both hydrolytic and transferase activity. it catalyses the transfer of glucosyl residues from sucrose to dextrans. these results showed that enzyme activity was . units/mg protein in control which was reduced to . units/mg protein in the presence of μg of igg, indicating a decrease of . % in glucosyltransferase activity under these conditions (fig. ). s. mutans is the well-established etiological agent in the pathogenesis of dental caries. the virulence traits by which infection is established includes biofilm formation by sucrose dependent and independent means, adherence, aciduricity, the purified enzyme was treated with different concentrations of antidextransucrase igg ranging from to μg. the enzyme activity was determined after incubation of min at °c as described under materials and methods. values are mean ± sd, n = fig. assay of cross reactivity of dextransucrase antibody with various mammalian tissues (a). immunoblots of tissues samples from liver, heart, spleen and kidney of rat (i), mice (ii) and rabbit (iii). purified dextransuccrase was used as the control. the tissue protein was resolved on sds-page by using % gels, transferred onto nitrocellulose membrane and detected by western blot analysis using purified dextransucrase antibody ( : ) dilution followed by hrpconjugated secondary goat anti-rabbit antibody ( : ) dilution. the blots were developed by ecl system. b the reactivity of dextransucrase antibody with tissues proteins of the heart, liver and gall bladder from humans by immunoblot analysis. purified dextransuccrase was used as control. acidogenicity and hydrophobic interactions (hasan et al. ; ferreira et al. ) . immune intervention against dental caries has been pursued for the last many years to develop a potent and effective vaccine that may help to combat this chronic infectious disease (zhang ) . several cell surface substances of s. mutans that have been used for vaccine preparation include glucosyltransferases, adhesins and glucan-binding proteins (giasuddin et al. ) . efforts are being made to raise antibodies against various s. mutans antigens and to examine their effect on caries formation (hajishengallis and michalek ) . in the previous studies, whole cells of the mutans streptococci were used as possible vaccine for the dental caries. surface substances of s. mutans including glucosyltransferases (gtfs), lipoteichoic acid, antigen a ( -kda protein), antigen c ( -kda protein), antigen d ( -kda protein), agi/ii ( -kda protein), agiii ( -kda protein) and gbp (glucan-binding proteins (koga et al. ; kuramitsu ) play crucial role in pathogen and host interaction. kim et al. ( ) have also reported the formation monoclonal antibody against clonal fragment of glucosyltransferase b in s. mutans gs- and showed its inhibitory activity against the enzyme. thus, these surface molecules have attracted much attention for the development of vaccine against dental caries. in the present study, we generated antibodies against dextransucrase principally involved in the metabolism of sucrose which is the main substrate of s. mutans in establishing the dental caries. dextransucrase was purified and antibodies against it were raised in rabbits. the raised antibodies were checked for affinity with dextransucrase using dot blot assay which showed significant reactivity and further validated by immunofluorescence using confocal microscopy which confirmed specific binding of dextransucrase antibody with the cells of s. mutans. dextransucrase helps s. mutans in the fig. a effect of dextransucrase antibody on biofilm formation by s. mutans under in vitro conditions. the cells were treated with antibody ( μg). data are mean ± sd n = . the bar represent p value < . compared with control. blank = only culture media, positive control = s. mutans culture without dextransucrase antibody, test = s. mutans culture with dextransucrase antibody. b biofilm formation assay performed in -well plate using crystal violet staining of biofilm. fig. effect of anti-dextransucrase antibody on the glucosyltransferase activity of dextransucrase enzyme. transferase activity was determined by measuring the amount of sugar content after treating the enzyme dextransucrase with antibody. standard = assay system contained glucose with no dextran, no enzyme and no antibody. control = enzyme + dextran test = enzyme + dextran + antibody. data are mean ± sd, n = metabolism of sucrose for its growth and producing waterinsoluble glucans with mixed α- , and α- , linkages (moye et al. ) which are involved in the attachment of s. mutans on the tooth surfaces and in the biofilm formation (bowen and koo ) leading to caries formation. immunotitration of dextransucrase with purified dextransucrase antibody showed a progressive increase in percentage inhibition of enzyme activity, although the antibody titre was high, presumably due to its polyclonal nature. also it was observed that the growth of s. mutans in the presence of different concentrations of antibody under in vitro conditions was inhibited, which correlated with the inhibitory effect on dextransucrase activity; however, the values were quantitatively distinct. this is in agreement to the studies of culshaw et al. ( ) on anticaries protection after immunisation of animals with intact gtf. transferase activity of dextransucrase also showed a significant reduction in presence of anti-dextransuccrase antibody which may help in reducing glucan formation. biofilm formation proceeds with the attachment of bacterial cells to the tooth surface and subsequent formation of multilayered cell clusters (challan et al. ; wen and burne ) . the biofilm is stabilised when the bacterial cells in the three-dimensional structure grow vertically and get connected through extracellular polysaccharides (ansari et al. ) produced by the metabolism of sucrose with the action of dextransucrase. song et al. ( ) have shown that biofilm formation is strongly dependent upon the surface adhesion and hydrophobicity of the material. more recently, nilsson et al. ( ) have shown that oxidative stress plays a role in the antibiotic tolerance of s. mutans biofilm. in our study, the efficacy of dextransucrase antibody on biofilm formation was studied using crystal violet microplate assay in which cells were grown in saliva coated wells for h which demonstrated that there was . % reduction in the biofilm formation in antibody-treated samples as compared with control suggesting that the dextransucrase antibody has inhibited enzymatic activity of dextransucrase and thus disrupted the bacterial adherence and subsequent aggregation of cell clusters leading to biofilm formation. although various types of vaccines have been developed and tried for caries prevention like whole cell vaccine, conjugate vaccine, synthetic vaccine and dna vaccine etc. and showed promising results for the protection against dental caries, however, it was reported that antibodies generated showed cross reactivity with human heart tissues and skeleton muscle tissues (ayakawa et al. ) . this was further confirmed by other investigators and these immunologically cross-reactive polypeptides were found in the cell membrane of s. mutans (giasuddin et al. ) . subunit vaccines have also been tried to overcome the cross reactivity but were reported having less immunogenicity (zhang ) . it is also assumed that besides s. mutans, other oral bacteria also play a role in the pathogenesis of dental caries although the mechanism is not established. nevertheless, the inhibitory effect of dextransucrase antibodies against several cariogenic factors of s. mutans may be helpful in formulating strategies to combat the disease (alam et al. ) . present data showed that the antibodies generated did not cross react with protein extracts from the liver, heart, lungs and kidneys of mice, rat and rabbit by western blot analysis. protein extracts from the human heart, liver and gall bladder were also checked for cross reactivity with the dextransucrase antibodies by western blot analysis which revealed negative results. we further investigated the cross reactivity of dextransucrase antibody with various bacterial strains and this specificity was tested by using total protein extract of various gram-positive and gram-negative bacterial strains. protein extracts form s. mutans, s. oralis, l. acidophilus, s. aureus, e. faecalis, s. typhimurium and e. coli were evaluated for cross reactivity by immunoblot analysis and the results have shown little reactivity with l. acidophilus and s. aureus other than s. mutans and there was no reactivity with the other bacterial strains. the reactivity with the l. acidophilus and s. aureus suggests a similar epitope recognised between two strains. since the dextransucrase antibody recognised the similar epitopes on the two oral strains, it can be predicted that anti-dextransucrase antibodies raised against s. mutans could recognise other species of the oral cavity, thereby strengthens the support to dextransucrase as a candidate vaccine against dental caries and shared epitopes between cariogenic strains can show better efficacy in clinical trials. thus, the data highlights the importance of antibody against dextransucrase which has inhibitory effect on the growth of s. mutans and worked effectively against biofilm formation. our preliminary observations also indicated that dextransucrase antibodies when added to the growth media inhibited acid production and reduced hydrophobicity of s. mutans (results not shown) which further corroborated the anticariogenic properties of the dextransucrase antibodies. moreover, examination of various animal and human tissues showed no cross reactivity with dextransucrase antibody which implies that they may have no harmful physiological effects in the humans, thus may prove a promising antigen for developing anticariogenic agent. further studies to characterise the cariogenic effects of antibody against dextransucrase are underway to ascertain these findings. the study was approved by central animal ethics committee panjab university chandigarh and experiments were performed in compliance with the guidelines of committee for the purpose of control and supervision of experiments on animals (cpcsea). human tissues were obtained from the histopathology department of postgraduate institute of medical education and research chandigarh in compliance with the standards of institutional ethical committee. methods statement authors declare that all experiments and procedures performed with animals were performed in accordance with relevant guidelines and regulations of the institutional animal ethics committee of panjab university, chandigarh, india. synthetic antigen-binding fragments (fabs) against s. mutans and s. sobrinus inhibit caries formation anti-biofilm activity of a self-aggregating peptide against streptococcus mutans immunochemistry of the streptococcus mutans bht cell membrane: detection of determinants cross-reactive with human heart tissue flagellin-pac fusion protein inhibits progression of established caries inhibition of streptococcus mutans and other oral streptococci by hop biology of streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms protein quantitation and analysis of purity immunogenicity and prediction of epitopic region of antigen ag i/ii and glucosyltransferase from streptococcus mutans assessment of the roles of luxs, s-ribosyl homocysteine, and auto inducer in cell attachment during biofilm formation by listeria monocytogenes egd-e novel technologies for the prevention and treatment of dental caries: a patent survey antimicrobial resistance-danger signs all around immunogenic and protective potential of mutans streptococcal glucosyltransferase peptide constructs selected by major histocompatibility complex class ii allele binding colorimetric method for determination of sugars and related substances a dna vaccine encoding a cell-surface protein antigen of streptococcus mutans protects gnotobiotic rats from caries sublingual immunization with the phosphate-binding-protein (psts) educes oral colonization by streptococcus mutans dental caries vaccine availability: challenges for the st century current status of a mucosal vaccine against dental caries inhibition of major virulence pathways of streptococcus mutans by quercitrin and deoxynojirimycin: a synergistic approach of infection control in vitro susceptibility of streptococcus mutans to chlorhexidine and six other antimicrobial agents a monoclonal antibody specific to glucosyltransferase b of streptococcus mutans gs- and its glucosyltransferase inhibitory efficiency surface proteins of streptococcus mutans passive immunisation against oral pathogen. from the division of clinical immunology at the department of laboratory medicine and center for oral biology at novum, institute of odontology. karolinska institute dental caries vaccine -a possible option? characterization of cell-associated dextransucrase activity from glucose-grown cells of streptococcus mutans virulence factors of mutans streptococci: role of molecular genetics cleavage of structural proteins during the assembly of the head of bacteriophage t recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy microbiology of dental decay and periodontal disease. medical microbiology, th edn. university of texas medical branch at galveston cariogenicity of streptococcus mutans glucan-binding protein deletion mutants characterization of proteins in rat and human intestinal surfactant-like particles growing and analyzing static biofilms fueling the caries process: carbohydrate metabolism and gene regulation by streptococcus mutans effect of salts on waterinsoluble glucan formation by glucosyltransferase of streptococcus mutans oxidative stress response plays a role in antibiotic tolerance of streptococcus mutans biofilms effects of material properties on bacterial adhesion and biofilm formation effects of local immunization with streptococcus mutans on induction of salivary immunoglobulin a antibody and experimental dental caries in rats dot blot assay for detection of antidiacyltrehalose antibodies in tuberculosis patients functional genomics approach to identifying genes required for biofilm development by streptococcus mutans protective efficacy of a targeted anti-caries dna plasmid against cariogenic bacterial infections salivary iga enhancement strategy for development of a nasal-spray anti-caries mucosal vaccine second-generation flagellin-rpac fusion protein, kfd -rpac, shows high protective efficacy against dental caries with low potential side effects anticaries vaccine based on clinical cold-adapted influenza vaccine: a promising alternative for scientific and public-health protection against dental caries dental caries and vaccination strategy against the major cariogenic pathogen, streptococcus mutans publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements shabeer ahmad rather is a recipient of senior research fellowship from icmr new delhi. we thank to mr. naveed parvaiz of department of zoology panjab university, chandigarh, india, for providing tissue samples of mice and rat and dr. chetan for rabbit tissues samples. we appreciate the help of professor praveen rishi of department of microbiology panjab university, chandigarh, india, for providing bacterial strains s. aureus, l. acidophilus, s. typhimurium, e. coli and e. faecalis. authors' contributions s.a.r wrote the main manuscript and performed experiments, a.m contributed in concept design and/or analysis and interpretation of data and revising it critically for important intellectual content and s.c.s provided culture facilities and reviewed the article for important inputs. the article was reviewed for final submission by all authors.competing interests the authors declare that they have no conflict of interest. key: cord- -s tfvtao authors: nan title: oral abstracts date: - - journal: vox sang doi: . /vox. _ sha: doc_id: cord_uid: s tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are billion hbv-infected people including million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was / ( %), whereas that of anti-hbc-negative components was / ( %), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was %, and the rate was higher without anti-hbs( . %) and lower with anti-hbs( . %). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs < miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to . %, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since , we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every th was designated as 'blood donation day' in . to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june , agreements for blood donation had been signed with enterprises and organizations. every year more than thousand donors agree to donate on a regular basis, and so far about thousand donors have been registered in the 'registered donor system'. the campaign 'every th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~ : - platelet products are contaminated with bacteria, and septic transfusion reactions occurred in : - , transfusions. clinical awareness of the danger and a aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since , and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s- system for red cells, demonstrating in vivo recovery after days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted b-h - no abstract available. b-h - how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately documents are in the repository from around countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in . there is one standard with progressively more rigorous steps of achievement as follows: level : minimum quality and operational requirements level : intermediate quality and operational requirements level : full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in while the malawi piloting will be completed in . comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. c-h - diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcΥr and c receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: ( ) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. ( ) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? ( ) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since , at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. d-h - distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in , the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt , to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt as a priority for action in improving quality management and haemovigilance. d-h - the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa- to - and - , which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa- system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa- system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa- and - , respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in , during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in , and the first workshop of the piwp-ar was organized in taipei in . in may , the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of cases that were diagnosed and studied as naitp ( ), ptr ( ) and ptp ( ) in asian platelet immunology laboratories were reviewed. of the cases, were japanese in japan, were chinese in china, were in india, were chinese in taiwan, china, were in south korea, were in thailand, were in kuwait, and case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa- (in kuwait), of anti-hpa- ( in china and in japan), of anti-hpa- (three in japan, one in china and one in taiwan, china), of anti-hpa- (all in japan), of anti-hpa- ( in china and in japan), of anti-hpa- new(antihit a )(in japan), of anti-hpa- (in japan), of anti-hpa- bw (in japan), of anti-hla( in china, in japan, in korea, in thailand, in taiwan, china and in india), of anti-a (in japan) as well as cases of anti-cd (nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities ( in china and in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa- is mainly found in japanese individuals, while anti-cd (nak a ) isoantibody that occurred in cd type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa- antibodies were found in asian population (all in kuwait), while anti-hpa- is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd deficiency is - %. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa- to and hpa- . the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa- , - , - , and - are common among caucasians while hpa- b is extremely rare. in the chinese however, hpa- b is extremely rare while uncommon in malays. hpa- b and hpa- bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa- b is the most common cause for nait in the asian population as compared to alloanti-hpa- a among caucasians. several cases of nait due to anti-hpa- bw have also been reported in asia. in contrast, nait due to anti-hpa- b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes a-s - setting up a patient blood management programme wood e , engelbrecht s , and robinson k transfusion research unit, monash university, melbourne, australia australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial h. massive blood transfusion is generally defined as the replacement bytransfusion of more than units of red cells over h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this ( %) patients received rh d positive platelet transfusions. in ( %) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was . years and included males and females. average number of rh d positive platelets transfused per patient was . units and total number of platelet units transfused was . anti d was detectable in ( . %) patients post transfusion and included one male and three female patients. of the female patients one was a year old child who received units of random donor platelets and second a year old women who presented in the emergency department as a case of trauma. the third female was year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of blood components were issued during the study period. transfusion feedback form were received for ( . %) blood components, as follows: prbc- ( %), platelets- ( . %), ffp - ( . %). patient identification number /wristband check was not done in ( . %) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in ( . %) cases. cross checking of component unit with request form was missed in ( . ) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in ( . %) episodes, monitoring of pulse in ( . %) episodes and patient's temperature was monitored in ( %) episodes. signature of nurse was missing in ( . %) and that of medical officer in ( %) of the form. adverse transfusion reaction was documented in / forms, whereas the transfusion reactions notified at the blood bank during the same period were . of ( . %) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was min for rbc components, min for platelets and min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = . ). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = ), hpa and hna (n = ) and high frequency antigens (hfa, n = , ) within years. specificities are grouped into single multiplex reactions (mpx, n = ) with up to snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached % for k/k, . for kp a . % for jkand . % for fy a/b/x/ . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel* . (r g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g r)null. serology for js a/b of some few kel* . positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were , and , respectively. call failures (no result) were observed in less than % of all mpxs. genotyping for rhd, rhce ( mpx), gypa and b (mnss, mpx) on more than samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately % again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna ( mpx) showed expected results among samples with the exception of hna- a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping ( mpx), currently, more than , blood donors were analyzed, and delivered: kk, kp(a+bÀ), js(a+bÀ), kel +kel +, lu(a+bÀ), lu +lu À, yt(aÀb+), co(aÀb+), kn (aÀb+), lw(a+b+), and > others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aÀb+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the - tcc ( s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about % of the total cost for serological methods, and about % for molecular methods. the cost of labour is about % for serological methods and < % for molecular. equipment cost contributes to < % of the cost using serological methods and about % using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman â snp genotyping assays in -well plates at ll final reaction volume, on a lightcy-clerii real-time instrument. identification of the cc polymorphism was by probes targeted to the c>g and t>c of the rhce allele while probes targeted to c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with of the ( %) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee ( / , . %), fya-/fyb+ ( / , . %), ss ( / , . %). the co allele responsible for co b was identified in the heterozygous state in four of evaluable samples giving an allele frequency of . % in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd . compared to usd . for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october , in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected donors aged < years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table . table : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of : according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy c> t. the results were confirmed in . % of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a -> log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, % plasma/ % intersol, or pbs. each sample ( . ml) was treated with lm amotosalen and j/cm uva (n = ). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample ( . ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from to bp was evaluated over cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in % plasma/ % inter-sol ( ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in . log to > . log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > % plasma > % plasma. exposure of platelets to amotosalen and uva showed an average of . log to . log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day- post-collection (n = ). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at °c with agitation and samples were taken on day and post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, % v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; mg/ml) for h at °c/ % co . protein secretion was inhibited using brefeldin ( lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd + /cd + ) and neutrophils (cd + / cd + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for days significantly increased monocyte mip- b expression ( . -fold; p = . ), whereas exposure to day platelets did not result in any significant differences in mip- b expression. as expected, lps stimulation significantly increased monocyte production of both il- ( . -to . -fold) and mip- b ( . -to . -fold). however, lps-induced monocyte il- production was significantly reduced by exposure to prt-treated or untreated platelets stored for days (prt-treated: . -fold, untreated: . -fold; p < . ) and days (prt-treated: -fold, untreated: . -fold; p < . ). furthermore, il- production was significantly lower following exposure to day prt-treated platelets compared to untreated platelets (p = . ); however there was no significant difference following exposure to day platelets. lps-induced mip- b production was not significantly differentfollowing exposure to either day or day prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip- , mcp- , mip- a, il- a, il- b, il- , il- , il- , or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for days results in significant changes in the il- production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of samples ofclinical patients and samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega software. moreover, spss . software was applied to compare the difference between these two groups. results: of clinical patients, hcv genotype a, b, a, a, b, a and n was ( . %), ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %), respectively. of blood donors, hcv genotype a, b, a, a, b, a and n was ( . %), ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %), respectively. the proportion of hcv b was higher in clinical patients than in blood donors(v = . , p = . e- ), while the proportion of a and a subtypes were higher in blood donors than in clinical patients(v = . , p = . ; v = . , p = . e- ). one possible cause was the transmission modes varied with different hcv subtypes. hcv b is more related with blood transfusion while a and a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from , the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about years and hcv a was transmitted into guangdong later than b. conclusions: in guangdong province, hcv b and a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv b, a and a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in - . in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were cases in - . among them, patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of patients ( , , , , patients in , , , , , respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells b-pl the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past years has seen the discovery of five new blood groups. at the isbt meeting in , fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd -deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg and abcb respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt . this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim . the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd whose function in complement regulation is well-understood, resulted in production of an alloanti-cd , demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. c-s - teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in , an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with % of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h n ) virus in , isolation of novel bat-associated reoviruses in , emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h n ) virus is evolving, and there has been a recent report of human infection with avian influenza a(h n ) virus as well. additionally, there are imported eids such as influenza a(h n ) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. % of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in to designate june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv- . this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from . % to . % in the general population, but is relatively low in blood donor population ( . %). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from < to iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high ( , - , cp/ml and , - , , iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may to december , donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was . days (table) . eleven ( %) of these were male donors, and the median age was years (range - years). the pcr for hav rna was positive in all of depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of blood components (rbc units, pc units and ffp units) were delivered to hospitals. twenty six products were transfused to patients, and the rest blood components ( rbcs, pc, and ffps) were recalled immediately and discarded. twelve recipients ( %) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were and years old developed symptoms on and days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were s. in korea, most people under years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. c-s - cable rt , pistorius c , andersson m , maponga t , lopez t , preiser w and tedder r hav seroprevalence was highest in the black donors ( %), lower in coloured donors ( %) and lowest white donors ( %). all were hav igm negative. there was an age-related increase from . % ( / ) in those - years old to % ( / ) in those > years. although no active hev infection was identified by pcr on pooled samples, % of donors were hev igg positive. rates were highest in whites at %, followed by coloured at % and lowest in black at %. again prevalence increased with age from . % in those - years to . % in those > years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t c and t c) and prec/bcp region (mutations including a t/g a, g a, t a, c t, t a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of samples ( . %)were subgenotyped successfully. the predominant subgenotypes were b , c , d and a which accounted for . %, . %, . %, and . % respectively. deletions and mutations (t c and t c) in pres region were found in ( / , . %) samples. five of these samples ( . % of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < . ). mutations in polymerase region were found in samples ( %, / ), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in samples ( . %, / ). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = . ). more a t/g a mutations were found in c than b genotype while the opposite was observed for g a mutation (p's < . ). conclusions: subgenotype b was the most frequent strain circulating in hbv infected chinese blood donors, followed by c . hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a t/g a mutations was higher in genotype c than in genotype b while the opposite is the case for g a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. c-s - a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least % of tested products meet or exceed the required platelet count. in practice, this means that up to % of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day , , , , and . assay of mp was by flow cytometry using mab to label cd a, cd , cd , and cd e. thrombin generation (tg) and cd b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day . results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day , red cell mp (rmp) were increased by . at day , and by . by day . small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally < % as many as rmp. levels of pmp rose rapidly from day and peaked at day . lmp began rising at days, increasing to . at day and . at day . emp changed little over days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was < % as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets ( - , per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd b expression increased from day to day in both lr and nlr pc, but in lr pc, it was only - % magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd ligand (scd l, scd ) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd l. method: the pmps were isolated by centrifugation of the platelet-free plasma from apheresis platelet concentrates (a-plts) at , g for h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd l during days storage (correlation was significant at the . level); pmn respiratory burst are declined by removing pmp with . lm pvdf membrane filtration or depletion pmp with cd monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from -day stored a-plts compared with -day stored a-plts (p < . ). conclusion: platelet-derived microparticles carry concentrated cd l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of whole blood donors and platelet donors with vary donation history from to times (mean at . ae . ) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g : - time, g : - times and g : > times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g : . ae . , g : . ae . and g : . ae . ng/ml respectly, p = . ) and mean platelet volume (mpv) was decrease (g : . ae . , g : . ae . and g : . ae . fl respectly, p = . ) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = . , p < . ). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either -or -days old; n = each) were pooled and equally split to obtain matched pairs (day ). one unit was washed with . % saline by repeated centrifugation then resuspended in ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce units of paediatric-sized washed or unwashed rccs. all units were stored at - °c and samples were taken on days , , , , and of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < . considered significant. results: the washing process resulted in reductions in red cell count ( . %), haemoglobin ( . %) and haematocrit ( . %) compared to unwashed rccs. overall, washing and subsequent storage of -and -day old rccs significantly reduced the ph (p < . ), lactate production rate (p < . ), and , -diphosphoglycerate concentration (p = . ). although the atp content of the rcc decreased during storage, it was not changed by washing (p = . ). haemolysis in the rccs was increased by the washing process, but remained < . % on day for all products. extracellular potassium was significantly reduced by washing (p < . ), but increased during storage in both washed and unwashed red cells (p < . for both). washing significantly reduced the number of microparticles in the supernatant of both -and -day-old rcc compared to unwashed rccs (p = . and . respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both -and -day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein- , scd p, rantes, anaphylatoxins (c a, c a, and c a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < . for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. d-s - burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically - or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h n and h n virus can cause acute community acquired pneumonia with to % mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than days after symptom onset. during the pandemic of h n influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august to october , , a total of persons were successfully contacted. a total of screening and whole blood donation appointments were made. in the former ( . %) attended screening but only could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, ( . %) had attended and donated. . l ( units) and l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited patients with severe h n infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients ( . %) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group ( . % vs . %; p = . ). convalescent plasma treatment was associated with significantly lower day , , and viral load, compared with the control group (p < . ) and corresponding lower serum levels of il , il , and tnf (p < . ). between and , patients were randomized to receive h-ivig ( patients) or ivig ( patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day and posttreatment viral load when compared to the control (p = . and p = . respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level days after treatment. subgroup multivariate analysis of the patients who received treatment within days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or: . , % ci, . - . ; p = . ]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of . higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b (tgf-b ) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw . mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase- (cox- ). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw . cells were grown in medium supplemented with % of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps ( ng/ ml added after h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox- in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b content ranging from approximately - ng/ml, and ppp the lowest ( - ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox- detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox- synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox- synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. d-s - tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of : ~ individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists rare blood phenotypes that are divided in two categories as shown in table . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since , our blood center has established moabs ( human and murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk- blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than , donors with rare blood phenotypes (category i, ; category ii, ) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since , a total of units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. d-s - the china national rare blood group screening program started in . the program has screened more than , , donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was / in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, . the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent years, units ( unit = ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt ). five cases were reported by daniels et al, (transfusion, ; : ) and two by reid et al, (transfusion ; (suppl) : s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a -year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was . g/dl, another two rbc bags were transfused, and his vital signs became stable. on day , he was transfused one rbc bag without a hemolytic transfusion reaction. on day , after receiving ml of rbcs, the patient vomited and had cola-colored urine (t-bil . mg/dl, ldh u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: + on day , + on day and negative on day . thereafter his anemia improved gradually by iron medication and he was discharged on day . aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at c ( +), by albumin iat ( +), peg-iat ( +), and papain-iat ( +) with all panel cells, and by peg-iat with samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg and igg with a titer of by peg-iat before transfusion rising to on day ; his serum demonstrated hemolysis after day . no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. d-s - background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. ( ) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program ( ) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. ( ) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of . we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a % increase in blood demand from year to with almost % blood being transfused to patients aged > . therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years - . statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of , donations made by , male and , female donors were analyzed. on average . % of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center ( . % vs . %, p < . ). male were more likely to come back for repeated donations than female ( . % vs . %, p < . ). a total of , donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by july . by the end of year , donations (consisting of males and females) were made with more than . % collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in , , donations were collected which already exceeded the planned collection target of , by year . interestingly, same . % donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a -prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about % of people visiting that vicinity were youths aged to ( . %) and young adults aged to ( . %). the main purposes of the visit were for work related activities ( %), attending school ( . %) and shopping ( . %). on weekends, % of these age groups visited the vicinity for leisure activities or to church. another % who visited there were adults age - years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of units of blood a day within the months of its operation (jan ); and now, has a daily average collection of units of blood. a-s - setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of . billion people, blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july , with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december . terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february and till date reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. a-s - background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in . in addition, the allelic frequency of hpdel was calculated to be . by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between and , in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of , patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january and december , were examined. healthy blood donors: a total of , blood donors who visited the japanese red cross blood centers in the tokyo area between june and august were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of mg/dl followed by simplified sandwich elisa with the detection limit of lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the , patients who had experienced nhtrs ( . %). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, ; ffp, ; rbcs, ; mixed, ] . nineteen of them ( %) experienced anaphylactic trs accompanied by severe hypotension and the other patients ( %) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in patients ( %). in addition to the igg antibody, the anti-hp ige antibody was detected in patients ( %). hpdeficient individuals were identified among japanese blood donors with a prevalence of / , ( . %). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp . the anti-hp antibody was not detected in / ( %) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < . ), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion labora- methods: a study on all reports of mislabeled and miscollected specimens from january to december was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during - was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january and december a total of , samples were received for abo grouping. of incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was . % or in samples. of cases resulted from wrong labeling during collection, cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in incidents, labeling was performed away from the bedside, in cases name labels of a different patient were found in the correct patient's medical file, in cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest % had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in under the ministry of health (moh). since its inception in the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january till december were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: ( ) incident reporting ( ) root cause analysis ( ) identification of corrective actions results: during the study period , requests for blood and blood components were received and total of , , blood components were issued within the hospital. a total of , reported errors were analysed. pre-analytical errors comprised a large majority ( , ; . %), most of them being errors of inadequate patient information on request forms ( ; . %) followed by sampling errors ( ; . %). analytical errors comprised ( ; . %) and post analytical errors accounted for ( ; . %) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is % self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from to in the last four financial yearsa % reduction. in addition, order fulfilment has improved from % to %, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. a-s - wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in . it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last years. evidently, blood shortages are largely preventable by adopting a proactive approach. a-s - . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from . in year to . in year . the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology ( . reduced to . ), surgical ( . reduced to . ), orthopedic ( . reduced to . ) and neurology ( . reduced to . ). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. a-s - fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to . and specialized services to . million population) reduced the regional rc inventory by % ( rcs to rcs; optimal hospital inventory index of . ). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at % (average shelf-life of transfused rcs reduced from . to . weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump ( and ) included in table . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by % (average). chronically transfused hematology and renal patients showed the highest reduction on rcu ( - %, p value < . ). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction ( %) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. a-s - no abstract available. a-s - burnouf t , tzeng ys , deng sc , wang ch , tsai jc and chen tm taipei medical university, taipei, taiwan tri-service general hospital, taipei, taiwan tatung university, taipei, taiwan background: approximately % of diabetic patients develop chronic ulcers, and % of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [ ]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged - with nine non-healing lower extremity ulcers (median size of cm ; range - cm ) were enrolled. their median duration of diabetes and ulcer was . years (range - years) and . months (range - months). none of the patients had received conventional skin grafting in the past. prp was prepared from ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating ml of plasma (tgd- ; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after to days with equal volumes ( to ml) of autologous prp and autologous thrombin to form the platelet gel within - s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well ( % healing rate). the interval between skin graft and complete wound healing ranged from to weeks in the eight successful cases. no ulcer recurrence was noted during the - months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated ( °c/ min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over bovine corneas (bcecs) using standard procedures and grown in a dmem-f medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) % fbs, . % dmso, ng/ml rhu-egf, lg/ml insulin, lg/ml transferrin, and nm cholera toxin (termed ' % fbs medium'), or with (ii) . %, %, . %, or % pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in , or -well plates at °c in a controlled atmosphere containing % co , with medium changes every two days. viable cells were counted for days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin , anti-na/k atpase alpha- subunit, anti-zo- and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained - mg/ml total proteins, and a range of approximately - . , . - , . - . , and . - . ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with . - % pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the ' % fbs medium' could be achieved only using % hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f medium supplemented with % hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor and rantes) and cytokines (e.g. interleukin- b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd / cd l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd + t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd + t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor (pf ) and the erythrocyte duffy-antigen molecule, which binds pf and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. c-s - graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor (pf ) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september . only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf /heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the ' t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. c-s - autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd + t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd + t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp + regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. c-s - seguchi s , maeda t , kanaumi y , kawamura s , kodama m , kawai t , okazaki h and miyata s national cerebral and cardiovascular center, osaka, japan the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor (pf ), resulting in the production of anti-pf /heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august and may , patients from hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf /heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the patients, patients were ultimately diagnosed with hit ( . %). optical density values of anti-pf /heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients ( . ae . vs . ae . for igg/a/m, p < . ; . ae . vs . ae . for igg, p < . ). the incidence of thromboembolic events was significantly higher in hit patients ( . %) than that in non-hit patients ( . %; p < . ). among the hit patients, patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within h and the other after days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. c-s - lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, repeat donors were typed for hla-a, -b and hpa- ,- , - , - , - , - and - using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, female)showing platelet refractoriness to random donor platelets [ h corrected count increment (cci) < ml/m , percentage of platelet recovery (ppr) < %] before. patients phenotyped for both hla-a,b and hpa- ,- , - , - , - , - and - . apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show h ppr > %. the mean , h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion , h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in % of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. c-s - xia w , xu x , ye x , fu y , deng j , liu j , ding h , chen y , shao y , wang j , li h and santoso s guangzhou blood center, guangzhou, china department of biotechnology, guangdong food and drug vocational college, guangzhou, china he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from healthy blood donors were typed for cd deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd deficiency. transfection in mammalian cells (hek- t) with cd mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of / blood donors failed to express cd on their platelets surface. in / individuals no cd expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd deficiency (platelets and monocytes) and type ii cd deficiency (platelets only) were approximately . % and . %, respectively. nucleotide sequencing analysis of type i cd deficient individuals revealed eight different mutations; four of them were not described so far. however, - del attgtgcctatt and - delac appeared to be the most common mutations related to type i cd deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor ) as well as in three cases of ptr (patients - ) and one case of fnait (patient ). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd cell lines. in all ptr patients, transfusion with platelets derived from cd negative donors resulted in good increment ( h, ppr > %). table shows the mutations found in these five gpiv defective individuals. conclusions: more than . % of cd type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. c-s - do we really need ffp? the evolving role of pf and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within h of collection (fp ) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp . in addition, the activity profiles of coagulation proteins in fp prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp , if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp , similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from h to up to days, if stored at °c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. c-s - background: in , the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of % of clinical plasma sourced from male only donors. methods: work began in early to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july showed that the blood service were issuing . % male clinical plasma; the group ab rate was . %. the results improved dramatically by november in groups o, a and b ( . %, . % and . % respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march , the results in group ab had improved to . % (chart below) and the directive was given to manufacture male clinical plasma only as routine. january was the first month ever where % of all clinical plasma was sourced from male donors only. in / , . % of clinical plasma was male onlythe . % constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by %, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide ( th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after days of storage at - °c. the new t&b production method for the separation of -ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. c-s - blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is %. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of - mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. c-s - wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p antigen) at nbc or at blood mobiles organized by nbc from november until july were retrieved and analyzed using spss . . results: a total of donors were nat detected and negative for anti-hiv and p antigen (none in , in , in , in , in , in and in introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. - years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar ). multi-stage random cluster sampling approach was followed and college and university students were targeted through university based blood donor organisations (bdos) out of a total bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version . results: a majority ( %) of the students had heard about blood donation through family/friends and a minority ( %) through the internet, although % preferred internet as spare time activity. majority (+ %) of the students had access to the internet and mobile phone. more than % of the respondents had donated blood: % donated for family, relatives or friends, % donated voluntarily as an act of altruism, and % donated voluntarily once and then stopped donating, but % of these respondents still considered themselves as volunteer blood donors. % indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people ( %), % followed friends invitations, in % cases respondents family or friends had received blood transfusions, % followed the example of fellow students. restriction for blood donation: % generally feared donation, % had a specific fear of the needle, % had no confidence in the public (health) sector, % condemned blood selling practice, % had no confidence in the donation procedure (hygiene), % experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. d-s - murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. d-s - managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that - % of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly % of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 's, most operations were performed to correct congenital heart disease. during the 's and 's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is - % and post bypass is > %) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. d-s - the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna- to . hna- , - , and - a alloantibodies have been implicated in the pathogenesis of trali, and especially hna- a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna- , - and - , those against hna- a and hna- a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna- a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna- , - , - , and - ). more recently, the molecular basis of hna- antigen has been elucidated, and stable cell lines expressing hna- antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna- antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. d-s - one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about % of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. d-s - when seven sera from donors were screened for neutrophil specific antibodies, % samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in . % male,~ . % nulliparous and~ . % parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn , also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn , also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein , mzf , is a transcription factor that trans-activates both ccn and ccn , in distinct cell types. for example, we have shown that mzf can stimulate ccn production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf also regulates the synthesis of ccn in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn in stromal fibroblasts, which in turn results in enhanced loading of ccn into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn expression, through mzf- , and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. d-s - background: foxp + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd + cd À t cells were purified from splenocytes of d mice and stimulated by anti-cd /cd in the presence of tgf-b for days, which were termed 'itreg'. and tdcs derived from bone marrow of d mice were induced by gm-csf, il- and tgf-b and harvested after -day cultivation. then, itregs were expanded by tdcs at the ratio : and collected after days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd t proliferation, induction of foxp + tregs from foxp À t cells and suppression of th cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d mice after immunized with bovine type ii collagen (cii) on day and day , termed collagen-induced arthritis (cia). and itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd + th subsets were analyzed. results: after -day induction in vitro, the percent of foxp + itregs increased from . ae . % to . ae . %. and these itregs could be expanded effectively by tdc in additional -day co-incubation, which continued to express higher levers of foxp ( ae . %) and increased . ae . fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd t cells converting to foxp + itreg and resistant to th cell differentiation more effectively. during the -weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il- , il- and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd + foxp +) and a decrease in the percentage of th cells (cd + il- +) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may the world health assembly passed a resolution, wha . , on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in . blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. a-s - background: blood transfusion service of nepal red cross society was established in the year . since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of centers in districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than , units of blood were served to the needy and around % of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. a-s - background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over million living on islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of . %, human immunodeficiency virus (hiv) of . %, and hepatitis c virus (hcv) . %. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of . million donations determined that id-nat could prevent infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $ million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $ million per year (or~ % savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that whole blood units andup to , blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $ million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. a-s - yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur - days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and out of may suffer from severe infection with significant neurological sequelae. in , transfusion-transmitted wnv infections were described. since , all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, transfusion-associated transmissions of wnv have been documented (mmwr ) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that million dengue infections occur annually and approximately . billion people live in dengue endemic countries (who ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be . - / , blood transfusions in the year , assuming a ration of asymptomatic-to-symptomatic infections of : to : . subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in . a-s - australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b , hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv- ), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~ agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between and through to its full implementation across the acute hospitals in oxfordshire in / , and its maintenance and further development since then. there was an improvement from . % to % of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of min to s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last years there have been substantial efforts to minimise or eliminate trali. despite these activities, in the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over , transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. blood) . that hna- a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna- ,- , and - were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna- a, - b, - c were . , . and . , respectively. hna- null allele was found in one sample. the gene frequencies of hna- a and - b were . and . , respectively. hna- a and - b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between to days, and about days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having ≥ symptoms of any kind. we calculated odds ratios (or) and % confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaÀ patients. results: of patients with samples and interviews - days following transfusion % were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaÀ recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. th we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were % and %, respectively. nobody has ever been bitten by kissing bug. % of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna- , - , and - ) were capable to induce trali reaction. among them, hla class ii and hna- a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from nulliparous females, parous females and male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna- and - were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, ; werth et al, ). hna- antibodies were analyzed by antigen capture assay (bayat et al, ). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of donors ( males and females) showed higher prevalence of hla antibodies production in female compared with male donors ( . % vs . %). we found in out of females donors alloantibodies against hla class i ( . %) and hla class ii ( . %). analysis among female additional immunosuppression may be used in patients with low adamts due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in , and expanded in to include all tma. of hospitals currently participate in the registry. of cases in patients were registered from sites; eight were excluded for incomplete data, leaving episodes in patients. results: median age was years ( - years), % were female, % were relapses. of % were idiopathic and % had ≥ identified precipitant, including infection %, autoimmune %, malignancy %, medication %, pregnant/postpartum %, solid organ transplant %, and allograft %. adamts levels were available in cases ( idiopathic and secondary). adamts was < % in / ( %) idiopathic and / ( %) secondary cases. precipitants in cases with adamts < % included infection (n = ), autoimmune (n = ), medication (n = ), and pregnancy (n = ). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in cases (table ) . of / ( %) received pex, by centrifugation in / , membrane filtration in / ( %), and unknown method in / ( %). plasma infusions were used in / cases (without pex in ). data on additional therapy was available for cases (table ) . rituximab was given in / cases with ≥ precipitant, including infection (n = ), non-haematological malignancy (n = ), haematological malignancy (n = ), autoimmune (n = ), medication (n = ), and pregnancy (n = ). death rates were lower in idiopathic than in secondary cases ( % vs %, p = . ). conclusion: our data confirm that very low adamts does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (< months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as > ml/kg of blood components during the surgery. analysis was done using spss software version . median levels were compared between mt and non-mt group using mann whitney test. results: between august to july , pediatric ldlt were performed in a single center in south india. thirty five ( . %) of them were females and ( . %) males; ( %) of the recipients blood group were o positive, ( %) b, ( %) a and ( %) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in , metabolic /hereditary liver disease in , hepatic tumors in and acute liver failure in recipients. of ( . %) patients received prc, ( %) ffp, ( . %) plt and ( . %) cryoprecipitate. sixteen ( . %) patients received massive transfusion (mt) with a median peld score of (À to ) compared to (À to ) recipients without mt (p < . ). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb ( . vs . mg/dl), platelet count ( vs per mm ), fibrinogen ( vs mg/dl), and a higher bilirubin ( . vs . g/dl). transfusion requirements of ffp was higher in acute liver failure ( ae . ml/kg) compared to metabolic liver disease ( . ae . ml/kg) and biliary atresia ( . ae . ml/kg); (p = . ). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. d-s - distler pb , slaper-cortenbach i and ashford p iccbba, san bernardino, united states of america university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:( ) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').( ) hyphenated class names will be replaced ( ) tc and therapeutic terminology will be replaced ( ) modifiers will be replaced with attributes providing the same information ( ) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes. d-s - kordelas l , rebmann v , ludwig a-k , radtke s , beelen dw , giebel b and horn p department of bone marrow transplantation, university hospital essen, essen, germany institute for transfusion medicine, university hospital essen, essen, germany university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a -years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il- , il- and il- was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord- - vlz dn authors: dimitrov, dimiter s. title: human monoclonal antibodies against hiv and emerging viruses date: journal: national institute of allergy and infectious diseases, nih doi: . / - - - - _ sha: doc_id: cord_uid: vlz dn nan human monoclonal antibodies against hiv and emerging viruses dimiter s. dimitrov throughout the centuries, viral diseases have killed hundreds of millions of people and continue to do so. more than million died in the past from smallpox caused by the variola virus. influenza killed about million people during the pandemic. more than million have been infected with hiv since the first cases of the epidemic were reported in , and more than million have died from aids. although vaccines remain the most cost-effective way to prevent viral infections, in many cases (e.g., hiv) vaccines are not available and, in other cases, available vaccines have to be modified frequently to counteract virus escape (e.g., influenza). prophylaxis and treatment in such cases are critically important to fight viruses. a number of studies have shown the importance of neutralizing antibodies in recovery and protection from viral infections ( , ) . sera from humans or animals containing antibodies have been widely used for prophylaxis and therapy of viral and bacterial diseases since the late s ( ) ( ) ( ) ( ) . serum therapy of most bacterial infections was abandoned in the s after antibiotics became widely available ( ) . however, polyclonal antibody preparations have continued to be used for some toxin-mediated infectious diseases and venomous bites ( ) . serum immunoglobulin (ig) has continued to be also used for viral diseases where there are few treatments available, although mostly for prophylaxis either prior to an anticipated exposure or following an exposure to an infectious agent ( ) ( ) ( ) . antibody products licensed in the united states for prevention or treatment of viral diseases include human ig for use against hepatitis a and measles; virus-specific polyclonal human ig against cytomegalovirus, hepatitis b, rabies, respiratory syncytial virus (rsv), vaccinia, and varicella-zoster virus; and the humanized monoclonal antibody (mab) synagis ( ) . polyclonal ig has also been used with various success for diseases caused by other human viruses including parvovirus b ( ) ( ) ( ) ( ) , lassa virus ( , ) , west nile virus ( , ) , some enteroviruses ( , ) , herpes simplex virus ( ) , crimean-congo hemorrhagic fever virus ( ) , junin virus ( ) , sars-cov ( , ) , and hiv ( ) ( ) ( ) ( ) ( ) ( ) . although serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot-to-lot variation, and uncertain dosing have limited their use ( ) . mabs including chimeric animal-human, humanized, and fully human mabs (hmabs) possess lower or absent immunogenicity, toxicity, and lot-to-lot variation. the molecular mechanisms of therapeutic efficacy of such antibodies are easier to dissect, and they can be engineered to further improve their therapeutic properties. recently, some mabs have shown clinical success. the humanized mab synagis (palivizumab), which is still the only mab against a viral disease approved for clinical use by the u.s. food and drug administration, has been widely used for prevention of rsv infections in neonates and immune-compromised individuals, and very recently has been further improved ( ) . however, it is not effective for treatment of an already established infection; for example, there were no significant differences in the clinical outcomes between the placebo and the palivizumab groups for children hospitalized with rsv infection ( ) ; in addition, resistance can develop relatively quickly: a recent study found f gene-resistant mutations in an animal model of the rsv infection (cotton rat) weeks after infection including a completely resistant virus ( ) . the development of hmabs for prophylaxis and treatment of diseases caused by hiv and emerging viruses is still in an initial stage. this chapter reviews hmabs with potential for prophylaxis and treatment of diseases caused by hiv- , sars-cov, and henipaviruses (hendra [hev]) and nipah [niv]). mostly igg are discussed unless specifically noted otherwise; other isotypes also could be useful but are not so frequently used and other formats including fabs and single-chain variable region fragments (scfvs) are noted when described. finally, possible implications for development of effective vaccines are discussed. the demand for new treatment options against hiv is becoming increasingly important as the side effects and the expansion and spread of drug-resistant virus within the infected population limit the clinical benefits provided by available anti-hiv drugs. despite the promise presented by synagis and its improved variants ( ) , developing effective antibodybased therapeutics against hiv presents an especially difficult challenge. therapies based on small molecule-based drugs eventually fail because of the expansion of a drug-resistant virus population in the infected individual. antibody-based therapies are not immune to this problem. further, hiv- replicates and spreads within the densely packed cellular environment (reaching about cells per ml) of the lymphoid tissues of the gut, spleen, and lymph nodes. antibodies may have difficulty preventing the cell-to-cell spread of virus in this seemingly impenetrable lymphoid environment. in fact, passive administration of anti-hiv antibodies as human immune plasma or polyclonal antibody preparations conferred, at best, only modest clinical impact ( , ) . unfortunately, these trials were complicated by the relative absence of highly effective concentrations of hiv-neutralizing activity in the polyclonal preparations used. preparations containing high concentrations of hmabs that exhibited potent hiv- -neutralizing activity in vitro (nhmabs), on the other hand, resulted in measurable decrease of plasma virus concentration ( ) . however, the in vivo potency of this preparation was insufficient to completely block virus replication, and resistant virus rapidly emerged. despite this discouraging result, these studies suggest that antibodies with enhanced in vivo potency could have a more profound clinical impact. a major question is whether antibody-based therapeutics can provide long-term clinical benefits for patients with established infections that are comparable to or better than drugs currently in clinical use. a specific feature of antibodies compared to other drugs is that hiv has evolved a number of strategies to escape neutralization. such evasion strategies of the virus against polyclonal antibodies elicited during infection and strategies used by the immune system to generate broadly neutralizing hmabs have been extensively reviewed ( , ( ) ( ) ( ) ( ) ( ) ( ) . thus, a short answer to this question is perhaps it is possible if we can outsmart the virus by engineering potent antibody-based therapeutics against which the virus has not yet evolved protective strategies. here, we discuss our ongoing efforts to improve the potential clinical utility of already known hmabs and identify novel, more potent antibodies against hiv. hiv entry into cells is initiated by attachment of the viral envelope glycoprotein (env) to a host cell receptor (cd ). conformational changes follow, which enable enhanced exposure of a co-receptor (typically ccr or cxcr ) binding site and binding of the viral glycoprotein gp to the co-receptor. subsequent conformational changes finally result in fusion of the viral and cell membranes. in some cases, cd is not required and the env directly interacts with a co-receptor. because the env mediates hiv entry and is the only viral surface protein exposed to the surrounding environment, it is a major target for neutralizing antibodies and a potent immunogen ( ) . env-specific antibodies are generated as early as a few weeks after productive infection or immunization. they do not typically neutralize current virus isolates but rather neutralize earlier isolates ( ) . such antibodies are isolate-specific and lack broad neutralizing activity because the virus has evolved to hide conserved epitopes and escape neutralization by a number of mechanisms. as a result, most of the antibodies generated in natural infection or immunization are non-neutralizing or neutralize few isolates. more than mabs have been reported as recognizing epitopes on gp and gp , but only a small number exhibit neutralizing activity against primary isolates from different clades, denoted as broadly cross-reactive neutralizing hmabs (bcnhmabs). using phage display or b cell immortalization, several bcnhmabs were identified from hiv-infected patients whose sera contained a high titer of such antibodies. six major classes of such antibodies relevant to the binding location and properties of their epitopes have been identified: ( ) antibodies that bind to the region containing the cd binding site (cd bs) on gp ; ( ) antibodies binding better to gp complexed with cd than to gp alone (cd i antibodies); ( ) carbohydrate-binding antibodies; ( ) gp v or v -binding antibodies; ( ) gp antibodies targeting the membrane-proximal external region (mper); and ( ) antibodies binding to other epitopes on gp . the best characterized and very potent cd bs antibody is b , a hmab selected from a phage-displayed antibody library constructed from the bone marrow of an hiv- -infected donor ( , ) . the cd binding site is masked by v /v variable loops and further shielded by n -glycan in the region. the limited accessibility of these conserved epitopes in the context of the oligomeric env could explain why most of the antibodies that are specific for the cd binding site bind weakly to env oligomers and exhibit weak to moderate neutralizing activities against primary hiv- isolates. recently, two novel nmabs (m , m ) were identified by sequential antigen panning (sap) against purified env ectodomains (gp s) of another phage-displayed antibody library derived from the bone marrow of three long-term nonprogressors with high titers of bcnhabs ( , ) . these antibodies cross-react with envs from primary isolates and exhibit differential neutralizing activity with b to isolates from different clades. the env undergoes significant conformational changes after binding to cd leading to exposure of structures that contain epitopes or portions of epitopes targeted by cd i antibodies. the best characterized cd i antibody, b, is only weakly neutralizing as igg , but its neutralizing activity increases significantly after its size is reduced to a scfv ( ) . the neutralizing activity of the most potent and broadly neutralizing cd i antibody, x ( ) , also increases in many cases with decreasing its size to scfv or fab. one should note however, that for some isolates and in some assays including assays based on spreading infection in peripheral blood mononuclear cells (pbmcs), igg x is more potent than fab x likely due to an increase in avidity. thus, an interplay between avidity and size could be important in determining the neutralizing activity of cd i antibodies. many gp -specific antibodies have been identified of which three with linear epitopes, f , e , and z , exhibit broad neutralizing activity and have been extensively characterized ( ) . they can bind peptides containing stretches of the mper of gp , which is relatively conserved. using a competitive antigen panning methodology, several new crossclade reactive anti-gp mabs have been identified (m , m , m , m , m , and m ) that neutralize a variety of primary isolates in pbmc/infectious virus-based assays and bind to conformational epitopes distinct from those of other anti-gp antibodies ( ) . there are several lines of evidence that antibodies can inhibit hiv- infections. first, it has been demonstrated that antibodies can exert selective pressure in humans suggesting that they inhibit infection ( ) . second, hiv- concentration in plasma of patents decreases following administration of hmabs ( ) . third, a triple combination ( g , f , e ) of nmabs at higher doses can delay viral rebound after cessation of antiretroviral treatment ( ) . in addition, a number of experiments have shown that antibodies can prevent infection in monkeys (see ref. for a recent review). naturally occurring antibodies with potent and broad neutralizing activity could be further improved. recently, scfv x was further improved in potency and breadth of neutralization by using the sap methodology and a library of x mutants; two scfvs, m and m , which exhibited on average two-to threefold lower ic than scfv x were identified ( ) . the scfv x mutant library was also screened by surface plasmon resonance technique on immobilized envs. one scfv was selected on the basis of its higher kinetic association rate, and its activity is being evaluated. because cd i epitopes may be available for limited time after cd binding to gp and before their interaction with a co-receptor, cd i antibodies with faster binding kinetics would bind efficiently and are likely to have better inhibitory activity against hiv- . we have been developing various other constructs including fusion proteins that are being evaluated for neutralizing activity. hiv has evolved a number of strategies to escape host immune surveillance, prominently by modifications of its env. thus, naturally occurring whole antibodies against its env may have little chance of significantly affecting viral replication and disease progression, as also evidenced by the lack of sustained significant effect in the few clinical trials that have tested such antibodies. this is mostly due to the rapid generation of resistant mutants, which remains a fundamental problem not only for antibodies but also for other antiretroviral drugs. antibodies against components of the entry machinery, engineered antibody fragments and their derivatives, and other antibodybased inhibitors that do not occur naturally and against which the virus has not developed defense mechanisms may be better able to control virus replication, although mechanisms of inhibitory escape such as generation of resistant mutants and difficult access could still be operating. the challenge is to fight these mechanisms and simultaneously ensure a relatively long half-life and biological effector functions. several directions of research appear promising in this aspect. one direction involves engineering antibody with higher binding affinity to conserved epitopes and with smaller size than the currently known antibodies. another uses the unique features of some anti-env antibodies (e.g., the mimicry of receptors) to engineer novel binding entities with high-binding affinity to many isolates. a third consists of engineering novel fusion proteins containing antibody-binding fragments. a fourth relies on using antibody effector functions, including antibody-dependent cellular cytotoxicity (adcc) and complement, to increase the efficacy in vivo . yet another direction of research aims to develop antibody-nanoparticle conjugates, or nanoliposomes in particular, that are able to irreversibly inactivate virus and cells expressing viral proteins. the development of novel approaches may also be fruitful in the future, as well as combining existing ones to develop antibody-based hiv inhibitors using other, yet undiscovered principles. whether these or other research directions will lead to clinically useful antibody-based inhibitors of hiv infections remains unknown; however, even if new engineered antibodies fail to inhibit hiv infections in a clinically useful way, there is still a basis for optimism in this endeavor because the new approaches can prove useful elsewhere. finally, one should note that all bcnhmabs have undergone a long maturation process and differ from the germline sequences by tens of mutations. one could speculate that generation of such antibodies in vivo following immunization is very unlikely because of the lack of b cells expressing surface-associated ig that is close in function to those bcnhmabs. this may represent a challenge in developing effective aids vaccines, and further studies are required to find novel approaches for elicitation of bcnhmabs in vivo . the sars-cov ( - ) caused a worldwide epidemic in and , and infected more than , humans with a fatality rate of about %. although there are no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging sars-cov or a related virus remains of high importance. sars-cov infection leads to generation of potent neutralizing antibodies that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. antibodies that neutralize the virus in in vitro assays were detected in sars-covinfected patients ( ) ( ) ( ) ( ) ( ) ( ) , and in mice ( ) , hamsters ( ) , and monkeys ( ) infected with the virus. these antibodies also protected uninfected animals from sars-cov infection, e.g., passive transfer of immune serum to naive mice prevented virus replication in the lower respiratory tract following intranasal challenge ( ) . patients infected with sars-cov were also treated with convalescent patient plasma containing polyclonal antibodies ( , ) , improvements of the antibody preparations were suggested ( ) , and batches of virus-inactivated hyperimmune globulins containing five to six times higher titers of sars-cov-specific antibodies than convalescent plasma were produced ( ) . in an amazing pace of research, several groups have recently developed hmabs to the sars-cov spike (s) glycoprotein that neutralize the virus and have potential for therapy and prophylaxis of sars (reviewed in ref. ) . recently, an improved method for epstein-barr virus transformation of human b cells has been developed based on cpg oligonucleotide (cpg ) that increases the b cell immortalization efficiency from - % to - %, and used for selection of hmabs specific for sars-cov proteins ( ) . one of the selected antibodies (s . ), which was specific for the s glycoprotein on the viral spikes, was about -fold more efficient in neutralization than convalescent serum. s . prevented the cytopathic effect of the sars-cov at ng/ml ( ) , and inhibited entry of pseudovirus with s glycoprotein from urbani isolate with about the same ic . however, it did not affect to any significant extent pseudovirus entry mediated by the gd isolate s glycoprotein and even enhanced the entry of virus pseudotyped with the s glycoprotein from the palm civet isolate sz ( ) . in a mouse model of sars-cov infection, this antibody prevented viral replication in the lower respiratory tract (at doses of and µg), and reduced it in the upper respiratory tract at the highest dose ( µg) used. unfortunately, data for the in vivo neutralizing activity of other nhmabs selected in this study ( ) , including the most potent antibody (s . ), which has a neutralizing concentration ( ng/ml) -fold lower than that of s . , have not been reported. the high neutralizing activities of these two hmabs in igg format indicates possibilities for their use alone or in combination for prophylaxis and treatment of sars. phage display technology has been increasingly used to produce high affinity hmabs from both naïve and immune libraries. an advantage of using a naïve library is that b lymphocytes from an infected or immunized host are not required. recently, two human nonimmune scfv libraries containing about members were developed from b cells of unimmunized donors, and used for selection of antibodies against a purified s fragment containing residues through ( ) . one of the selected antibodies, igg r, can neutralize % of the virus in a microneutralization assay at a concentration as low as . nm. it also blocked formation of syncytia, which could contribute to the spread of the virus in vivo , although at significantly higher concentration ( nm) . its epitope overlaps the binding site of the sars-cov receptor ace suggesting a possible mechanism of neutralization by preventing the virus attachment to its receptor ( ) . when r igg was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced to below assay limits ( ) . one should note that the conditions used for evaluation of the neutralizing activity of different antibodies in this study are not exactly the same as in the study described previously and later; thus comparing the activity of different antibodies should be done with caution unless they are tested side by side under exactly the same conditions. three neutralizing hmabs were also selected from another large naïve antibody library ( , ) . they bound a recombinant s fragment comprising amino acid residues to that also binds the receptor ace -the receptor-binding domain (rbd; ref. ). the most potent of these hnmab, igg cr , required the residue n for binding ( ) . this antibody exhibited in vitro % neutralizing activity at about µg/ml. more importantly, this antibody showed neutralizing activity in ferrets. in one set of experiments, ferrets were inoculated either with virus at two doses (low: tcid ; high: tcid ) or with virus preincubated with the antibody at . mg/ml for the low dose and . mg/ml for the high dose. animals exposed to the virus-antibody mixture had almost undetectable sars-cov in the lung, showed no lung lesions on day or , and did not shed virus in their throats unlike control animals treated with irrelevant antibody. in a second set of experiments, the antibody at mg/kg was administered hours before challenge with virus and reached - µg/ml serum concentration in three of the animals (< µg/ml in the fourth one). in the three ferrets with high antibody concentration virus shedding in the throat was completely abolished, while in the fourth one it was comparable to that of the control group. the cr -treated animals had . logs lower mean virus titer than the controls and were completely protected from macroscopic lung pathology. note that the antibody dose used ( mg/kg) was less than the one ( mg/kg) used for prevention of rsv infections in infants, which is administered once a month. these results suggest a potential use of cr for prophylaxis of sars-cov infections in humans if it can reduce the virus replication to the same extent as in ferrets. however, one should note that currently there is no available animal model of the sars-cov infection that results in death as in humans. two other hmabs ( and ) were derived from transgenic mice with human ig genes and evaluated in a murine model of sars-cov infection ( ) . one of these antibodies [ ] bound within the rbd of the s protein at amino acid residues through , and the other one ( ) to a region including residues through . in a microneutralization assay based on protection to cytopathic effects the ic for was about . µg/ml ( ) . mice that received mg/kg of these antibodies prior to challenge with the sars-cov were completely protected from virus replication in the lungs, and doses as low as . mg/kg offered significant protection. these antibodies have potential as therapeutics and research tools, and further studies are planned to evaluate the nhmab for potential clinical use ( ) . we have recently identified a novel cross-reactive potent sars-cov-neutralizing hmab, m , by using a fragment containing residues through as a selecting antigen for panning of a large human antibody library constructed from the b lymphocytes of healthy volunteers ( ) . this fragment was previously identified to contain the rbd ( - ) , which is a major sars-cov neutralization determinant ( , ( ) ( ) ( ) ( ) ( ) ( ) . it potently inhibited s-mediated cell fusion (ic = . µg/ml), pseudovirus entry (ic = . µg/ml), and replication of infectious virus in mice (complete protection at . mg per mouse; zhu et al., unpublished) . interestingly, this antibody also inhibited entry mediated by the s glycoprotein from the / gd isolate, which has a thr/ser mutation compared to the middle/late phase / isolate tor , and is not neutralizable by other known potent hmabs including r and s . . it bound with high (pm) avidity to the rbd in a biacore chip and competed with ace suggesting a mechanism of neutralization that involves competition with the receptor for binding to the s glycoprotein (zhu et al., unpublished) . the epitope of this antibody was identified by crystallography and proposed as a possible vaccine immunogen (a retrovaccinology approach for design of vaccine immunogens [ ] ). neutralizing antibodies directed to s inhibit sars-cov entry either by interfering typically with the s rbd-receptor interactions ( ) or by other mechanisms including binding to other portions of s. recently, a human scfv, b , was identified, which recognizes an epitope on s protein located within amino acids to ( ) . this antibody recognized sars pseudovirus in vivo and competed with sars sera for binding to sars-cov with an equilibrium dissociation constant, k d = nm. the b also had potent neutralizing activities against infection by pseudovirus expressing sars-cov s protein in vitro . other mechanisms of sars-cov infection inhibition could include steric hindrance that indirectly prevents virus attachment to receptors and binding to entry intermediates. mechanisms that could operate in vivo (and for lack of data are not discussed here) are related to the antibody biological effector functions conferred by the antibody fc, e.g., adcc. niv and hev are closely related emerging paramyxoviruses that comprise the henipavirus genus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the broad species tropisms and the ability to cause fatal disease in both animals and humans distinguish hev and niv from all other known paramyxoviruses (reviewed in ref. ). they are biological safety level pathogens and are on the niaid biodefense research agenda as zoonotic emerging category c priority pathogens that could be used as bioterror agents. there are currently no therapeutic modalities for treating niv or hev infections, and a vaccine for prevention of disease in human or livestock populations does not exist. although antibody responses were detected in infections caused by these viruses, the development of hmabs specific for hev and niv have only just been realized. we recently reported the identification of potent neutralizing hmabs targeting the viral envelope glycoprotein g by using a highly purified, oligomeric, soluble hev g (sg) glycoprotein as the antigen for screening of a large naïve human phage-display library ( ) . the selected seven fabs, m - , inhibited, to various degrees, cell fusion mediated by the hev or niv envs and virus infection. the conversion of the most potent neutralizer of infectious hev, fab m , to igg significantly increased its cell fusion inhibitory activity-the ic was decreased more than -fold to approximately µg/ml. the igg m was also exceptionally potent in neutralizing infectious hev; complete ( %) neutralization was achieved with . µg/ml and % neutralization required only . µg/ml. the inhibition of fusion and infection correlated with binding of the fabs to full-length g as measured by immunoprecipitation, and less with binding to sg as measured by elisa and biacore. m and m competed with the ephrin-b , which we and others recently identified as a functional receptor for both hev and niv ( , ) , indicating a possible mechanism of neutralization by these antibodies. the m , m , and m antibodies competed with each other suggesting that they bind to overlapping epitopes that are distinct from the epitopes of m and m . in an initial attempt to localize the epitopes of m and m , we measured their binding to a panel of g alanine scanning mutants and identified two mutants, p a and q ,k a, which significantly decreased binding to m , and one, g a, which decreased binding of m to g. these results suggest that m - are specific for hev or niv or both, and exhibit various neutralizing activities; they are the first hmabs identified against these viruses and could be used for treatment, prophylaxis, diagnosis, and as research reagents and aid in the development of vaccine. recently, we matured in vitro m to a very potent crossreactive antibody, m . , which neutralized both infectious niv and hev with an ic of ng/ml (zhu et al., unpublished) . we developed a cell line that produces large amounts of igg m . . this antibody will be tested in an animal model of henipavirus infection and if successful, which is very likely, it could be clinically useful for humans. interestingly, the antibodies against sars-cov and henipaviruses were identified from naïve libraries, and in contrast to hiv have only few mutations compared to the respective germline sequences. this correlates with the ability of various vaccines to elicit potent cross-reactive neutralizing antibodies against these viruses in contrast to hiv. one could speculate that humans have in their native repertoire antibodies that can bind with relatively high affinity envelope glycoproteins from these emerging viruses but not to the hiv env. further experiments are required to test this hypothesis, which if true could lead to novel approaches for design of effective vaccines against hiv and other viruses. the hmabs directed to the sars-cov s glycoprotein and the henipavirus g glycoprotein are currently in an advanced stage of development and offer the best hope as potential therapeutics. these antibodies specific for sars-cov, hev, and niv have potential for further development into a clinically useful product for prophylaxis and perhaps treatment of the diseases caused by these infections. they are very potent, and the viral infections to which they are specific are acute, such that only control or dampening of virus replication for a relatively short period of time (few weeks) is likely to be required after which the host immune system could control virus replication. in addition, these antibodies could be cross-reactive. thus, the problem of neutralization resistant mutants able to evade their inhibitory activity and the immune response is not as significant as for chronic infections with a high level of virus replication, e.g. hiv infections. a note of caution is that careful examination of candidate antibody therapeutics is required because of the possibility for infection enhancing effects, as e.g., for ebola, and animal model-dependent effects as well as in some cases, although rare, toxicity. a recent study also reported the possibility that neutralizing antibodies can enhance entry of sars-cov by a mechanism that involves antibody interactions with conformational epitopes in the s rbd ( ) . in addition, it is known that in some cases antibodies that do not neutralize in the assay currently used for evaluation of their in vitro activity could exhibit potent neutralizing activity in vivo ; thus new approaches should be developed and antibodies tested also for their effector functions mediated by the fc including antibody-dependent direct cytotoxicity and complement-mediated immune responses. however, only further exploration of these antibodies and their extensive evaluation in animal models, likely in com-bination with other antiviral drugs (antibodies or small molecules), would allow identification of the best candidates for potential therapeutics. the rapid progress made in the last few years toward the development of potent neutralizing hmabs against emerging viruses and viruses of biodefense importance is a basis for further more accelerated development of neutralizing antibodies in the next years and their testing in animal models. it is likely to see novel and even more potent antibodies against the sars-cov than the currently existing ones. they could be used alone or in combination with the existing antibodies in animal models of viral diseases and for evaluation of toxicity in human clinical trials. the currently available hmabs against niv and hev are likely to be tested in animals. if successful, which is very likely, they can undergo evaluation in human clinical trials. although the interest of big-and medium-size companies to such antibodies appears to be relatively minor, small companies and start-ups could be interested in developing such antibodies provided there is a continued governmental support by programs like bioshield. five years from now, it is likely to have at least several hmabs of potential clinical use in case of outbreaks or terror attacks. these antibodies could be used in combination with other therapeutics to increase potency and cope with resistance. several key issues are listed below: • continuation of research and development funding at the same or accelerated pace is of critical importance for development of potent and clinically useful therapeutic antibodies. • phage display techniques as well as novel methodologies will be critical for the development of fully human antibodies. • cloning, expression, and purification of novel antigens for screening of human antibody libraries is of critical importance. • crystal structures of antibody complexes with virus envelope glycoproteins and their use for further improvement of the antibodies and understanding of their interactions is of critical importance. • development of appropriate novel animal models 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paramyxovirus key: cord- -wq ik authors: relford, roberta l.; dimarco, anthony title: new diagnostic tools for infectious disease date: - - journal: consultations in feline internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: wq ik nan diseases caused by infectious agents are frequent in veterinary medicine. the signalment, history, and a thorough physical examination lead the clinician to suspect that an infectious disease may be present and lay the foundation for development of a differential diagnosis list and choice of the appropriate laboratory tests. clinical signs associated with infectious disease can be varied and depend on the infecting agent and the organ system involved. the first step in the laboratory evaluation is to establish a minimum database, including a complete blood count, serum biochemistry panel, and urinalysis. this minimum database, along with ancillary diagnostics such as radiographic imaging, aids in identifying specific organ involvement and can lead to a presumptive diagnosis that an infectious disease is present. further testing often is required to identify specifically which infectious agent is involved to determine an appropriate treatment plan for the patient. this chapter discusses the laboratory tools available for infectious agent identification. laboratory diagnostic tools can be divided into two main categories: ( ) direct identification of the infecting agent/ antigen (table - ), and ( ) indirect identification by detection of antibodies directed specifically against the infecting agent/ antigen ( table - ). the most common laboratory methodologies used to identify an infectious agent include visualization of the organism via cytology/biopsy, isolation of the agent in microbiological culture, immunodiagnostics/serology, and nucleic acid technology. cytology is the fastest and most inexpensive way to identify the presence of an infectious organism. microscopic examination of body fluids, fine-needle aspirates of solid organs, and imprints or scrapings of superficial lesions are just a few examples of ways in which infectious agents can be collected and identified morphologically. one of the limitations of cytology is whether the organism occurs in sufficient numbers in circulation or in the tissues or fluids to be identified. to help identify low numbers of organisms, intracellular organisms, and viruses, special stains with affinities for certain physical characteristics of the agents can be applied to the cytology sample. routine special stains include gram stain, periodic acid-schiff (pas), and acid-fast stain. a major advance in cytology over the years has been the development of immunocytochemistry. this methodology uses agent-specific polyclonal or monoclonal antibodies that react with unique antigens on various pathogenic organisms. the resulting antibody-antigen complexes then are detected by either fluorochromes that emit fluorescence or a chromogen that provides a color change. for example, anticoronavirus antibodies can be applied to fluid samples or granuloma aspirates from cats with suspected feline infectious peritonitis to detect the presence of coronavirus within macrophages or monocytes in the sample. immunohistochemistry is used classically to characterize and identify tumors for prognosis or to identify markers for therapeutic intervention, and also can be used for organism identification. polyclonal anti-mycobacterium bovis antibody has been shown useful as a single screening method for the detection of a number of different microorganisms in skin biopsies. cytology and biopsy samples also can be used as a source for cells, dna, and rna extraction. cells and organisms can be extracted from cytology slides, formalinized tissue, and paraffin-embedded tissue and then evaluated via flow cytometry with immunomarkers or polymerase chain reaction (pcr). as more antibodies are made available, the menu of organisms that can be identified will expand. these techniques are offered at many universities and reference laboratories. microbiological culture typically is performed to identify bacterial or fungal organisms. challenges associated with culture include adequate sample collection, biological behavior of the organism, and interpretation of the results. the first challenge is collection of the specimen. the affected organ may not be readily accessible and the sample must be collected without contamination. at the laboratory, the great diversity of infectious microorganisms and their varied biological behaviors can make accurate identification difficult. to help overcome these challenges, many microbiology laboratories now use automated systems that can run a wider range of biological tests and compare the behavior of the sample organism on these tests with a database containing information about the reaction patterns of known pathogens. with the addition of more tests and computer algorithms to compare with a large database, the probability of correct identification of the organism is increased. once identified, the susceptibility of the organism to numerous antimicrobial agents can be determined. laboratories now routinely provide the minimum inhibitory concentration (mic), which is defined as the lowest concentration of the antimicrobial drug that inhibits the growth of the organism. these values then can be compared with the levels attainable in serum or tissue to determine the most appropriate antimicrobial agent for the treatment of that particular organism in that particular site. some new assays combine culture and cytology. for example the inpouch tv culture system by biomed diagnostics (white city, or) for the identification of tritrichomonas spp. uses a clear pouch containing culture media to increase low numbers of organisms rapidly in a sample. the sealed pouch then is placed directly on a microscope for reading (see figure - ). the advantages of this system include less likelihood of contamination of the sample, reduced exposure of laboratory staff to a zoonotic organism, long shelf life of the culture system, and improved sensitivity compared with wetmount preparations. fastidious, intracellular, and viral organisms do not lend themselves readily to growth-based technologies, and cytological detection is limited by the shedding characteristics and intracellular location of some agents. whole organisms or their antigens can be detected immunologically in a wide range of specimens including serum, whole blood, feces, cerebrospinal fluid, body cavity effusions, synovial fluid, cell aspirates, and tissue samples. because these assays are directed toward an antigen of the infectious organism, they usually are not speciesspecific and reagents from human assays often can be used on feline samples. however, when reagents validated for human assays are used, they should be revalidated with feline samples to ensure that interfering factors or cross-reactive antibodies are not present. immunodiagnostic assays include fluorescent antibody (ifa), enzyme-linked immunosorbent assay (elisa), latex agglutination, and immunoblotting. fluorescent antibody and immunoblot assays are performed in commercial laboratories, whereas numerous patient-side rapid elisa and latex agglutination kits are available for antigen detection. the antigen elisa assay comprises capturing the antigen of interest by using an antibody immobilized on a solid phase (e.g., a microtiter plate, a membrane, or microparticles) and forming a sandwich with a second antibody that is coupled to an enzyme. formation of this sandwich occurs only if the corresponding antigen is present in the sample. after binding, the enzyme is catalyzed to give a color reaction. the most widely used elisa for antigen determination in cats is the feline leukemia virus (felv) antigen assay. as with other antigen serology assays, this technique is limited based on the level of antigen load, the location of the antigen (i.e., intracellular or extracellular), and cross-reactivity with other antigens. recent advances in elisa assays have been directed toward improving the specificity of the antibody to the organism in question or enhancements in the detection system. initially, nonspecific, outer surface proteins were used to develop the assay antibodies. this often resulted in cross-reactivity with multiple organisms and false-positive test results. many tests now use purified, unique proteins to produce highly specific antibodies for the assays. these improved tests have a high diagnostic sensitivity and specificity (greater than per cent) and often are discordant with less specific whole-cell ifa tests. , additional improvements in elisa diagnostic assays include enhancements to the conjugate that allow shorter incubation times, superior reaction detection through the use of enzyme stabilizers, and longer conjugate stability and shelf life. some detection systems have been changed to a non-enzyme-based conjugate for color detection using colloidal gold or colloidal carbon. latex agglutination assays use latex microspheres coated with specific antibodies directed against an infectious agent. if the organism or antigen is present, the antibody-coated particles bind to the organism and cause agglutination. latex agglutination tests are available for cryptococcus neoformans, toxoplasma gondii, histoplasma capsulatum, and sporothrix schenckii, in addition to numerous bacterial pathogens. one of the most rapidly expanding areas of organism identification and antigen detection has been in the area of nucleic acid-based testing. until the introduction of nucleic acid amplification by the pcr, detection of an organism's dna or rna often was impossible because of the small amount of antigen present in a sample. pcr technology takes advantage of the normal function of polymerase enzymes. these enzymes are present in all living things and are responsible for copying, proofreading, and correcting errors in genetic material. polymerase enzymes "photocopy" or amplify minute quantities of genetic material to a volume sufficient for it to be detected. to have a useful pcr assay, a nucleic acid sequence unique to a particular organism or class of organisms must be identified. this unique sequence is the material amplified and detected if the organism in question is present in the sample. during the first step, dna is extracted carefully from the sample containing the suspected infectious agent. the extracted dna then is heat-stressed to cause it to uncoil from its normal double-helix structure and separate into individual strands. the second step involves the addition of the two unique primers that flank each side of the sequence of interest. the primers aid in identification and amplification of the unique dna sequence in question. when the primer finds the matching section of nucleic acid sequence, this sequence is copied to produce a small fragment of dna (the amplicon) specific to the organism. this series of steps is repeated to times, with the product of each round serving as additional template for subsequent rounds of amplification. the final result is a logarithmic amplification of the original nucleic acid sequence. detection of the multiplied nucleic acid sequence is performed during the amplification process with fluorescence emission with each amplification step (real-time pcr), after the completion of all amplification steps with the use of a secondary detection method, or from electrophoretic separation of the final dna fragment and detection by application of a labeled complimentary nucleic acid probe. pcr has opened a new spectrum of infectious disease tests, and numerous laboratories have started offering pcr tests for veterinary pathogens. the challenges faced today are not technological but in establishing a standardized process for accuracy, reproducibility, and quality control of such a sensitive and powerful assay. pcr testing is being offered at an increasing number of facilities. however, few laboratories have provided the important validation data that are necessary to evaluate and compare methods for diagnostic accuracy. the absence of standardization in testing protocols, primer selection, laboratory contamination control measures, quality control monitoring, and validation methods has led to increasing lab-to-lab variability and poor lot-to-lot reproducibility. until protocols are defined and universally standardized for pcr among laboratories, the clinician should request information regarding quality control testing and controlled data supporting the primers in use at that specific laboratory. the introduction of pcr also has opened the door for another area of debate: the issue of whether infection necessarily means "disease." the ability of pcr technology to detect very small numbers of organisms has led some to consider the level of infection required to overcome the body's immune response and cause disease, suggesting that the organism detected by pcr may not always be the infectious agent causing the animal's current illness. this is further complicated by the fact that pcr cannot distinguish between viable and nonviable organisms, reinforcing the need to establish a comprehensive clinical profile in diagnosis. in addition, diagnostic guidelines must be established to aid in determining how many organisms are needed to cause disease, or additional data must be generated to better understand the rate of clearance of infectious agents after effective treatment. unlike the other methods discussed, pcr testing essentially is limited to reference laboratory testing because of the complexity of the materials, equipment, and technique, and the lack of an in-clinic pcr device. however, like elisa and other previously complex methods, new technology eventually will provide in-clinic pcr capability, accompanied by standardized methods. identification of the organism always has been the gold standard for diagnosing an infectious disease. however, because many organisms elude detection because of their small numbers or occult location within the body, direct identification of the organism is not always possible. recent advances have extended the range of direct antigen detection (see discussion above on pcr). however, reliance on the detection of antibody in the serum as an indirect method for diagnosing many infectious diseases is prevalent. the methods for detecting antibody include elisa, ifa, complement fixation (cf), hemagglutination inhibition (hi), serum virus neutralization (svn), and western blot analysis. in the elisa and ifa antibody assays, a specific antigen from the infectious agent in question is fixed to a solid surface (microtiter plate or glass slide, respectively) and the patient's serum is added. if antibodies to this organism are present in the patient's serum, they will bind to the antigen. an enzymeconjugated or fluorescent-labeled anti-species antibody then is used to detect the bound antibody. commercial laboratories usually use antigen bound to microwell plates. membranebound antibody is used in the small, self-contained in-clinic test kits used by most veterinary hospitals. a fluorescent microscope and trained technicians are required to perform ifa testing and these tests are available only in commercial laboratories. the hemagglutination inhibition test is used to detect antibody against some viruses that possess a hemagglutinating antigen (ha), which is capable of agglutinating erythrocytes. the ha is placed in a microtiter well and incubated with the patient's serum. if virus-specific antibodies are present in the test serum, they will bind to ha and prevent agglutination of erythrocytes added in the last step. unfortunately, some serum samples have interfering factors that bind to ha and produce false-positive results. in addition, the ha may have varying affinities for different species of erythrocytes and produce different results with sheep, rabbit, mouse, or guinea pig red blood cells. serum virus neutralization (svn) and complement fixation (cf) assays determine whether antibody is present by evaluating some normal immunoglobulin functions. the svn assay evaluates the ability of antibodies in a patient's serum to prevent the infection of culture cells or embryonated eggs with a known specific virus. the patient's serum first is inoculated with the virus. then the virus-serum mixture is injected into a cell culture to detect virus infectivity. virus-specific antibodies in the patient's serum inactivate the virus and prevent infection of the cells. the cf assay assesses the ability of antibodies to bind to a specific antigen and complement to form an antigenantibody-complement complex. this complex ties up the complement and prevents it from lysing red blood cells added as a substrate. complement fixation assays also can be used to detect antigen. all of the antibody tests discussed so far measure antibody to whole cell antigens or viruses. the western blot assay separates the infectious agent into its composite proteins by gel electrophoresis. the proteins are transferred to blot paper and are incubated with the patient's serum. if antibodies to virusspecific proteins are present, they will bind to the protein bands on the blot paper and can be detected by a labeled secondary anti-species antibody. antibody binding to a combination of bands usually is required to confirm a diagnosis. the main limitation of using antibody detection for diagnosis is that, in most diseases, the presence of antibody against an infectious agent cannot differentiate among patients with previous exposure having lingering antibodies, patients with current active infection, or patients with antibodies generated by previous vaccination. until the recent release of a whole virus vaccine against feline immunodeficiency virus (fiv), the presence of serum antibodies against fiv was definitive proof of infection because virus-infected cats remain persistently infected for life. unfortunately, antibodies produced in response to this whole virus fiv vaccine are indistinguishable from antibodies generated by true fiv infection and are detected with all fiv antibody assays (ifa, elisa, and western blot). this severely limits the utility of fiv antibody detection in fiv-vaccinated cats. to combat this type of interference, researchers are looking for antibodies produced in response to infection but not as a result of immunization. for some pathogens, antibodies have been identified that are directed against specific proteins that are present only on the organism when it is alive within the host. new assays use these live-pathogen specific peptides instead of whole cell proteins to detect antibodies directed only against those specific peptides. an example of this type of peptide is the recently identified invariable, immunodominant region (ir ) on a variable region of the lipoprotein (vlse) of borrelia burgdorferi. the ir peptide is highly antigenic and stimulates antibody production that rises rapidly during experimental infection and drops rapidly with successful treatment. several theories have been proposed to explain this phenomenon, including the possibility that the variable region of the vlse protein is changed rapidly in the organism and older variant molecules are degraded rapidly. this high turnover rate also would ensure that the protein would be rare in dead organisms. therefore acute infections treated immediately showed a rapid rise and drop in antibody against this antigen. it has yet to be determined whether antibody levels in chronic cases will respond similarly, and whether the ir is exposed to memory cells over time, resulting in antibody levels that persist following infection and treatment. vaccination against borrelia spp. apparently does not induce antibodies to ir , because the dna that encodes the ir sequence is not present in the laboratory strains of b. burgdorferi used for vaccine production. rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal bcg antibody multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor setting comparison of four different methods for detection of cryptosporidium species comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern tennessee seropositive to ehrlichia canis detection of giardia lamblia and cryptosporidium parvum antigens in human fecal specimens using the colorpac combination rapid solid-phase qualitative immunochromatographic assay characterization of a borrelia burgdorferi v se invariable region useful in canine lyme disease serodiagnosis by enzyme-linked immunosorbent assay antibody response to ir , a conserved immunodominant region of the vlse lipoprotein, wanes rapidly after antibiotic treatment of borrelia burgdorferi infection in experimental animals and in humans dogs vaccinated with common lyme disease vaccines do not respond to ir , the conserved immunodominant region of the vlse surface protein of borrelia burgdorferi key: cord- -zn na authors: slifka, mark k.; amanna, ian j. title: passive immunization date: - - journal: plotkin's vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: zn na nan passive immunization, passive immunity, and passive immunotherapy all refer to the transfer of antibodies to an unprotected individual for the prevention or treatment of disease. the first formal demonstration of passive immunization for successfully treating diphtheria and tetanus dates back to animal studies published in deutsche medizinische wochenschrift (german medical journal) in . the technique was quickly adapted to clinical use and as early as the mid- s, diphtheria-specific antitoxin was used successfully in the hospital setting to reduce mortality during diphtheria outbreaks. [ ] [ ] [ ] indeed, in emil von behring was awarded the first nobel prize for physiology or medicine for the discovery of this important medical intervention. the significance of this clinical advance cannot be overstated; behring estimated that , lives were saved each year using diphtheria-specific passive immunotherapy in germany alone. in the s, the mortality rate of hospitalized cases ranged from % to %, and the work of emil von behring and his colleague, shibasaburo kitasato, provided the only hope for diphtheria patients in the preantibiotic era. according to behring, the discovery of passive immunization would not have occurred if it were not for his earlier work that focused on characterizing the protective mechanisms of active immunization against diphtheria , and through the work of his collaborator, kitasato, on the mechanisms of vaccine-mediated immunity against tetanus. when guinea pigs were infected with corynebacterium diphtheriae, the animals routinely died of the disease. however, when behring vaccinated animals and they mounted neutralizing antibodies to diphtheria toxin, he found that they were protected from a normally lethal dose of c. diphtheriae. to determine if protection was now an intrinsic property of the immune host that could be transferred to a susceptible host, he injected naïve guinea pigs with diphtheria toxin and then successfully treated them with immune serum from vaccinated animals. likewise, injection of clostridium tetani or purified tetanus toxin was typically lethal, but through a method developed by paul ehrlich, animals could eventually become immune to high doses of tetanus toxin by sequentially inoculating them with lower, nonlethal doses of tetanus toxin. kitasato used this approach to demonstrate that the blood of vaccinated, tetanusimmune rabbits could be transferred to naïve mice and fully protect them from a normally lethal dose of virulent c. tetani or from filtered c. tetani culture supernatant containing tetanus toxin. behring and kitasato may have said it best in the final sentence of their landmark study, "the result of our experiments remind us forcibly of these words: blut ist ein ganz besonderer saft [blood is a very unusual fluid]." technology has advanced substantially in the more than years since behring and kitasato's first formal demonstration of protective passive immunotherapy. in those early days, it was infeasible to use human immune serum to treat diphtheria, so the first large-scale production of polyclonal diphtheria-immune serum was prepared by vaccinating dairy cows. to this day, commercial antisera used to treat a broad range of toxins are still produced in animals (table . ). passive immunotherapy with animal-derived antibody preparations should only be used under close medical supervision or the resulting host immune response to the foreign immunoglobulins and serum proteins may trigger serum sickness, urticaria, and/or anaphylaxis following administration. fortunately, the advent of several innovative technologies that reduce the need for animal-derived antibodies have forged new paths in terms of safety, feasibility, and the protective efficacy afforded by passive immunization. following the discovery of monoclonal antibody technology, , further refinements have been made, including use of various display techniques (e.g., phage display, yeast display) to screen large antibody libraries. other technological advances include the development of chimeric monoclonal antibodies in which the murine antibody is "humanized" by genetically replacing the heavy chain region of the molecule with the human immunoglobulin counterpart and the use of transgenic mice in which the endogenous murine immunoglobulin genes have been replaced by human immunoglobulin genes. this latter approach has the advantage that hybridomas from immunized transgenic mice produce fully human monoclonal antibodies without requiring further genetic modifications. recently, development of epstein-barr virus (ebv)-transformed human memory b cells for the production of monoclonal antibodies has led to yet another surge in the production of new human monoclonal antibodies with rare antigenic specificities to uncommon pathogens and these can be produced directly from immune human subjects. , before the era of antibiotics, antibodybased therapy was the only option available for combating many bacterial diseases. even today, there are only a handful of antiviral drugs available and no therapeutic options exist for most viral diseases. however, new antibody-based therapies are continuing to be developed with the potential to provide protection against a broad array of bacterial and viral pathogens. in this chapter, we describe the role of passive immunity in the protection of the naïve host, discuss the parameters involved with successful immunotherapy, and provide examples of protective efficacy in animal models as well as in human clinical studies. age who were born to mothers who received pertussis vaccination during pregnancy, , thus lending further support to the current recommendations for the vaccination of pregnant mothers against b. pertussis. the age limit of younger than months was chosen as this is the age at which primary pediatric vaccination is recommended and analysis beyond this age might be confounded by the protective effects of direct vaccination of the child. nevertheless, the protection afforded by maternally derived igg against respiratory infections involving viral (e.g., influenza) or bacterial (e.g., b. pertussis) pathogens together demonstrate the broad impact that maternal vaccination and the subsequently increased transfer of maternal antibodies can have on the health of young infants. before vaccines and antibiotics revolutionized modern medicine, antibody-based therapies represented the only effective medical treatment for many life-threatening diseases including diphtheria, scarlet fever, bacterial meningitis, and bacterial pneumonia. , today, most commercial forms of antibodybased immunotherapy for infectious disease still rely on polyclonal antibodies of human or animal origin, with the notable exceptions of the monoclonal antibodies, palivizumab and raxibacumab (see table . ). the main advantage of using polyclonal antibodies for passive immunotherapy is that this approach will include antibodies to multiple epitope specificities that may work in an additive or synergistic manner with the potential contribution of multiple immunoglobulin isotypes and subclasses that have different biological functions (table . ). on the other hand, there are several potential challenges to using polyclonal antibodies for immunotherapy including low antigen-specific activity, supply limitations (especially for rare diseases), variability between manufacturing lots, and safety as well as quality control issues that are often associated with the use of human blood products. in ungulates. these differences also indicate that care should be taken when choosing an appropriate animal model for studying the role of maternal antibodies against infectious disease as the mechanisms may be more species-specific than typically realized. in a comprehensive study involving the analysis of antibodies to viruses using samples from , patients, the relationship between maternal immunity and infant immunity is clear (fig. . ). the prevalence of antibodies to each viral antigen among infants less than month old is remarkably similar to those observed in the -to -year old adults who represented the main age group of the mothers. for instance, immunity to common childhood diseases such as measles and mumps was comparable between newborns and their mothers. immunity to less-common viral pathogens, such as influenza b, was relatively low among infants and adults in the cohorts examined in - , but higher among those sampled in - , - , and - , coinciding with an influenza b epidemic that had occurred in . this shows that the prevalence of maternal antibodies is dynamic and that recent outbreaks involving a specific pathogen will result in a higher frequency of pathogenimmune mothers and a concomitant increase in the number of infants who are likewise bestowed at least transient immunity to that particular microbe. as expected, maternal antibodies wane rapidly during the first months of life and then exposure to pathogens over the following months and years results in an accumulation of different antibody specificities as children reach adulthood (see fig. . ). the overall protective efficacy of maternal antibodies is perhaps most pronounced among children with genetic immunodeficiencies such as severe combined immunodeficiency (scid), resulting in the lack of functional t and b cells or agammaglobulinemia, in which patients lack functional b cells while still having the ability to mount pathogen-specific t-cell responses. the clinical presentation of scid is not apparent at birth but relatively uniform diagnosis occurs at a mean of . months of age, which is also about the age that maternal antibodies have reached their lowest levels (see fig. . ). likewise, agammaglobulinemic patients also begin to present with symptoms of immunodeficiency around this same age. maternal antibodies represent an immunological "doubleedged sword" in the sense that they are known to interfere with live attenuated virus vaccines such as the mmr (measles, mumps, rubella) [ ] [ ] [ ] and rotavirus vaccines, , whereas direct immunization of mothers in the third trimester of pregnancy can significantly increase protection of infants against common respiratory viruses such as influenza. [ ] [ ] [ ] indeed, maternal vaccination may result in a % to % reduction in influenzarelated hospitalizations among infants younger than months of age. [ ] [ ] [ ] likewise, the importance of maternal vaccination against bordetella pertussis (i.e., whooping cough) was recognized as early as the s to s with studies showing higher antibacterial antibody responses and potential protection from exposure to whooping cough among infants born to vaccinated mothers. [ ] [ ] [ ] [ ] recent studies verify these earlier results, demonstrating a % to % vaccine efficacy against whooping cough among infants younger than months of nonlymphoid tissues and to penetrate mucosal sites of infection is likely to explain why it is often considered the best immunoglobulin isotype for routine passive immunization and has shown clinical benefit ranging from reduced clinical symptoms to nearly complete protection from lethal infection in a number of infectious disease models (table . ). over the last century, it has been well established that high specific antibody titers and early timing of antibody transfer in relation to disease onset are the two most important parameters involved with determining the protective efficacy of passive immunization ( fig. . ). in one account of the early days of clinical diphtheria-specific immunotherapy developed by behring and ehrlich, initial failures in patients after treatment with weak or unstandardized diphtheria-immune serum brought ehrlich to describe three points that he believed were important for successful immunotherapy: (a) treatment has to be initiated at the onset of disease; (b) the more the disease has progressed, the higher the serum quantities necessary for cure; and (c) depending on the severity of the case, certain minimal doses can be specified. later studies confirmed these results: if diphtheria immunotherapy was initiated on the first day of disease, there was % mortality (n = ). however, if therapy was delayed to , , or days after disease onset, then the accompanying diphtheria case-fatality rate subsequently increased to . % (n = ), . % (n = ), and . % (n = ), respectively. these results are similar to those observed during antibiotic-based therapy of bacterial sepsis. in an ideal setting, it is recommended that antibiotics be administered within hour of diagnosis of severe sepsis or septic shock as these drugs provide clinical benefit only if administered early in the course of disease and are generally ineffective during late-stage disease. the importance of high-dose immunotherapy given at the earliest sign of disease is not unique to bacterial anti-toxin therapy. the same rules apply to preventing or treating viral infections as well. during a measles epidemic in - , % of exposed individuals (n = ) who received no passive immunization contracted measles. if convalescent serum was administered within days of exposure, then the attack rate was reduced to % (n = ) whereas if therapy was not initiated until to days postexposure, approximately % of contrast, monoclonal antibodies are, by definition, limited to a single epitope specificity but they have several advantages over polyclonal antibodies since they can be manufactured in vitro at large scale, with inherently high specificity and lot consistency (table . ). for example, the combination of . mg of two tetanus-specific human monoclonal antibodies has the same neutralizing capacity observed with administration of to mg of polyclonal tetanus immunoglobulin. likewise, administration of . mg of a vaccinia virus-specific monoclonal antibody provides the same level of protection afforded by mgs of vaccinia immunoglobulin (vig). although neutralization escape mutants are a valid concern when using monoclonal antibody therapy, , this has not yet been a major problem during clinical use of palivizumab for respiratory syncytial virus (rsv). initially, sequencing of rsv isolates demonstrated that there were no mutations in the neutralizing epitope of the f protein. subsequent studies identified rsv escape mutants in approximately % of breakthrough cases, indicating that selective pressure for escape mutations is still relatively uncommon under current conditions of use. this suggests that monoclonal antibodies can remain effective when used clinically in the long-term, as long as they are specific for a stable epitope for that particular pathogen. the functional characteristics of the immunoglobulins used for passive immunization is an important consideration in determining protective efficacy in vivo. for example, serum igg molecules equilibrate into extravascular space whereas igm is largely confined to intravascular space. igm molecules also have a short half-life ( days ) and are typically of low affinity, which is why igm is not an optimal choice for passive immunotherapy. serum iga is monomeric and, although it also equilibrates into extravascular space, it has only a -day half-life and does not appear to contribute significantly to functional iga in the lungs of mice. , human igg on the other hand, has an average half-life of approximately days (except igg , which has a -day half-life), , is typically of high affinity, and transudation across mucosal barriers can protect against pathogens that invade through mucosal routes. interestingly, serum igg (and serum iga) responses elicited in response to vaccination against neisseria meningitidis correlate strongly with the levels of antibacterial antibodies present in the saliva at month and year after vaccination, indicating that circulating serum antibodies may be an important contributor to the antibodies released in mucosal secretions. indeed, after intravenous administration of an hiv-specific monoclonal antibody into rhesus macaques, serum antibody titers of to µg/ml resulted in mucosal antibody titers of to µg/ml in vaginal fluids and provide complete protection against intravaginal challenge with shiv (chimeric simian immunodeficiency virus expressing hiv envelope). influenza virus is another mucosal pathogen with strict tropism to the respiratory tract, but influenza-specific serum antibody titers correlate with protection in humans. in mice, the relative roles of influenza-specific polymeric iga and igg were compared in terms of antiviral protection in the upper respiratory tract versus the lung after influenza challenge. when polymeric iga was transferred hours prior to influenza infection, this prevented pathology in the upper respiratory tract but was not effective in the lung, whereas transfer of igg prevented pathology in the lung, but required higher doses to protect against infection of the upper respiratory tract. the authors concluded that different antibody isotypes may function preferentially at different anatomical sites in vivo. these results are in contrast to experimental influenza infection in humans in which inactivated influenza vaccinederived igg is believed to be a major contributor to protection of the nasal compartment. overall, the ability of igg to enter efficacy of passive immunity decreases with disease progression. full protection from symptomatic disease is best achieved through prophylactic administration of antibody therapy prior to exposure or infection. however, antibody therapy may also be highly effective at early points postexposure, prior to the onset of disease symptoms. passive immunity is generally less effective when administered after the onset of symptomatic disease, and typically shows little to no clinical benefit once severe late-stage disease has occurred. to iu/ml, the postexposure incidence of measles increased from % to % despite either lot being administered within days of exposure. likewise, the timing of passive immunotherapy is also important for enteric (e.g., polio) and respiratory pathogens (e.g., rsv). an outbreak in involving polio patients showed that if convalescent serum was administered within to days of meningitis, then paralysis was reported in . % of patients (n = ). if treatment was delayed until to days after meningeal disease onset, . % reported paralysis (n = ), and if treatment was delayed for more than days, then paralysis was noted in . % of polio patients (n = ). for rsv, polyclonal rsv-immunoglobulin reduced the incidence of rsv-associated hospitalization by contacts subsequently contracted measles (n = ). in a study published in involving cases of measles exposure, % of the individuals who received immunotherapy within to days of exposure contracted measles compared to % for those whose treatment was delayed to to days postexposure. the dose used in these studies was also critical: % of patients who received . ml/kg of gammaglobulin contracted measles whereas only % of patients who received . ml/kg of gammaglobulin contracted the disease. the titer of virus-specific antibodies will often differ between lots of polyclonal immunoglobulin preparations (see table . ). in another study, when the measles-specific titer of gammaglobulin from different lots decreased from iu/ml for animal studies, prophylaxis is defined as antibody administration prior to experimental infection and treatment is defined as antibody administration after infection. for clinical studies, prophylaxis is defined as antibody administration prior to disease onset and treatment is defined as antibody administration after disease onset. b evidence provided through maternal immunization studies. c anecdotal results are defined as small studies that indicate passive immunization may provide clinical benefit but are too limited in scope to be conclusive. d not available, although two studies , demonstrated prophylaxis by direct mixing of mumps virus and antibody prior to inoculation. confirmed lassa fever who received immune serum within days of hospitalization survived ( of ; %). however, if treatment was not initiated until more than days after hospitalization, then only of ( %) patients survived, similar to the untreated group in which only of ( %) patients with virologically confirmed lassa fever survived. although passive immunity against toxins and systemic infections such as measles , , - and smallpox [ ] [ ] [ ] is well established, the impact of this approach for the prevention or amelioration of disease caused by respiratory and enteric pathogens may not be as well recognized. however, several studies support the role of passive immunity against mucosal pathogens (see table . ) , including examples such as influenza (respiratory virus), haemophilus influenzae (respiratory bacterium), rotavirus (enteric virus), and escherichia coli (enteric bacterium). influenza is a significant cause of morbidity and mortality throughout the world, including both seasonal transmission and pandemic outbreaks. [ ] [ ] [ ] [ ] the clinical correlation between homotypic influenza immunity and vaccine-associated protection was recognized early in the development of the influenza vaccine. [ ] [ ] [ ] early animal studies confirmed this result, with passive transfer of antibodies (both systemic and mucosal delivery) able to protect naïve animals against subsequent challenge, or provide therapeutic benefit when administered postexposure. [ ] [ ] [ ] more recent animal studies with defined monoclonal antibodies continue to support and extend these earlier results. , passive immunization against influenza in humans has also been successful. in a comprehensive retrospective metaanalysis of eight passive immunization studies performed during the spanish influenza outbreak ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , a significant % decrease in mortality ( % confidence interval [ci], - %; p < . ) was observed. subset analysis of studies that recorded early (treatment initiated within days of pneumonia complications) versus late intervention (> days) showed a significant advantage for early treatment, with mortality decreasing from % ( of ) to % ( of ) with earlier intervention, consistent with general considerations for effective passive immunity against infectious diseases (see fig. . ). in a recent double-blinded, randomized controlled study during the influenza pandemic, the use of hyperimmune intravenous immunoglobulin (ivig) (from recovered convalescent donors) was compared to normal ivig in the treatment of severe infection in subjects. the hyperimmune treated group (n = ) demonstrated more rapid viral clearance than the control group (n = ), with a greater than % drop in viral loads by day posttreatment. in those patients receiving immunoglobulin within days of symptom onset (n = ), all who received hyperimmune ivig survived ( of ), whereas only % of patients receiving normal ivig survived ( / , p = . ). h. influenzae type b (hib) is an extracellular gram-negative bacterium that initially infects the host via the respiratory tract and represents another important human pathogen that can be controlled through passive immunization. several early reports described the use of concentrated rabbit immune serum as a successful adjunct therapy to sulfonamide treatment for patients suffering from hib meningitis. - indeed, a full course of serum therapy (in addition to antibiotics) was able to reduce mortality to % ( of ) when compared to % mortality rate ( of ) in those patients only receiving sulfonamides. a more recent study established the prophylactic % among children with a history of prematurity or bronchopulmonary dysplasia. prophylactic administration of a neutralizing monoclonal antibody, palivizumab, was shown to significantly improve clinical outcome by reducing rsvassociated hospitalizations of children with congenital heart disease by %. among premature infants or those with bronchopulmonary dysplasia, rsv-associated hospitalizations were reduced by %. a third palivizumab study confirmed these results by showing a % reduction in hospitalizations among premature infants and infants with chronic lung disease. another monoclonal antibody, motavizumab, demonstrated a further % relative reduction in rsv hospitalizations compared with patients receiving palivizumabbased prophylaxis. in contrast, once rsv infection has been established, the use of palivizumab, motavizumab, or rsvimmunoglobulin shows no clinical benefit, although rsvimmunoglobulin may provide limited protection in the most severe cases. passive immunotherapy can be highly successful for severe, even life-threatening human diseases such as smallpox, or hemorrhagic fever caused by arenaviruses including junin or lassa fever virus (see table . ). successful intervention, however, typically requires initiating treatment before or very shortly after symptom onset. when convalescent serum from smallpox survivors was administered to smallpox patients during the late stages of confluent or hemorrhagic smallpox, there was no clinical benefit observed in comparison to untreated controls ( % vs. % mortality, respectively). when vaccinia-immune gammaglobulin (vig) was administered to smallpox contacts prior to disease onset in addition to postexposure vaccination (i.e., standard of care), the number of smallpox cases was reduced by % compared to contacts who received postexposure smallpox vaccination alone. likewise, administration of vacciniaimmune serum of animal origin along with postexposure vaccination resulted in of cases ( %) of smallpox among close contacts compared to of cases ( %) among controls who received smallpox vaccination alone. during a smallpox outbreak in , of patients ( %) died while undergoing standard clinical care. to determine if addition of passive immunotherapy would reduce mortality after smallpox diagnosis, cases of smallpox were treated with convalescent serum or blood, with no smallpoxassociated deaths reported ( of ). approximately patients were described as having severe or hemorrhagic smallpox at the time of treatment and yet all survived. this appears to be the result of using convalescent serum obtained at the peak of the humoral immune response shortly after recovery from smallpox and the use of an optimized dosing schedule with higher doses administered to patients with the more severe disease manifestations. argentine hemorrhagic fever is caused by infection with the junin virus and untreated cases result in % to % mortality. [ ] [ ] [ ] convalescent serum is protective in animal models of junin infection [ ] [ ] [ ] and when administered within days of symptom onset, the mortality rate among human cases drops to % to %. , likewise, in % to % of hospitalized cases, lassa fever virus causes severe disease including diffuse capillary leakage and hemorrhagic diathesis. prophylactic administration of immune serum protects guinea pigs , and nonhuman primates , from subsequent lethal challenge, indicating that antibodies play a clear role in protection against this virulent viral pathogen. in one small clinical study, if passive immunotherapy was administered within to days after admission to the hospital, of ( %) patients survived whereas if immunotherapy was initiated to days after hospitalization, of ( %) patients survived. in another study, patients with virologically with any new scientific advance, there is controversy. in , when behring demonstrated that immune serum therapy could protect against diphtheria, it went against the current dogma at that time in which the cellular theory of phagocytosis was believed to be the primary mechanism of host protection. there were also skeptics who, as early as , discussed why antibody immunotherapy would not work. however, the science not only prevailed but today a number of passive immunotherapy products are in clinical use (see table . ) and an ever-increasing number of human diseases benefit from the use of this technology (see table . ). some believe that antibody plays a more important role in protection against cytopathic viruses and extracellular bacteria, but that t cells must be required for protection against infection by noncytopathic viruses and other intracellular pathogens. although this is partially refuted by the protective efficacy of maternal antibodies and ivig therapy in scid patients who do not have functioning t cells, it is important to bear in mind that antibody-mediated protection by passive immunotherapy in immunocompetent individuals does not function in isolation, but instead works best in conjunction with other immune defenses, including host t cells, b cells, natural killer (nk) cells, etc. although the role of antibody-mediated protection against intracellular bacteria and chronic viral infections was thought to be relatively minor, there are examples in each of these instances in which passive immunity provides substantial clinical benefit. as noted previously, prior to the advent of antibiotics, passive immunotherapy was the only option for clinical treatment of most bacterial infections including francisella tularensis, a facultative intracellular bacterium that causes tularemia, a severe disease associated with up to % mortality in untreated cases. , when streptomycin became available, a comparative study in was performed with tularemia patients who received only symptomatic treatment, who received immune equine serum, who received hyperimmune equine serum, and who received streptomycin. the untreated tularemia cases required an average of . months to recover and only three modes of therapy showed substantial improvement-treatment with immune serum within days of disease onset ( . months until recovery), treatment with hyperimmune serum ( . months until recovery), and treatment with streptomycin ( . months until recovery). two clinical cases were extensively described, with the following summary: "the clinical responses to each agent [i.e., immune serum, and streptomycin] were similar, prompt amelioration of the symptoms of intoxication-headache, mental dullness or lethargy, sense of prostration and severe malaise; reduction of fever and of the sizes of the buboes, acceleration in the healing of ulcers and in the resolution of pulmonary exudates." in other words, passive immunotherapy appeared in many ways to mimic antibiotic therapy in terms of protective efficacy. however, it was noted that treatment with equine serum caused serum sickness in % of the patients and had a more variable outcome than the antibiotic approach, leading to the recommendation that streptomycin would be the agent of choice for future treatment of this disease. with the recent development of polyclonal and monoclonal antibodies that show protective efficacy against tularemia in animal models, [ ] [ ] [ ] it may be possible to incorporate both passive immunotherapy and antibiotic treatment into clinical practice not only for tularemia, but for other bacterial diseases, especially in cases in which antibiotic resistance is becoming more widespread. , mycobacterium tuberculosis is another intracellular bacterium that, despite the availability of antibiotics, remains one use of human immunoglobulin in at-risk populations. santosham and colleagues administered hyperimmunoglobulin (n = ), or saline placebo (n = ) to infants at , , and months of age and examined the rates of invasive hib. for the first days following the passive immunization protocol, none of the treated infants experienced invasive hib ( % incidence), compared to of placebo-treated children ( . % incidence, p = . ). rotavirus represents an enteric viral pathogen wherein protective passive immunotherapy has been demonstrated. [ ] [ ] [ ] [ ] [ ] [ ] in one example of postexposure treatment in infants, oral administration of hyperimmune antibody (in addition to standard supportive care) was able to efficiently reduce rotavirus shedding compared to placebo controls; treated patients (n = ) exhibited no evidence of viral shedding by day posttreatment as compared to % of controls (n = ). in a separate study, prophylactic passive immunity using orally administered bovine colostrum from immunized animals was tested in a blinded and randomized trial among infant children ( - months old) admitted to a hospital, typically for respiratory conditions. following admission, infants were given a -day course of the bovine colostrum or placebo. infants who received placebo contracted symptomatic rotavirus at a rate of % ( of ) whereas no symptomatic rotavirus disease was observed in the colostrum-treated infants ( of ; p < . ). analysis of rotavirus vaccine failures also indicates that maternally derived antibodies play a role in passive immunity to rotavirus infection. in a study involving vaccinated infants, a strong inverse correlation was observed between maternally derived rotavirus antibodies and the ability of infants to seroconvert following vaccination with a live rotavirus vaccine. this is an important demonstration not only of passive immunity to an enteric pathogen, but also has broader implications on the timing of vaccine administration, especially in developing countries where preexisting immunity is relatively high, and rotavirus vaccine immunogenicity appears impaired. e. coli is a significant enteric pathogen wherein prophylaxis through passive immunity has been demonstrated in several clinical studies. - tacket and colleagues were able to passively protect human subjects against experimentally induced e. coli diarrhea with specific bovine antibody. using heatinactivated or glutaraldehyde-inactivated e. coli for vaccination, pregnant cows were hyperimmunized with a large number of enterotoxigenic o serogroups. milk collected during the first days of lactation was purified, concentrated, lyophilized, and formulated for oral administration. as a control, a similar preparation was made using rotavirus as the immunizing antigen. subjects received daily treatment ( times daily) for days, with e. coli challenge administered days into the treatment regimen. of the subjects who received the e. coli antibody prophylaxis, all remained diseasefree following challenge, compared with clinical diarrhea in of placebo subjects (p < . ). using a closely related clinical protocol, otto and colleagues also demonstrated good efficacy with hyperimmune bovine colostrum tablets. in the first study conducted in this trial, of ( %) of placebo subjects contracted diarrhea following challenge, but this was reduced to only of ( %) in treated subjects (p = . ). in a second study investigating the impact of omitting buffer to the oral prophylaxis, the authors also examined dose sparing. in these studies, the standard dose still conferred significant protection with of ( %) treated subjects contracting diarrhea, compared with of ( %) of controls. interestingly, if the dose was reduced by one-half then disease incidence increased to of subjects ( %), indicating a key role played by treatment dose in achieving successful passive immunotherapy. developed at similar rates among all three groups (p = . ). however, because administration of immunoglobulin is typically only performed during the first months after transplantation and antiviral antibody half-life is estimated to be approximately days, it is not surprising that the protective effects of passive immunotherapy were only maintained through the first year. nevertheless, the inadvertent discovery of the protective role of antibodies in preventing ebv-induced non-hodgkin lymphoma represents a potential breakthrough in clinical management of this vulnerable patient population. despite decades of research aimed at finding a vaccine or a cure for hiv infection, this virus remains a scourge of global proportions. early attempts at passive immunotherapy using first-generation hiv-specific monoclonal antibodies were not highly effective [ ] [ ] [ ] and this approach was not further pursued until a new generation of highly potent and broadly neutralizing antibodies were identified. , in particular, a recent phase i clinical trial involving a single administration of a broadly neutralizing antibody, bnc , has renewed interest in the study of passive immunotherapy for hiv prevention and therapeutic intervention. bnc is an anti-cd binding site antibody that neutralizes of hiv strains comprising six different clades and was tested in a doseescalation study among hiv-positive patients with different levels of viremia. at a dose of or mg/kg, patient viral load was reduced by up to . log (average decline: . log ) in of individuals. the subject that did not respond to antibody treatment at mg/kg was infected with a resistant strain of hiv. although the effect of antibody therapy on viremia was mainly transient after a single administration, the viral load remained lower than their preexisting set point in of patients at days and one subject exhibited viremia levels that remained near the limits of detection throughout the -day study. it is currently unclear if hiv viremia in these patients will eventually rebound to their original levels. similar results were observed during antibody-based therapy of shiv-infected rhesus macaques in which most animals showed a rebound in viral replication after the transferred monoclonal antibodies declined to undetectable levels but a subset of animals maintained virological control in the absence of further infusions. combinations of antiretroviral drugs are currently the standard of care for treatment of hiv infection and it is unlikely that one dose of a single monoclonal antibody will be sufficient to have a long-term clinical benefit among a broad patient base. however, there is growing optimism that combining a cocktail of potent, broadly neutralizing monoclonal antibodies with antiretroviral drugs and/ or agents that activate latent virus reservoirs could theoretically provide long-term reduction in viral load and reduce the rates of transmission. with substantial advances in monoclonal antibody technologies and an increasing appreciation for the role of antibodies in the control of infectious disease, the development of sophisticated new passive immunotherapies is likely to continue at an accelerated pace. antibiotic resistance among clinically relevant bacteria including multidrug-resistant (mdr) and xdr m. tuberculosis, methicillin-resistant staphylococcus aureus (mrsa), and dominant strains of antibiotic-resistant salmonella typhi and other gram-negative bacterial species is a growing concern. , , [ ] [ ] [ ] this, coupled with the knowledge that fewer new antibiotics are moving through the drug pipeline, , may further motivate research into the development of antibody-based therapies to overcome these challenges to clinical intervention against microbial disease. one drawback to passive immunization is that antibody half-life in vivo of the most common human diseases and it is estimated to infect up to one-third of the world's population. the development of strains of extensively drug-resistant (xdr) tuberculosis (tb), some of which are resistant to all current antibiotic therapies, , is also a growing concern, especially as there are few antibiotic drugs in the pipeline. , there is considerable debate over the role of antibodies in controlling tb, with many believing that antibody plays little or no role in protective immunity (reviewed in references and ). in a comprehensive historical review by glatman-freedman and casadevall, the clinical benefit of antibody-mediated immunotherapy, albeit quite variable, provides evidence to suggest that antibody plays a role in protection against tb. in studies reported by paquin in , a group of patients with pulmonary tb confirmed by the presence of bacterium in their sputum showed clinical benefit. after months of passive immunotherapy, % of patients showed reduced cough, reduction in bacterial load in sputum, clearance of pulmonary infiltrates, reduction in hemoptysis, improved appetite, and weight gain. , at months after initiating treatment, all the treated patients were alive and more than half were discharged from the hospital. in contrast, more than untreated tb patients from another ward in the hospital had died within months of starting the study. experimental proof of antibody-mediated protection against tb was also published in by fisch. , after lethal tb challenge of guinea pigs, administration of immune serum was performed on days , , and , with further doses administered every other day for weeks and once a week after that. fisch reported that of treated animals were alive after . months ( % survival). if treatment was delayed until day postchallenge, then of ( %) animals survived but showed signs of illness. if no antibody treatment was performed, then of ( %) of the animals survived past day . the same approach was used to treat patients with pulmonary tb. all of the patients treated at the earliest stages of disease improved rapidly after passive immunotherapy and were tuberculin negative at the end of the study. of the patients treated at the "incipient" stage of disease, % no longer had bacilli in their sputum and were considered cured and % showed substantial improvement in disease symptoms. the patients with advanced tb showed only modest or no improvement after therapy and it was concluded that immune serum was only beneficial in early but not advanced cases of disease. ebv is a common human pathogen that causes a chronic infection and is a leading cause of posttransplant non-hodgkin lymphoma resulting from the uncontrolled proliferation of ebv-infected b lymphocytes in patients undergoing immunosuppressive therapies. in a large retrospective study involving , kidney transplant patients, the effect of prophylactic treatment for cytomegalovirus (cmv) on posttransplant incidence of non-hodgkin lymphomas was examined. the standardized incidence ratio (sir) for non-hodgkin lymphoma was expressed as the number of lymphoma cases per , persons and calculated after normalizing for age, sex, and geographical origin. the , patients who did not receive cmv prophylaxis had a sir = . , which remained unchanged (sir = . , p = . ) among the , patients who received antiviral drugs (acyclovir or ganciclovir). in striking contrast, the patients who received anti-cmv immunotherapy showed a complete absence of lymphomas during the first year after transplantation (sir = , p = . vs. antiviral treatment). the most common anti-cmv immunoglobulin products were shown to contain antibodies against ebv and it is believed that this is the mechanism of action for the protection afforded during the first year posttransplantation. in the subsequent years of follow-up, new cases of lymphoma sufficient for protection or therapeutic intervention of acute or remittent disease, active immunization through improved vaccine design may still be needed to train the host immune system to maintain long-term levels of protective immunity. importantly, examples of successful passive immunization approaches may provide a useful framework for developing new and improved vaccines that elicit the most protective antibody responses. references for this chapter are available at expertconsult.com. often provides only transient protection unless repeated administrations are performed. this may change as new technologies that increase the half-life of monoclonal antibodies are employed. for example, the fc region of an anti-rsv monoclonal antibody, motavizumab, was mutated to increase its binding to the neonatal fc receptor (fcrn), resulting in serum antibody pharmacokinetics in human subjects that increased from 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protection against yersinia pestis human immune response to a plague vaccine comprising recombinant f and v antigens administration of antibody to the lung protects mice against pneumonic plague passive immunity to yersiniae mediated by anti-recombinant v antigen and protein a-v antigen fusion peptide roles of v antigen in promoting virulence and immunity in yersiniae les épidémies de peste en extrême-orient. xiiie congrès international de médecine sur la peste bubonique (sérothérapie) prophylaxis and therapy for chikungunya virus infection chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes case reports of neuro-chikungunya in southern thailand antigenic sites of coxsackie a virus inducing neutralizing monoclonal antibodies protective in mice a murine model of coxsackievirus a infection for anti-viral evaluation effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration vaccination for the prevention of maternal and fetal infection with guinea pig cytomegalovirus rat cytomegalovirus vaccine prevents accelerated chronic rejection in cmvnaive recipients of infected donor allograft hearts complete protection of mice against lethal murine cytomegalovirus challenge by immunization with dna vaccines encoding envelope glycoprotein complex iii antigens gh, gl and go passive immunization during pregnancy for congenital cytomegalovirus infection prophylaxis of primary cytomegalovirus disease in renal transplant recipients. a trial of ganciclovir vs immunoglobulin cmv-hyperimmune globulin for preventing cytomegalovirus infection and disease in solid organ transplant recipients: a meta-analysis the development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type epitope determinants of a chimpanzee dengue virus type (denv- )-neutralizing antibody and protection against denv- challenge in mice and rhesus monkeys by passively transferred humanized antibody development of a humanized antibody with high therapeutic potential against dengue virus type ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies therapeutic intervention of ebola virus infection in rhesus macaques with the mb- monoclonal antibody cocktail treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee preventive effect of igg from ebv-seropositive donors on the development of human lympho-proliferative disease in scid mice a mouse monoclonal antibody against epstein-barr virus envelope glycoprotein prevents infection both in vitro and in vivo active and passive vaccination against hantavirus pulmonary syndrome with andes virus m genome segment-based dna vaccine dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome a non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome by andv the use of human immune serum globulin (gamma globulin) in infectious (epidemic) hepatitis in the mediterranean theater of operations i. studies on prophylaxis in two epidemics of infectious hepatitis prevention of infectious hepatitis with gamma globulin the prevention and attenuation of infectious hepatitis by gamma globulin gamma globulin in the prevention of infectious hepatitis; studies on the use of small doses in family outbreaks preclinical evaluation of two human anti-hepatitis b virus (hbv) monoclonal antibodies in the hbv-trimera mouse model and in hbv chronic carrier chimpanzees hepatitis b immune globulin as a prophylactic measure for spouses exposed to acute type b hepatitis liver transplantation in hbsag-positive hbv-dna-negative cirrhotics: immunoprophylaxis and long-term outcome liver transplantation in hbs antigen (hbsag) carriers. prevention of hepatitis b virus (hbv) recurrence by passive immunization human monoclonal antibody hcv effectively prevents and treats hcv infection in chimpanzees sexual transmission of the hepatitis c virus and efficacy of prophylaxis with intramuscular immune serum globulin. a randomized controlled trial human monoclonal antibody mbl-hcv delays hcv viral rebound following liver transplantation: a randomized controlled study immunotherapeutic potential of neutralizing antibodies targeting conserved regions of the hcv envelope glycoprotein e clinical evaluation (phase i) of a human monoclonal antibody against hepatitis c virus: safety and antiviral activity successful passive and active immunization of cynomolgus monkeys against hepatitis e role of immune serum globulins in pregnant women during an epidemic of hepatitis e passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein d and monoclonal antibodies to glycoprotein b effect of antibody alone and combined with acyclovir on neonatal herpes simplex virus infection in guinea pigs l or more. effect on viral, opportunistic, and bacterial infections. the national institute of child health and human development intravenous immunoglobulin clinical trial study group intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv- infection in a transgenic mouse model antibody-based protection against hiv infection by vectored immunoprophylaxis hiv therapy by a combination of broadly neutralizing antibodies in humanized mice hiv- suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice passive immunotherapy in aids: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus antibodies vs. transfusions of seronegative plasma delay of hiv- rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies safety and efficacy of hiv hyperimmune globulin for prevention of motherto-child hiv transmission in hiv- -infected pregnant women and their infants in kampala passive immunotherapy in the treatment of advanced human immunodeficiency virus infection a murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection in vivo mechanisms of vaccine-induced protection against hpv infection the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory metaanalysis protection of mice against japanese encephalitis virus by passive administration with monoclonal antibodies passive protection of mice, goats, and monkeys against japanese encephalitis with monoclonal antibodies protection of monkeys against machupo virus by the passive administration of bolivian haemorrhagic fever immunoglobulin (human origin) protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the h protein mimicked by peptides which are not recognized by maternal antibodies correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus a protective therapy for mumps mumps; use of convalescent serum in the treatment and prophylaxis of orchitis serum prophylaxis of epidemic parotitis use of human blood in protection against mumps intravenous immunoglobulin therapy for pure red cell aplasia related to human parvovirus b infection: a retrospective study of patients and review of the literature guidelines on the use of intravenous immune globulin for hematologic conditions the relation of the meninges and choroid plexus to poliomyelitic infection passive immunity in poliomyelitis. iv. protection of rhesus monkeys against cerebral challenge experimental poliomyelitis in monkeys seventh note: active immunization and passive serum protection evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. iv. final report of results based on clinical diagnoses evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. . reanalysis of results based on laboratory-confirmed cases resume of the results of therapy with convalescent serum in poliomyelitis intramuscular use of convalescent serum in treatment of poliomyelitis preparalytic poliomyelitis: observations in one hundred and six cases in which convalescent serum was used recherches sur la vaccination antirabique evaluation of human rabies immune globulin and homologous and heterologous antibody antiserum in the prophylaxis of rabies comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin use of hyperimmune anti-rabies serum concentrates in experimental rabies laboratory data supporting the clinical trial of anti-rabies serum in persons bitten by a rabid wolf management of rabies in humans development of a humanized monoclonal antibody (medi- ) with potent in vitro and in vivo activity against respiratory syncytial virus quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats studies of passive immunotherapy for infections of respiratory syncytial virus in the respiratory tract of a primate model isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (fab rsvf - ) that exhibits therapeutic efficacy in vivo respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children active and passive immunization against rift valley fever virus infection in syrian hamsters topological mapping of antigenic sites on the rift valley fever virus envelope glycoproteins using monoclonal antibodies passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp and vp serum igg mediates mucosal immunity against rotavirus infection a gastrointestinal rotavirus infection mouse model for immune modulation studies rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection prophylaxis of german measles with immune serum globulin prevention of rubella by gamma globulin during an epidemic in barrow, alaska, in trial of high-titre human rubella immunoglobulin rubella epidemic, : effect on , pregnancies passive immunization against rubella: studies on the effectiveness of rubella-immunoglobulin after intranasal infection with rubella vaccination virus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection passive immune globulin therapy in the siv/macaque model: early intervention can alter disease profile passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies fc receptor but not complement binding is important in antibody protection against hiv protection of macaques against pathogenic simian/human immunodeficiency virus . pd by passive transfer of neutralizing antibodies determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies highly potent hiv-specific antibody neutralization in vitro translates into effective protection against mucosal shiv challenge in vivo antibody-mediated immunotherapy of macaques chronically infected with shiv suppresses viraemia passive transfer of modest titers of potent and broadly neutralizing anti-hiv monoclonal antibodies block shiv infection in macaques neutralizing antibody directed against the hiv- envelope glycoprotein can completely block hiv- /siv chimeric virus infections of macaque monkeys pre-and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model passive immunization of mice with monoclonal antibodies raised against tickborne encephalitis virus efficiency of use of immunoglobulin preparations for the postexposure prevention of tickborne encephalitis in russia (a review of semi delayed humoral immunity in a patient with severe tick-borne encephalitis after complete active vaccination combination therapy of vaccinia virus infection with human anti-h and anti-b monoclonal antibodies in a small animal model disparity between levels of in vitro neutralization of vaccinia virus by antibody to the a protein and protection of mice against intranasal challenge protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies postexposure prevention of progressive vaccinia in scid mice treated with vaccinia immune globulin prophylactic effect of antivaccinia gamma-globulin against post-vaccinal encephalitis experience of anti-vaccinia immunoglobulin in the united kingdom complications of smallpox vaccination. effects of vaccinia immune globulin therapy prevention of varicella by zoster immune globulin the use of serum gamma globulin antibodies to control chicken pox in a convalescent hospital for children congenital varicella syndrome: the evidence for secondary prevention with varicella-zoster immune globulin a humanized murine monoclonal antibody protects mice either before or after challenge with virulent venezuelan equine encephalomyelitis virus antibody prophylaxis and therapy against west nile virus infection in wild-type and immunodeficient mice efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection development of a humanized monoclonal antibody with therapeutic potential against west nile virus possible benefit of intravenous immunoglobulin therapy in a lung transplant recipient with west nile virus encephalitis treatment of west nile virus encephalitis with intravenous immunoglobulin lethal d yellow fever encephalitis in mice. i. passive protection by monoclonal antibodies to the envelope proteins of d yellow fever and dengue viruses the duration of passive immunity in yellow fever persistence of yellow fever immunity on the use of immune serum at various intervals after the inoculation of yellow fever virus into rhesus monkeys notes on laboratory infections with yellow fever observations on laboratory and hospital infections with yellow fever in england immunoprotection against systemic candidiasis in mice rats clearing a vaginal infection by candida albicans acquire .e section general aspects of vaccination specific, antibody-mediated resistance to vaginal reinfection a vaccine and monoclonal antibodies that enhance mouse resistance to candida albicans vaginal infection vaccine and monoclonal antibody that enhance mouse resistance to candidiasis immunoglobulin g blocking antibodies to the fungal pathogen cryptococcus neoformans human immunoglobulin g (igg ) and igg , but not igg or igg , protect mice against cryptococcus neoformans infection more is not necessarily better: prozone-like effects in passive immunization with igg phase i evaluation of the safety and pharmacokinetics of murine-derived anticryptococcal antibody b in subjects with treated cryptococcal meningitis enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency efficacy of monoclonal antibodies against defined antigens for passive immunotherapy of chronic gastrointestinal cryptosporidiosis prophylactic effect of bovine anti-cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with cryptosporidium parvum demonstration of passive immunity in experimental monkey malaria model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum the importance of human fcgammari in mediating protection to malaria gamma-globulin and acquired immunity to human malaria antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes monoclonal antibodies to toxoplasma cell membrane surface antigens protect mice from toxoplasmosis generation of a neutralizing human monoclonal antibody fab fragment to surface antigen of toxoplasma gondii tachyzoites protection against experimental toxoplasmosis by adoptive immunotherapy impact of trough igg on pneumonia incidence in primary immunodeficiency: a meta-analysis of clinical studies high-vs low-dose immunoglobulin therapy in the long-term treatment of x-linked agammaglobulinemia history of immunoglobulin replacement use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology effect of intravenous gammaglobulin therapy in igg deficient and igg sufficient children with recurrent infections and poor response to immunization with hemophilus influenzae type b capsular polysaccharide antigen intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection. the national institute of child health and human developments intravenous immunoglobulin study group polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and metaanalysis supplemental immune globulins in sepsis: a critical appraisal prevention of infection in multiple trauma patients by high-dose intravenous immunoglobulins the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis immunity in mumps: i. experiments with monkeys (macacus mulatta). the development of complement-fixing antibody following infection and experiments on immunization by means of inactivated virus and convalescent human serum an experimental study of parotitis key: cord- -pv re x authors: mestecky, jiri; russell, michael w. title: specific antibody activity, glycan heterogeneity and polyreactivity contribute to the protective activity of s-iga at mucosal surfaces date: - - journal: immunol lett doi: . /j.imlet. . . sha: doc_id: cord_uid: pv re x an explanation of the principles and mechanisms involved in peaceful co-existence between animals and the huge, diverse, and ever-changing microbiota that resides on their mucosal surfaces represents a challenging puzzle that is fundamental in everyday survival. in addition to mechanical barriers and a variety of innate defense factors, mucosal immunoglobulins (igs) provide protection by two complementary mechanisms: specific antibody activity and innate, ig glycan-mediated binding, both of which serve to contain the mucosal microbiota in its physiological niche. thus, the interaction of bacterial ligands with iga glycans constitutes a discrete mechanism that is independent of antibody specificity and operates primarily in the intestinal tract. this mucosal site is by far the most heavily colonized with an enormously diverse bacterial population, as well as the most abundant production site for antibodies, predominantly of the iga isotype, in the entire immune system. in embodying both adaptive and innate immune mechanisms within a single molecule, s-iga maintains comprehensive protection of mucosal surfaces with economy of structure and function. large surface areas of mucosal membranes (∼ - m ) are in constant contact with a highly diverse microbiota [ ] [ ] [ ] [ ] [ ] [ ] estimated to comprise ∼ , - , species and genera [ , ] and exceeding the total number of nucleated cells by an order of magnitude [ , , , ] ( nucleated cells vs. ∼ bacterial cells). more than . % of all commensal bacteria are found in the gastrointestinal tract, particularly in the large intestine [ , ] . through evolution, the selective pressure arising from environmental antigens of microbial and food origin has resulted in a strategic, functionally advantageous distribution of cells involved in antigen uptake and processing, and the initiation of immune responses in mucosal tissues [ , [ ] [ ] [ ] . the mucosal immune system contains this antigenic onslaught without compromising the integrity of the mucosal barrier [ ] or exhausting the immune system, in part through the induction of mucosal (oral) tolerance [ , ] . in addition to mechanical barriers and humoral effectors of innate immunity [ , , ] , mucosal antibodies and mucosal t cells provide antigen-specific protection [ , ] . the characteristic distribution of antibodies in blood and external secretions, including the intestinal fluid, reflects the functional adaptation of various ig isotypes to different immune compartments. given that mucosal membranes are the most important site of antigen encounter, it should not be surprising that most antibody production takes place in mucosal tissues, particularly the intestine, rather than in the bone marrow, spleen, and lymph nodes [ , [ ] [ ] [ ] [ ] , and that the daily synthesis of iga far exceeds that of igg, igm, igd and ige combined [ ] [ ] [ ] [ ] . importantly for mucosal protection, more than two-thirds of total iga production ends up in the external secretions [ , ] . quantitative studies of the origin of mucosal antibodies, particularly in the intestinal tract, demonstrate that > % is of local origin and only trace amounts are derived from the circulation [ , , ] . the mucosal microbiota, epithelial cells, and the mucosal immune system constitute a stable and interdependent "tripod" that maintains mucosal homeostasis by complex mechanisms [ , , , [ ] [ ] [ ] [ ] [ ] . for example, epithelial cells display surface receptors that are selectively exploited by bacteria adhering to their apical surfaces [ , , [ ] [ ] [ ] , and express the basolateral membrane receptor (polymeric ig receptor; pigr) that transports locally produced polymeric (p) iga into the external secretions [ ] . bacteria table examples of glycans as adhesion sites and receptors for selected bacteria and viruses that colonize, or infect, mucosal surfaces (adapted from [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] endogenous to the intestinal tract, oral cavity, and probably also the respiratory and genital tracts, are coated in vivo with s-iga [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] that limits their epithelial adherence and penetration, thereby confining them to the mucosal surfaces. numerous models have demonstrated the role of antibodies, especially s-iga, in protecting the intestinal and other mucosal tracts. this has most convincingly been demonstrated in vivo in germ-free, colostrumdeprived newborn piglets [ ] [ ] [ ] , which, unlike humans, mice, rats, or rabbits, are born without transplacentally acquired ig. in the absence of maternal as well as endogenous antibodies, milkdeprived piglets die of septicemia (usually e. coli) within - days after birth, whereas milk-fed animals survive [ ] . furthermore, piglets fed milk or serum, survive oral challenge with e. coli, whereas control animals deprived of ig, irrespective of its source, succumb to the infection. in mice in which pigr is copiously expressed on hepatocytes (not the case in humans, pigs, or dogs) and piga from the circulation is selectively transported into the bile and thence into the gut lumen [ , ] , pathogen-specific piga hybridoma antibodies derived from "backpack tumors" [ ] [ ] [ ] [ ] protect mice against oral challenge with salmonella enterica serovar typhimurium, vibrio cholerae, or rotavirus [ , , [ ] [ ] [ ] . in contrast igg hybridoma antibodies of the same specificity are not protective, due to the lack of receptor-mediated transport of igg into the intestine. numerous such experiments clearly demonstrate protection in vivo dependent on the presence of antigen-specific iga antibodies that interfere with pathogen adherence to or penetration through the mucosal barrier, or neutralize biologically active antigens such as viruses or toxins [ , , , [ ] [ ] [ ] [ ] [ ] . likewise many in vitro studies of specific antibody-mediated inhibition of bacterial adherence to epithelial cells corroborate these findings [ , [ ] [ ] [ ] . however, agglutination and inhibition of the adherence of e. coli with type fimbriae to colonic epithelial cells that express a corresponding receptor can be mediated by iga independently of specific antibody [ , , ] . s-iga and iga myeloma proteins of both subclasses agglutinate e. coli, and mannose (man) inhibits this agglutination. furthermore, adherence of e. coli to human epithelial colonic cells can be inhibited by s-iga as well as by iga myeloma proteins. analysis of the carbohydrate composition and complete primary structure of the oligosaccharide side-chains reveal that the most active piga myeloma protein contain several man-rich n-linked glycan chains [ ] . thus, man-dependent adherence of e. coli to epithelial cell receptors mediated by type fimbriae is competitively inhibited by similar glycans on s-iga and iga myeloma proteins acting as decoy receptors. consequently, we have proposed that iga proteins exhibit protective functions through antibodydependent specific immunity as well as glycan-dependent innate immunity [ ] . this concept was confirmed in vitro for other microbial ligand-glycan receptors [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to e. coli, many other bacteria such as helicobacter pylori, streptococcus pneumoniae, clostridium difficile, shigella flexneri, pseudomonas aeruginosa and neisseria gonorrhoeae, and some viruses (table ) interact with epithelial receptors via their glycan moiety. thus, it has become obvious that the nand o-glycans of s-iga provide a link between innate glycan-mediated and adaptive specific antibody-dependent protection (fig. ) . this concept, of paramount importance in iga-mediated mucosal defense, prompts additional considerations. first, it has been shown that bacteria indigenous to the oral cavity and intestinal tract are coated in vivo with iga [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, it is not known whether this coating depends on specific antibody-antigen or glycan-mediated interactions. considering the enormous numbers of bacteria (∼ /g of intestinal content) [ ] , their diversity (∼ , - , species of genera) [ ] , and the large number of potential antigenic determinants on many bacterial structures, it is unlikely that such coating is based exclusively on specific recognition by s-iga antibodies. secondly, in the large intestine iga -producing cells are dominant in contrast to other mucosal tissues [ , ] , and antibodies to antigens (e.g., endotoxin) of gram- fig. . model of a human dimeric s-iga molecule with assigned adaptive (specificantibody) activity and innate (glycan-dependent) activity [ ] . asterisk-possible nglycosylation sites within the cdr segment of the vh region of ␣ chains [ , , ] . negative bacteria are associated predominantly with the s-iga subclass [ ] [ ] [ ] . thirdly, in addition to glycans on the h chain of iga [ ] [ ] [ ] [ ] [ ] , secretory component (sc), the extracellular segment of pigr, is extremely rich in glycans comprising n-linked chains [ , [ ] [ ] [ ] that also act as highly effective inhibitors of adherence for some bacterial species (e.g., shigellae, s. pneumoniae [ , , [ ] [ ] [ ] [ ] ). finally, prevention of the adherence of enormously diverse and variable mucosal microbiota is likely to be at least partially independent of specific antibody activity, reflecting the immediate need for protection against a broad spectrum of daily encountered microorganisms. thus, in concert with the postulated fab-mediated "polyreactivity" of s-iga antibodies [ ] [ ] [ ] [ ] [ ] [ ] , glycan-mediated interactions are likely to further enforce protective functions of s-iga. skeptics of these concepts may argue that mucosal defenses in iga-deficient individuals should be significantly compromised. indeed, the majority of such patients display a higher incidence of respiratory and intestinal infections [ ] [ ] [ ] . currently, iga deficiency is defined as < mg iga/ ml of serum, regardless of s-iga which is not taken into account although it is usually also diminished. because complete absence of iga in sera or secretions of igadeficient patients is extremely rare, it is possible that even low levels of s-iga may provide some level of protection. furthermore, in most iga-deficient individuals s-iga is replaced by s-igm [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thus, in iga-deficient individuals, - % of total ig-containing cells in the intestines produce igm, in sharp contrast to normal individuals (in the large intestine, ∼ % of cells produce iga, ∼ % igm, and ∼ % igg) [ , ] . most importantly in this context, igm and iga display many common structural features, including: ability to form polymers; presence of j chain [ , ] ; ability to bind pigr (thereby forming sc-containing secretory igm) [ , , , ] ; homologies between primary structures of c , c and c␣ , c␣ domains and the c-terminal "tail-piece" [ , , ] ; and vh gene segment representation [ , ] , indicating their close evolutionary and functional similarity [ ] . this structural homology also extends to the glycan moieties: igm and iga molecules display a similar number and domain location of n-linked glycan side-chains, and both contain man-rich chains [ , [ ] [ ] [ ] [ ] [ ] . therefore, despite differences in the number of ag-binding sites (up to for igm, and - for iga dimers and tetramers), and ability to activate complement, it is clear that iga and igm are structurally, functionally, and evolutionarily closely related isotypes. structural studies of human polyclonal s-iga and monoclonal (myeloma) iga and iga proteins reveal considerable heterogeneity with respect to number, sites of attachment, composition, and primary structure of their glycan side-chains [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] , which is likely to be of enormous biological importance. because different microorganisms interact with epithelial cells through diverse glycan receptors, heterogeneity of iga-associated glycans affords a variety of structures that can effectively inhibit these interactions. glycan moieties in s-iga molecules are associated with h chains, j chain, and sc [ , [ ] [ ] [ ] [ ] [ ] ] , but man-rich n-linked glycans that inhibit the binding of type fimbriae to epithelial receptors occur only on the h chains [ , ] . however, other bacteria may interact with nor o-linked glycans on h chains or sc (table , section b) . although the majority of n-linked glycans are found in the fc region of the ␣ chains [ ] [ ] , , ] , there is great heterogeneity in the number and composition of individual glycan chains [ , ] and additional n-linked glycan chains may also be present in the fd fragment (n-terminal half of the ␣ chain comprising vh and ch domains), within the third complementarity-determining region (cdr ) [ , , ] . the authors of these novel and functionally important studies propose that a high rate of somatic mutation in the cdr taking place within intestinal iga-producing cells [ , , , , , ] generates a glycosylation-signaling sequence that alters the specificity of intestinal iga antibodies. thus, antigen-binding by fab segments of s-iga is determined by both specific antibody activity and glycandependent interactions. the heterogeneity of nand o-linked side-chains, with respect to their number, composition, and types of glycosidic bonds is further extended because many of them are incomplete, truncated forms [ , , , ] . most importantly, and in sharp contrast to the combinatorial possibilities of amino acids, glycans can generate a remarkably higher number of structures, due to the variety of glycosidic bonds. thus, a sequence of (out of ) amino acids can theoretically generate . × distinct hexapeptides, while there are potentially . × different hexasaccharides [ ] . specific antibody diversity is generated in an antigenindependent fashion during the differentiation of b lymphocytes by a number of mechanisms including recombination of multiple vj (for l chains) and vdj (for h chains) gene segments, combinatorial diversity of l and h chains, somatic hypermutation, gene conversion, and others [ ] . the result of these genetic events is the generation of b lymphocytes with surface membrane ig molecules that accommodate an enormous number of potential antigens, leading, after antigen-specific recognition, to b cell proliferation, differentiation, and the ultimate secretion of large amounts of antigen-specific antibodies. it is conceivable that analogous mechanisms operate in the generation of innate, glycanmediated mechanisms of protection. through random generation of enormously diverse glycan structures on mucosal glycoproteins, including s-iga, s-igm, sc, mucin, and lactoferrin, glycan configurations are generated that complement the equal heterogeneity of microbial adhesins. the protective effectiveness of these mechanisms may be further enhanced by subsequent somatic mutations within v regions of h and l chains, including the generation of glycosylation signals that lead to alterations of antibody specificities. parallel structural and functional exploration of the principles of adaptive (specific antibody) and innate (glycan) s-iga-mediated immunity is likely to generate novel approaches to the 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effect of antibodies in the intestinal tract of germ-free baby pigs the role of immune pig colostrum, serum and immunoglobulins igg, igm, and iga, in local intestinal immunity against enterotoxic strain in escherichia coli o in germfree piglets the effect of antibodies present in the intestinal tract of germfree piglets on the infection caused by the intravenous administration of the pathogenic strain escherichia coli phylogeny and comparative physiology of iga monoclonal secretory iga for protection of the intestinal mucosa against viral and bacterial pathogens monoclonal secretory immunoglobulin a protects mice against oral challenge with the invasive pathogen salmonella typhimurium passive immunization: systemic and mucosal biological activities of iga innate secretory antibodies protect against natural salmonella typhimurium infection mucosal immune defense: immunoglobulin a defense mechanisms involving fcdependent functions of immunoglobulin a and their subversion by bacterial immunoglobulin a proteases microbial pathogens and the intestinal epithelial cell intestinal iga: novel views on its function in the defence of the largest mucosal surface iga response to symbiotic bacteria as a mediator of gut homeostasis using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases chemical identification of a glycosphingolipid receptor for escherichia coli attaching to human urinary tract epithelial cells and agglutinating human erythrocytes anti-pili antibodies in breast milk secretory immunoglobulin a and g antibodies prevent adhesion of escherichia coli to human urinary tract epithelial cells iga oligosaccharide chains as receptors for bacterial lectins agglutination of e. coli by secretory iga-a result of interaction between bacterial mannose-specific adhesins and immunoglobulin carbohydrate? nonimmune binding of human immunoglobulin a to type ii group b streptococci the high lectin-binding capacity of human secretory iga protects nonspecifically mucosae against environmental antigens carbohydrate specificity of the surface lectins of escherichia coli, klebsiella pneumoniae, and salmonella typhimurium free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic escherichia coli specificity of s fimbriae on recombinant escherichia coli: preferential binding to gangliosides expressing neuac ␣ - )neuac the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces escherichia coli fimbriae recognizing sialyl galactosides the human polymeric immunoglobulin receptor binds to streptococcus pneumoniae via domains and glycans on secretory component participate in innate protection against mucosal pathogens secretory component: a new role in secretory iga-mediated immune exclusion in vivo novel functions for mucosal siga fab-independent antiadhesion effects of secretory immunoglobulin a on s-fimbriated escherichia coli are mediated by sialyloligosaccharides identification and characterization of binding properties of helicobacter pylori by glycoconjugate arrays salivary receptors for the proline-rich proteinbinding and lectin-like adhesins of oral actinomyces and streptococci coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque adhesion of viridans group streptococci to sialic acid-, galactose-and n-acetylgalactosamine-containing receptors a specific cell surface antigen of streptococcus gordonii is associated with bacterial hemagglutination and adhesion to alpha - -linked sialic acid-containing receptors specific inhibitors of bacterial adhesion: observations from the study of gram-positive bacteria that initiate biofilm formation on the tooth surface recognition of immunoglobulin a by oral actinomyces and streptococcal lectins circulating antibodies against faecal bacteria assessed by immunomorphometry: combining quantitative immunofluorescence and image analysis the role of b- cells in mucosal immune responses direct flow cytometry of anaerobic bacteria in human feces distribution of iga -, iga -, and j chain-containing cells in human tissues the human mucosal b-cell system immunoglobulin a subclass distribution of naturally occurring salivary antibodies to microbial antigens subclass distribution of iga antibodies to microbial and viral antigens iga subclasses of human colostral antibodies specific for microbial and food antigens heterogeneity of o-glycosylation in the hinge region of human iga secretory iga n-and o-glycans provide a link between the innate and adaptive immune systems location and structural significance of the oligosaccharides in human ig-a and iga immunoglobulins the moieties of iga, j chain, secretory piece and igd studies on human secretory immunoglobulin a. iv. carbohydrate composition carbohydrate moieties in human secretory component structures of the carbohydrate moieties of secretory component purified from human milk microdetermination of monosaccharides in glycoproteins by gas-liquid chromatography from natural polyreactive autoantibodies to a la carte monoreactive antibodies to infectious agents: is it a small world after all? diversity of antibody-mediated immunity at the mucosal barrier polyreactive binding of antibodies generated by polyclonal b cell activation. i. polyreactivity could be caused by differential glycosylation of immunoglobulins polyreactive binding of antibodies generated by polyclonal b cell activation. ii. crossreactive and monospecific antibodies can be generated from an identical ig rearrangement by differential glycosylation natural polyreactive secretory immunoglobulin a autoantibodies as a possible barrier to infection in humans natural polyreactive immunoglobulin a antibodies produced in mouse peyer's patches iga deficiency mucosal immunology mucosal immune defense: immunoglobulin a igm antibodies to streptococcus mutans in subjects with selective iga deficiency study of colostrum of a patient with selective iga deficiency human secretory immunoglobulin m. an immunochemical and immunohistochemical study immunoglobulin m: local synthesis and selective secretion in patients with immunoglobulin a deficiency the clinical condition of iga-deficient patients is related to the proportion of igd-and igm-producing cells in their nasal mucosa iga deficiency in children. immunoglobulin-containing cells in the intestinal mucosa, immunoglobulins in secretions and serum iga levels clinical findings and intestinal immunoglobulins in children with partial iga deficiency mucosal immunoglobulins immunoglobulin m and secretory immunoglobulin a: presence of a common polypeptide chain different from light chains secretory component of epithelial cells is a surface receptor for polymeric immunoglobulins structure, function, and evolutionary relationships of fc domains of human immunoglobulins a structure of the human iga subclasses and allotypes characteristics of igvh genes used by human intestinal plasma cells from childhood human iga-and igm-secreting intestinal plasma cells carry heavily mutated vh region genes structure of the high mannose oligosaccharides of a human igm myeloma protein. i. the major oligosaccharides of the two high mannose glycopeptides linkage and sequence analysis of mannose-rich glycoprotein core oligosaccharides by proton nuclear magnetic resonance spectroscopy localization and overall structure of a mannose-rich glycopeptide from a pathologic immunoglobulin characterization of the glycosylation of a human igm produced by a human-mouse hybridoma carbohydrate heterogeneity of human myeloma proteins of the iga and iga subclasses structural analysis of the n-glycans from human immunoglobulin a : comparison of normal human serum immunoglobulin a with that isolated from patients with rheumatoid arthritis the glycosylation and structure of human serum iga , fab, and fc regions and the role of n-glycosylation on fc␣ receptor interactions determination of aberrant o-glycosylation in the iga hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry analysis of o-glycan heterogeneity in iga myeloma proteins by fourier transform ion cyclotron resonance mass spectrometry: implications for iga nephropathy human serum iga is substituted with up to six o-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry effect of somatic hypermutation on potential n-glycosylation sites in human immunoglobulin heavy chain variable regions strong intrinsic biases towards mutation and conservation of bases in human igvh genes during somatic hypermutation prevent statistical analysis of antigen selection glycomics aims to interpret the third molecular language of cells immunoglobulins: molecular genetics virus infection of epithelial cells this work was supported in part by grants, u ai , the czech republic (vzmsm ), de , ai , and the john r. oishei foundation. key: cord- -zhwr h authors: oxford, john; gilbert, anthony; lambkin-williams, robert title: influenza vaccines have a short but illustrious history of dedicated science enabling the rapid global production of a/swine (h n ) vaccine in the current pandemic date: - - journal: influenza vaccines for the future doi: . / - - - - _ sha: doc_id: cord_uid: zhwr h vaccines for the swine flu pandemic of have been produced in an exquisitely short time frame. this speed of production comes because of years of hard work by virologists worldwide in pharma groups, research laboratories, and government licensing units. the present chapter presents the background framework of influenza vaccine production and its evolution over years. isolation of the causative virus of influenza in , followed by the discovery of embryonated hen eggs as a substrate, quickly led to the formulation of vaccines. virus-containing allantoic fluid was inactivated with formalin. the phenomenon of antigenic drift of the virus ha was soon recognized and as who began to coordinate the world influenza surveillance, it became easier for manufacturers to select an up-to-date virus. influenza vaccines remain unique in that the virus strain composition is reviewed yearly, but modern attempts are being made to free manufacturers from this yolk by investigating internal virus proteins including m e and np as “universal” vaccines covering all virus subtypes. recent technical innovations have been the use of vero and mdck cells as the virus cell substrate, the testing of two new adjuvants, and the exploration of new presentations to the nose or epidermal layers as dna or antigen mixtures. the international investment into public health measures for a global human outbreak of avian h n influenza together with a focus of swine influenza h n is leading to enhanced production of conventional vaccine and to a new research searchlight on t-cell epitope vaccines, viral live-attenuated carriers of influenza proteins, and even more innovative substrates to cultivate virus, including plant cells. when the influenza a virus first emerged from a presumed avian reservoir at the end of the ice age , or so years ago, there was a distinct difficulty in finding new human victims. for example, at that time, only a few hundred settlers were in the london region near the royal london hospital, now a community of four million people. at that time a traveler would have to walk a miles to find another small settlement, perhaps at stonehenge near salisbury. nowadays we have a truly global community of six billion people, linked so that two million people are moving each day by plane, while perhaps ten million are journeying in their homelands. influenza, like all viruses, is opportunistic. in , it had the unprecedented opportunity to spread at the end of the first global war. ten million soldiers began the move homewards, and every steamship was packed as they fanned out from france to england, europe, the usa, canada, australia, india, and se asia [ ] [ ] [ ] . how perfect for a virus spread by aerosol droplets, close contact, and contamination of towels, cups, and every day utensils. a virgin population, which had never before encountered the avian virus (h n ), was on the stage of this theater of infection. perhaps, a billion people were infected in the next months, and - million died, making this by far the biggest outbreak of infectious diseases ever recorded, with an impact many times greater than the so-called bubonic plague outbreaks in medieval europe. however, more than two billion people survived. the overall mortality was less than %, although in a few semi-closed societies of hunter-gatherers in the arctic, the mortality from the disease and subsequent starvation as young hunters died and husky dogs attacked and ate the survivors exceeded % [ ] [ ] [ ] [ ] . it is well to remember that when h n emerged in / and became pandemic in everyone except for the over s were fully susceptible. this is different from today where most people on planet earth have immune memory to the h n family of viruses and by definition to a/swine flu. this explains why the current h n vaccine is immunogenic. while most people in the world were infected, we are forced to view the innate protective power of our immune system with awe [ , ] . we are equipped with , genes, seven million years of evolution, and , years of specialization since our emergence from africa. in contrast, influenza is a miniscule eight-gene vehicle. a recent study [ ] of the reproductive number (r ) of the virus suggests that, unexpectedly, it may have been quite low, not exceeding three persons infected with a single case. the current pandemic a/swine h n virus is not so different. this would place pandemic influenza not far above the lowly group of viruses such as small pox and sars and not reaching the heights that measles has attained. however, this unexpected theoretical analysis, if it is not flawed, gives us more practical opportunities to break a chain of infection of a pandemic with antivirals, hygiene, and vaccines [ ] [ ] [ ] . we are experimenting with these approaches at the present moment. the new world of the twenty-first century, although harboring in some countries a few old-fashioned attitudes, akin to "influenza and pneumonia is the old person's friend" nevertheless has the capability for the first time to defend itself against mother nature and her threat of influenza. for the first time in history, intense surveillance by the world health organization (who), early identification of a new pandemic influenza virus by molecular diagnostics, application of vaccination and antiviral chemoprophylaxis, and possible quarantine and masks could actually prevent a pandemic arising. for the expressed intention of who and the world community of infectious disease researchers is to deflect the first wave of the first pandemic of the twenty-first century. in this endeavor, our huge resources of natural innate immunity, assisted by new vaccines, are already helping us. the formulation of the vaccines and their stockpiling alongside antineuraminidase (ni) antivirals has needed significant investment of time and money, and this started with a three billion euro investment from the usa and eu. we are presently gathering the fruits of this investment with the outbreak of a/swine (h n ) virus. baroness findlay of glandaff put the epidemiology of influenza h n situation succinctly in the house of lords report of pandemic influenza [ ] "we believe the risk of a pandemic of human-to-human transmissible virus is to be taken very seriously. we believe that it may not happen in the very short time. to explain why we came to this stance; we believe that the problem, if it does emerge is more likely to emerge in asia. asia is where fire fighting must be done today." the baroness had just heard the background science that china alone holds million domestic ducks, a possible trojan horse of virus persistence, which approximates to % of the world's domestic duck population. expert evidence from fao had summarized that china, indonesia, and vietnam represented the core of the problem, but only million dollars were available at that point in / to help, and biosecurity is not imposed strictly, while veterinary services are haphazard. the current pandemic virus emerged from pigs but a continuing threat is another reassortant event with h n most likely in a coinfected child in egypt or se asia where h n viruses are endemic and where swine h n viruses are spreading. we are not the first generation of virologists to recognize the influenza pandemic threat, but we are the first to have the knowledge of the avian and pig reservoir and the tools to deal with the problem in a scientific manner. the world capacity for influenza vaccine today of one billion doses did not arrive by accident: it came to us from the hard work and dedication of four generations of dedicated scientists and doctors. the intention here is to give just tribute to these pioneers and their new discoveries. using the vaccine methods developed over six decades, we can for the first time confront influenza as it emerges, surround it, and actually prevent a pandemic. we no longer need to be passive observers at a theater of infection. churchill coined the phrase "give us the tools and we will finish the job." well, we now have them and we will. such is the essence and spirit of this chapter. the serendipitous discovery of infection of ferrets, which produce clinical signs, and the cross-infection of a student from a ferret was the first technology foundation stone [ ] . ferrets are used today as a key model to investigate new vaccines. the two most important technologies, which form the granite-like foundation of influenza vaccine research, are the hemagglutination inhibition test (fig. ) and the cultivation of virus in embryonated hen's eggs (fig. ) , first reported in and , respectively [ , ] . if one adds two other vital scientific observations that of hobson et al. [ ] who correlated a hi titer of with protective efficacy in volunteers in and then the discovery of a single radial diffusion for standardization of the hemagglutinin (ha) content of vaccines by schild in , it is quite apparent that the technologies are all now well tried and tested [ ] . the elucidation of the structure of the fragmented influenza genome [ ] has quickly led to techniques, genetic reassortment, and correlation of functions with certain genes (fig. ) . from a practical viewpoint, some old much passaged viruses such as a/pr/ / (h n ) grew to extraordinary infectious titers in the egg allantoic cavity, exceeding a new wild-type virus by fold or more. why not create a reassortant in the laboratory with six replicative genes of a/pr/ / to give high replication while having the two new ha and neuraminidase (na) genes of the new epidemic virus? this technique proved to be a masterstroke and in the last quarter of a century three laboratories, csl in fig. the classic hemagglutination inhibition test. the test depends upon interaction of eight ha units of virus that would normally agglutinate . % turkey red blood cells. preincubation of this standard virus with dilutions of serum antibody abrogates the agglutinating property of the virus (vertical rows and ). no antibody is detectable in rows - , [ ] [ ] [ ] melbourne, nibsc in london, and ed kilbourne's laboratory in new york, have rushed each year to produce the new candidate vaccine viruses prefixed ivr-, nib-, and x-, respectively. the almost made-to-order technique of gene reassortment fig. inoculation of embryonated hen's eggs to grow influenza virus for vaccine. virus is inoculated through the shell of a -day-old embryonated hen's egg and more rarely in the research laboratory into the amniotic cavity (top). after days of incubation at c, the clear fluids are removed and titrated for ha by hemagglutination fig. the influenza genome is in eight fragments. the genome could be labeled with p extracted and separated on polyacrylamide gels with influenza was also central to producing host range mutants with attenuation genes for live vaccines. some of the starter and seed viruses for the current production of a/california/ / h n (swine) vaccine used this biological technology, while others used reverse genetics to make a gm starter virus. undoubtedly the simultaneous discovery of the reverse genetics [ , ] by the three laboratories in new york, wisconsin, and oxford was a masterstroke in technical advance, which has enabled mutations to be placed, at will, into the genomes of the negative-strand viruses. the conjunction of older and newer techniques with the licensing of the mammalian cell lines from monkey kidney (vero) [ ] , dog kidney (mdck) [ ] , or human tissue (per- ) has led directly to the newly emerging influenza vaccines of the twenty-first century. we are using all these techniques of the last years to produce the a/swine h n vaccines of for the current pandemic. the first experiments on the attempted immunization of animals were made in the usa by francis magill [ ] and in england by andrewes and smith in [ ] . the model is still vital today and the first experimental assessment of a/california/ / h n vaccine was made in this model. mouse lung suspensions or filtrates were used after inactivation with formaldehyde, and it was found relatively easy to protect mice against intranasal infection with influenza. immunization experiments in man were accelerated when allantonic fluid preparations of virus formed the starting material soon after the technique of allantoic inoculation of fertile hen's eggs was discovered [ ] . the first field trial demonstrating short-term protection by inactivated vaccine took place in the usa during a sharp epidemic of influenza in (commission influenza ) [ ] . progress with the development of purer, more potent vaccines has proceeded steadily since those early days, and technical advances with ultracentrifugation and chromatography, by methods producing richer cultures and chemical inactivation avoiding too great a modification of the surface ha and na antigens have all helped. to avoid the relatively high rate of local and general systemic reactions caused by the older egg-grown inactivated whole-virus vaccines, chemical treatment to disrupt the particle and to separate the wanted antigens (ha and na) from other constituents of the virus has led to a variety of different split or subunit vaccines . ether extraction [ , ] , deoxycholate treatment [ ] , and treatment with other detergents have been introduced. some methods have provided subunit vaccines causing fewer clinical side reactions than the older whole-virus particle vaccines, but drawbacks have appeared, including that of reduced antigenicity. adjuvants of oily emulsions promised potent vaccines with excellent antibody responses, and a few reactions were first encountered. however, a rare abscess at the site of inoculation caused much distress and this early approach had to be abandoned. in spite of attempts to develop safer materials, none have yet the whole virus is disrupted with detergent, which dissolves the lipid membrane releasing ha, na, and internal np, seen as "lamb tails" been developed commercially until very recently when mf and a have been formulated. thus, after years of work, the hope of an ideal inactivated vaccine free from the induction of clinical reactions and yet potent immunogenically has just been fulfilled with pandemic h n vaccines and swine h n vaccines. in , a major antigenic deviation of influenza a virus occurred with the appearance of a/cam/ (h n ) virus in australia. in the usa and europe, outbreaks of influenza occurred early in , which were due to the same virus; some communities previously receiving vaccine containing pr and weiss viruses (h n in the old classification and now reclassified h n ) were attacked. this time the vaccine did not protect against the new virus typified by the prototype a/fm/ / (h n ) [ , ] , and this led to realization of the enormous importance of the updated antigenic make-up of inactivated vaccine. yet other difficulties have become appreciated, one of which is the inappropriate antibody response occurring sometimes after inoculation, when the vaccine induces cross-reacting antibody to heterologous viruses or the first virus in the subtype which the vaccine first experienced, rather than that appropriate to the specific antigen, ha, of the vaccine virus. this response is probably allied to the phenomenon of "original antigenic sin." sometimes this aberrant response can be useful as with a/swine h n vaccine. it is likely that the over s will produce recall antibody to h n viruses which infected them in the s and that this virus is somewhat related to the current a/swine virus. the starting materials for almost all types of inactivated vaccine are allantoic fluids from fertile hen's eggs previously inoculated with a seed culture, the yield of which is enhanced using a recombinant virus, one parent of which is a high-yielding laboratory strain (a/pr / ) and the other acts as the donor of the requisite surface ha and na antigens from a wild-type virus [ ] . the a/pr/ / virus donates six genes and the wild-type virus two genes: the ensuing reassortant high growth viruses are called / reassortants. purification from unwanted egg material is accomplished by ultracentrifugation on a zonal ultracentrifuge [ ] . whole-virus particles thus separated are inactivated by formalin or b-propiolactone, the ha content being as high as possible commensurate with the necessity to avoid febrile reactions after inoculation. children were sensitive to the older egg-grown wholevirus vaccines; as many as % under years developed fever after . ml of vaccine and up to % of -year-old children were similarly affected after . ml [ ] . the precise constituent producing the fever was not clearly identified, but the viral proteins were believed to be concerned [ , ] . more modern whole-cell virus vaccines produced in cell culture are more purified and produce fewer side reactions. separation of the ha and na by means of detergents such as tween or triton n produced split-virus or subunit vaccine, and general experience suggested that these materials are less pyrogenic, but less immunogenic, than whole-virus vaccine [ ] . this was particularly well demonstrated by studies during the swine influenza campaign in the usa in , when many observers reported results, which ultimately led to the recommended use in children of two doses of split-type rather than whole-virus vaccines. such recommendations continue at the present time. in adults, too, the older egg-grown whole-virus vaccines gave a higher proportion of febrile reactions than split virus [ ] . however, this situation is changing as whole-virus vaccines produced in vero cells for example come to the fore. former methods for assays of the potency of inactivated vaccine depended on measuring the ha activities of the vaccines with erythrocyte suspensions using the salk pattern technique of miller and stanley [ ] . in retrospect, this technique was not hugely accurate especially for subunit and split viruses. in a major technological breakthrough, schild et al. [ ] proposed a method of assay based on single radial immunodiffusion (srd) (fig. ) . the ha antigen content of vaccines was estimated using srd tests in agarose gels containing specific hi antibodies. the srd method was modified and refined by wood et al. [ ] . it may be gradually replaced now by hpmc technologies. the srd technique was valid for both whole-virus and split-virus vaccines and was quickly adopted for international use and is still the gold standard. in this test, vaccine virus preparations and reference antigen calibrated in terms of micrograms of ha are disrupted with detergent, and dilutions of the treated antigens are introduced into wells in srd immunoplates. the size of the precipitation ring obtained for the vaccine is compared with that obtained with a reference antigen of calibrated ha content titrated on the same plate. the vaccine potency is measured in terms of micrograms of ha per vaccine dose. inactivated influenza vaccines frequently contain two or more virus strains and the ha content of each component ( mg) is assayed independently. it has been known for many years that the serological response to inactivated vaccine depends on the previous experience of the recipient to infection by viruses of the same subtype of influenza a virus as that present in the vaccine. although a single subcutaneous injection of (h n ) vaccine gave as good a response as two doses prior to , the advent of the new pandemic a/asian (h n ) virus produced a different effect. thus, holland et al. [ ] demonstrated that two doses at an interval of two or more weeks produced a better response to one dose and in this regard the vaccine-induced immune response was much inferior to that noted single radial diffusion (srd) test to standardize ha. vaccine antigen is pipetted into -mm wells in an agar plate containing specific anti-ha, -na, and -np antibodies. after a few hours incubation, a zone of precipitation is quantified and the area is proportional to the quantity of ha in the vaccine before the change in virus subtype. such an experience was again noted during the first year of circulation of a/hong kong (h n ) virus and also when the a/new jersey/ (hsw n ) vaccine was used in children and young adults. also, in the circumstances of - , when most persons under years of age had no previous antibody to the recirculating h n virus, a two-dose regimen for children and young adults produced a more satisfactory response than a single injection [ ] . to reiterate, in an "old" h n virus from the s was accidentally released from a laboratory and established itself as an epidemic virus. it was called a "pseudo pandemic." everyone over years had previous immunity. the contrast between the effects of a single dose of vaccine in persons infected with h n viruses at least years earlier was very striking. these data have immediate relevance today in terms of h n vaccine and of course with the a/swine vaccine. the world is full of immune virgins as regards h n but not in the case of a/swine h n . most persons have immune memory to the h n family, and therefore it comes as no surprise that vaccines induce high levels of hi antibody. several factors are of importance in the determination of the quantity and the precise composition of the antibody response to the surface antigens of the virus present in inactivated vaccine. first and foremost, the quantities of hi and ni antibodies induced by vaccine are broadly related to the quantity of antigen present in a single dose. second, the precise composition of the antibodies formed in response to influenza a virus is important. thus, reinforcement of previously acquired antibodies by the orientation of the b-lymphocyte response to the first infection by the particular subtype of virus experienced in childhood or later may take precedence over the strain-specific antibody response to the vaccine virus. third, the precise response is influenced by the route by which the vaccine is presented to the body's immune system. first then, several earlier studies reported a graded relationship between the quantity of antigen inoculated and the antibody response that results. this was so in the study of mostow et al. [ ] , who gave increasing doses of vaccine in a single injection containing - , chick cell agglutination (cca) units containing a/japan/ (h n ) virus groups of volunteers. the serum hi response was tested with four different h n viruses isolated - and also the homologous virus. with more than a tenfold increase in ha from the least to the highest dose, the geometric mean titer (gmt) of antibody increased only fivefold. similar results were obtained by potter et al. [ ] , who inoculated student volunteers with vaccines ranging in dosage from to iu and containing a/port chalmers/ (h n ) virus. the vaccine was a surface-antigen detergent-treated material [ ] adsorbed to aluminum hydroxide gel. gmt hi serum titers increased against homologous virus from -to -fold with the increase in dose of vaccine ha. three other h n strains and a/singapore/ (h n ) virus were also tested, and all three h n viruses showed graded hi antibody responses proportional in magnitude to increase in antigen dose, as did the homologous virus. the pandemic working group of the mrc committee on influenza vaccine [ ] gave graded doses of whole-virus vaccine containing the a/new jersery/ (hsw n ) strain to groups of volunteers in . those less than years of age, who did not possess significant serum hi antibody to the virus before immunization, showed a postvaccination antibody titer ranging from to gmt with a nearly eightfold increase in dose from to mg of ha. above this age, in those - with preexisting hsw antibody, there was an increase in antibody titer from to times (gmt) with a change in ha concentration from to mg. thus, the effect of increasing the potency of this vaccine on the antibody response was much greater in those sera, which indicated that they had been exposed to the antigen, presumably by infection with a related virus, than in those with no such exposure. both whole and detergent-split-virus vaccines showed a relatively poor hi response in volunteers less than years of age whose initial serum had no significant amount of prevaccination or postinfection hi antibody. in this group of subjects, two doses of vaccine gave a better antibody response than did one, but the resultant postvaccination gmt was half that obtained with a single dose of the vaccineover years of age. this historical data is very relevant to us today as we analyze the hi data from the current batches of a/swine h n vaccine where in the over years a single dose of vaccine is sufficient because of wide preexposure to members of the h n family of viruses. the younger groups had no prior immune memory to the h n family of virus, their experience being more orientated to h n and h n families. these examples underline the practical importance of a considerable degree of antigenic drift within a subtype comprising hi antibody response. also, the recall of antibodies induced by previous infection illustrates the general rule that an up-todate monovalent vaccine reinforces antibodies against former members of the subtype, while also inducing specific antibodies to the vaccine virus. this was clearly shown by direct comparison of monovalent and polyvalent vaccines such as the mrc committee on influenza vaccine's trials [ ] [ ] [ ] [ ] . the quantitative dose response already described for hi is also found with ni antibody but is less consistent. thus, potter et al. [ ] noted that there was a two-to sixfold increase in ni antibody as vaccine potency was increased from to iu of ha. yet the trial of a/new jersey/ (hsw n ) vaccine conducted by the pandemic working group of the mrc influenza vaccine committee [ ] found only a slight increase in ni antibody after an increased dose from to iu using iu of ha in the vaccine. nicholson et al. [ ] gave a whole-virus vaccine of the a/ussr/ (h n ) virus, which ranged in potency up to sixfold, and found, in those under , a threefold increase in ni antibody. however, in those over years of age, an increase in dose of vaccine had a less constant effect on ni antibody formation. one possible reason for the variation in the effect of different vaccines on the ni antibody is the lack of consistency in the na content [ ] ; however, another possibility may be that immunological priming to the ha in the vaccine can in some way suppress the immunogenicity of the na antigen, which may be physically associated with the ha. the second important variable in the immune response to inactivated vaccine arises from the relative amounts of cross-reactive and strain-specific antibodies that are generated. the differentiation of these require special techniques such as srd and the adsorption studies. webster et al. [ ] compared, in adults, the response to an a/port chalmers/ (h n ) subunit vaccine to homologous and heterologous h n viruses. most of the antibody was cross-reactive with a/hong kong/ virus but when higher doses of the vaccines were used, strain-specific a/port chalmers/ antibody was produced in addition to that against heterologous virus. oxford et al. [ , ] compared whole-and split-virus vaccines containing a/victoria/ or a/scotland/ viruses and using single radial hemolysis and adsorption techniques showed that in an immunized adult, cross-reactive antibody was induced much more frequently than specific antibody against homologous virus. they showed the same phenomenon in adults during infection with a/port chalmers/ virus, who frequently also developed antibody rises to a/hong kong antigens from . oxford et al. [ ] used similar techniques to analyze sera from children aged - years immunized with a surface-antigen vaccine containing a/victoria/ (h n ) antigens. most children produced a strain-specific serum antibody to the vaccine antigens, whereas adults similarly vaccinated tended to produce antibody cross-reacting with all variants of the h n subtype tested. postepidemic sera from those of various ages recently infected by a/texas/ -like strain showed crossreactive antibody in adults but in contrast mostly strain-specific responses in children. strain-specific antibody is considered to be more protective. the influence of the route of immunization with inactivated vaccine has been studied in the past by many observers. the chief alternative to the subcutaneous-intramuscular route is intradermal injection using a reduced amount of vaccine. the advantages of this route are economy and the avoidance of febrile reaction. the principal disadvantage is the fact that the antibody response is less consistent. it was shown by appleby et al. [ ] that the gmt after intradermal vaccine was less than half that obtained with subcutaneous vaccine, and this seemed logical in that only one-tenth of the vaccine dose was given intradermally. mccarroll and kilbourne [ ] found little difference in the antibody responses to intradermal and subcutaneous vaccines in equivalent doses. tauraso et al. [ ] reinvestigated the question using a two-dose regime before the arrival of the a/hong kong/ (h n ) epidemic. in the equivalent amount of . ml of vaccine, antibodies formed in higher titer after intradermal than subcutaneous vaccine. however, the titers after . ml of vaccine subcutaneously were little different from intradermal injection of . ml. it is considered advisable, however, in practice to limit intradermal vaccination when the vaccine is in short supply or when, in children or the aged, reactions after subcutaneous vaccine might pose problems. the nasal route of inoculation either by instillation of drops or by spray was first studied in detail by waldman et al. [ ] . compared with the subcutaneous vaccine in a dose of . ml, antibodies capable of neutralizing the virus a/taiwan/ (h n ) increased to a greater extent in sputum and nasal secretions after repeated nasal inoculation with a total volume of . ml vaccine. in contrast, the intranasal vaccine produced a much lower rise in serum antibody, the gmt being only onesixth that after subcutaneous vaccine. waldman et al. [ ] , using an aerosol spray, found that a better serum antibody response occurred with a small-sized particle spray than a larger one, but the nasal antibody response was better after the latter or with nasal drops. absorption studies showed that a majority of the secretory antibody (iga) response in nasal secretion was cross-reactive with heterologous viruses (a/hong kong/ h n ). phillips et al. [ ] compared subcutaneous or intradermal vaccine in nurses with vaccine dropped intranasally. the subcutaneous route produced the best serum antibody rises, and intradermal vaccine was superior to the intranasal route in terms of antibody response. the nasal antibody titers after immunization by either subcutaneous or respiratory routes paralleled those in serum. the fact that nasal antibodies increase after subcutaneous vaccine [ , ] is important because the lack of a good response in serum antibody in those given the same vaccine intranasally is a limitation hardly offset by local nasal secretory changes. challenge of immunized groups of persons by live-attenuated virus also supports the view that nasal antibodies play a supplementary role to serum hi antibody [ ] . [ ] . after a second dose of the same vaccine, fewer volunteers experienced reactions than seen after the first dose. later studies of the endotoxin content of various pools of inactivated type a or b vaccines using the limulus lysate test gave no hint of a parallel between the occurrence of general reactions and the endotoxin content [ ] . neurological illness is a recognized sequel to immunization with a variety of vaccines but had not previously been observed with any frequency after influenza virus vaccines. wells [ ] noted the rare instance of guillain-barré syndrome (gbs), which appeared in excess among the persons vaccinated with a/swine vaccine compared with the numbers in unvaccinated individuals. of , persons with gbs reported from october , , to the january , , had received vaccine before the onset of neurological symptoms. the overall risk of gbs was calculated as ten cases per million vaccinated. the rate of occurrence during the -week swine vaccine period was five to six times greater than in unvaccinated persons. however, the excess in number was greater in the second and third weeks after inoculation than either the first or subsequent weeks. as reported by langmuir [ ] , gbs was not associated with a particular variety of vaccine or age group. however, that numbers were slightly greater in those aged - than in middle aged or elderly persons, which appears to rule out the possibility that the syndrome was, in some way, related to the absence of antibodies to the swine virus before immunization, for most of those aged over would have been exposed to antigens of this virus many years before. after the swine influenza campaign was terminated, surveillance was continued, and during the period - , when . million doses of ordinary inactivated vaccine were estimated to have been used, the related risk of gbs was . times the incidence in unvaccinated persons. this risk was regarded as not significant [ ] . no clue to the cause of the marginally increased risk of gbs in immunized persons in has yet been obtained but could be virus strain related. no untoward effects have been noted in the billions of vaccines used since to the present day. at the time when a/hong kong/ (h n ) virus was spreading in asia, plans were made by the mrc committee on influenza vaccine to protect children in residential schools and other groups in a controlled manner. inactivated polyvalent vaccine containing two h n viruses (a/england/ and a/england/ ) and a b strain were compared with an h n a/hong kong whole or deoxycholate-treated virus vaccine in initial serological trials. antibody formation even in those without detectable serum hi antibody gave gmts over in those receiving a/hong kong vaccine intramuscularly. however, controlled trials in two boarding schools showed no convincing evidence of protection. in uncontrolled trials in other schools either the polyvalent or the a/hong kong vaccine were given or no vaccine at all. there were schools where epidemics of influenza occurred in january and february but no evidence of protection was found in those receiving a/hk vaccines. the only clue obtained concerning the vaccine failure was first that only one dose of vaccine had been given, and this is known to be inadequate to give a satisfactory antibody response in previously seronegative persons, and second, there was an interval between vaccine administration and infection of - months. these two factors may have combined to explain the absence of protection because of the inadequacy of the antibody response at the time of challenge. it would be fair to add that others [ , ] did obtain protection from a/hong kong/ whole-virus vaccine during the first outbreak of influenza due to this virus in the usa. the use of modern adjuvanted h n vaccine in two doses is anticipated to give protective effects. the current a/swine vaccines produce protective hi antibody (> ) in most persons over years of age following a single dose. as emphasized above, this reassuring situation is because most persons have prior immunity to the h n family of viruses which circulated between and and then again from to the present day. the use of living but attenuated virus as an immunizing agent developed slowly from the initial studies of mawson and swan [ ] in australia and the ussr. the major difficulty of the lack of a laboratory test to indicate that cultured virus had lost its pathogenicity, while retaining infectivity for man, meant that deliberate intranasal inoculation of volunteers furnished the only way to select a suitable strain for infection without causing clinical reaction. in spite of the widespread adoption of live vaccines selected by this method and given as an intranasal spray in the ussr, little interest was exhibited in most other countries. from onwards, trials took place in volunteers in england and wales to provide evidence of safety and immunogenicity of cultured viruses and the drawback of a reduced infectivity of well-attenuated viruses handicapped progress. the necessity to observe a match between the antigens of epidemic viruses and those present in the vaccine was a further drawback until the technique of reassortment of characters between two strains, one of which was of proven attenuation, was utilized to yield seed viruses with the desirable clinical and antigenic properties. other disadvantages of live viruses appeared during the intensive researches of the s particularly in the usa and in england [ , ] . it cannot yet be claimed that the ideal live-attenuated virus vaccine has been formulated, but reverse genetics and increased knowledge of virulence genes have now lead to a resurgence of interest. in the s, genetic studies were intensively pursued in attempts first to define the particular gene or combination of genes, donated by the attenuated virus that confers the property of attenuation upon the reassortant strain. it was found that the biological properties of excreted virus may be altered compared with those of the original virus in the vaccine and the manner of this alteration was also studied genetically. such work is essential in achieving the goal of an effective and safe vaccine virus for human use. experimental inoculations were carried out initially in small-scale tests in volunteers under semi-isolation to permit close observation (see below). multiple cultivation and passage of viruses either in animal hosts, such as ferrets and mice, or in developing chick embryos or tissue cultures had been practiced even before the use of temperature-sensitive (ts) or cold-adapted (ca) mutants was suggested. early workers in britain used the pr / virus as a host range mutant, which, although noninfective for man, has retained animal pathogenicity even after many passages in eggs. as a donor parent with good powers of multiplication in the laboratory, pr was mated with various strains of wild-type influenza a viruses to obtain recombinants with up-to-date surface ha and na antigens. this method was preferable to simple laboratory cultivation because some viruses failed to alter in pathogenicity after as many as serial passes in cultures [ ] , although other virus strains appeared to become attenuated with only a few passages in eggs. pr virus was chosen also by workers in belgium who prepared reassortants from a number of viruses, some of which were licensed for human use [ ] . to select recombinants with as high proportion of rna components as possible derived from the host range mutant pr , florent et al. [ ] used rna-rna hybridization to identify gene origins. later the gene constellation of four of the candidate vaccine viruses was determined, and florent [ ] found that some clones of beare and hall's [ ] recombinants of pr and a/englannd/ (h n ) containing five genes from pr were satisfactorily attenuated. however, one clone though containing six pr genes was nevertheless clinically virulent to volunteers. a further genetic study of pr host range recombinants using viruses tested clinically by beare and reed [ ] was made by oxford et al. [ ] . it was again found that recombinants from pr and a/england/ viruses could contain only the surface ha and na genes from wild-type virus and yet retain virulence for man. additional attempts to stabilize the attenuation of candidate viruses were made both by beare at the medical research council's laboratories at salisbury and the rit workers by rendering the virus resistant to an inhibitor present in normal horse serum. this property was present in the rit series of recombinants. it seems strange that stabilization has not been pursued since nor has cultivation of host range mutant viruses, such as pr , at abnormally low temperatures, such as c. this method was found by sabin [ ] to be preferable to normal temperatures when attenuating polio viruses, and it was exploited by both workers in the usa and ussr. marker tests, which can be equated with attenuation of virulence for man, were sought with relatively variable results. one such test used weanling rats that were inoculated intranasally first with virus and later with cultures of haemophilus influenzae. virulent virus induces bacteremia and meningitis, and using this method jennings et al. [ ] successfully separated a number of reassortant viruses and obtained some correlation with clinical virulence. yet the host range mutant parent pr / and rit , which are both attenuated in man, were classed as virulent by the rat. a new approach at that time used an avian (duck) virus, which was found to have only low pathogenicity for squirrel monkeys inoculated intranasally and was proposed as a donor of attenuation. a reassortant with a virulent human a/udorn/ (h n ) virus behaved as did the avian parent in the squirrel monkey, although immunizing the latter against the virulent parent. clinical trials have suggested that this virus is attenuated for man and is immunogenic but has not been investigated since [ ] . most work on the development of viruses with restricted multiplication at temperatures above the normal range for cultivation has been affected by chanock, murphy, and associates at the national institutes of health, bethesda [ ] . the technique used chemically produced mutation in virus rna by cultivation in the presence of the mutagenic agent -fluorouracil. after cultivation and plaquing at c, c, and c, mutant viruses with the requisite temperature sensitivity were obtained. intranasal inoculation of hamsters confirmed temperature restriction, in that much lower titers of virus were found in the hotter lungs than in the cooler upper respiratory tract. spread from inoculated volunteers to adults in contact was not observed, and no evidence of a change in virulence was found in viruses recovered from adult recipients of vaccine [ ] . however, in seronegative children, the a/hong kong/ -ts-l [e] virus produced mild febrile reactions and a virus that had lost its properties was recovered from some who were infected. a second series of ts- a was then developed by combining two defective ts viruses, each of which belonged to a different complementation group in respect of the genetic defect. the progeny exhibited greater temperature restriction than the ts- [e] line of viruses. it was termed a/udorn/ ts- a , and it was recombined with three further viruses; wild-type a/victoria/ / , a/alaska/ (h n ), and a/hong kong/ (h n ). these ts- a viruses were highly immunogenic and exhibited temperature restriction of multiplication in cell cultures and reduced replication in the hamster lung. the a/victoria/ / -ts- a recombinant retained its ts properties after inoculation into doubly seronegative children. unfortunately, when the a/alaska/ -ts- a virus was similarly tested in a single child after tests in adults had shown genetic stability, the nasal secretions of the vaccine yielded a ts-positive virus that produced plaques at c even though the child had shown no symptoms or fever. the recombinant a virus with a/hongkong/ (h n ) parent exhibited a capacity to infect % of doubly seronegative adults and was attenuated compared with the wild-type parent. nevertheless, it appeared possible that a virus such as the a/alaska-ts- a might, if transferred to contacts from an inoculated child, result in clinical illness, and clinical studies with this particular virus were not pursued. beginning with a strain of h n virus recovered in ann arbor, michigan, in by cultivation of throat washings in tissue cultures at c, maassab [ , ] evolved a virus, a/ann arbor/ / (h n ), which has acted as a donor of attenuation to other viruses by genetic reassortment. earlier passages were made in chick kidney tissue cultures followed by intranasal passages in mice and then a gradual adaptation to lower temperatures, in tissue cultures and in developing hens' eggs inoculated allantoically, led to a virus with good powers of multiplication at c. the ca variant was found to retain the infectivity of the original strain for both the mouse and the ferret, although it produced no deaths in mice and no fever or turbinate lesions in ferrets, whereas the original virus was pathogenic for both species. the virus proved to be temperature sensitive with a shut-off temperature of c [ ] . recombinants with wild-type viruses of both h n and h n subtypes were prepared, studied in the laboratory and in volunteers, and analyzed genetically. the original a/ann arbor/ / (h n ) virus was not, however, tested in fully susceptible persons presumably because of the difficulty in that period of finding seronegative adults. a few persons with low titers of serum neutralizing antibodies ( : to : ) were inoculated and as judged by antibody responses, became infected without undergoing clinical illnesses. more rigorous clinical studies have been pursued with recombinants, in particular, those with h n antigens, and details of the results have been brought together and earlier data summarized by kendal [ ] . the donor ca parent has been more recently reassorted with h n genes. it is clear that infectivity and immunogenicity were fully retained for seronegative adults of whom received h n recombinants. among those receiving three of four recombinants, clinical reactions were minimal or negligible but with the fourth, derived from the a/scotland/ parent, in of volunteers receiving . and in receiving . tcid , there were clinical illnesses. viruses reisolated from the vaccines retained ts properties and so did those given recombinants of a/victoria/ (h n ) and a/alaska/ (h n ). however, some loss of ca restriction was found in virus re-isolated from volunteers given the a/scotland/ recombinant. cold-adapted recombinants with a/ussr/ (h n )-like virus have also been studied in adult volunteers and found to be less immunogenic as judged by hi antibody responses. a better response was obtained by wright et al. [ ] in children in nashville given . tcid of strain cr (h n ) and none of children developed adverse clinical reactions even though eight became infected. all reisolated viruses retained the ts phenotype. the failure to elicit serum antibody response in adults given this same virus recombinant is puzzling. using the elisa enzyme-linked assay, murphy et al. [ ] found that by this more sensitive method antibody rises could be demonstrated and the results tallied better with the ability to re-isolate viruses from the inoculated volunteers than did the serum hi responses. the leningrad group of workers led by smorodinstev [ ] was the first to obtain a virus indirectly attenuated by cultivation at c. the group used strains selected by inoculating volunteers with several viruses derived from cultures repeatedly incubated at - c to speed up attenuation. approximately - months were required for the preparation and production of new strains even using genetic recombination to incorporate new surface ha and na antigens. although alexieva et al. [ ] found that cold cultivation was not successful in producing reliably attenuated viruses for use in children, the technique was adopted for general use. genetic studies of the leningrad viruses are described briefly by kendal et al. [ ] , and these parent ca viruses are currently the center of new interest for attenuated h n vaccines. usually, preliminary studies were made in the ussr in - -year-old seronegative adults who receive virus twice at intervals of - days administrated by nasal spray. viruses were attenuated by passage for varying periods at c and both donor viruses and recombinants proved temperature sensitive. in - , when h n viruses were circulating, , children aged from to received the ca a/leningrad/ (h n ) virus. some febrile reactions occurred but only in less than % of the children. further studies of recombinants with h n or h n antigens and the same leningrad h n parent after serial passages under cold conditions of cultivation ( c) were conducted in children, half of whom had no detectable serum antibody to the vaccine strain. no reactions occurred and over % of the children responded with antibody production. it is clear from the earlier papers by alexieva et al. [ , ] that intranasal administration of children aged - were too reactogenic and that this is the reason why the peroral route has been chosen for routine administration in the ussr. a japanese virus recovered in , a/okuda/ (h n ), was found to be attenuated for children and served as a donor of attenuation both in japan and in england. zhilova et al., japanese workers, [ ] developed a recombinant virus (ko- ) from ultraviolet-irradiated a/okuda/ and wild-type a/kumamoto/ / (h n ). serial passaging in eggs in the presence of normal horse serum was followed by plaque purification and later clinical tests in a few children. the m (membrane) gene was found to have been donated by the okuda parent. from reassortants with other human viruses, a candidate wrl virus was selected and underwent clinical trials without harmful clinical effects [ ] but has been little investigated since that time. cultivation of influenza viruses in mammalian cells rather than eggs initially encouraged two manufactures to invest in cell culture fermenters for vaccine production [ , ] . many more groups are now using these technologies to produce the current a/swine h n vaccine. capacity can be increased to cope with a surge in demand for a pandemic virus vaccine. moreover, the final vaccine has the theoretical advantage of the absence of egg proteins. the cell culture vaccine virus is also easier to purify. where clinical isolates of influenza viruses are cultivated in mammalian cells and eggs in parallel, different antigenic variants may be selected [ ] . the biological variants have amino acid substitutions in the receptor binding site in proximity to an antigenic site on the ha, and an amino acid change in this region can alter antigenicity. of the two virus subpopulations that can be selected, the virus which is grown on mdck (or vero) cells rather than in eggs appears more closely related to the wild-type clinical virus. there is some indication that cell-grown virus vaccines offer greater protection in animal models than the corresponding egg-grown vaccine. these are all powerful arguments in favor of the new generation of influenza vaccines being cultivated currently in vero [ ] or mdck [ ] or per cells. alongside years of experience producing an immunogenic and safe vaccine, the world capacity for influenza monovalent vaccine manufacture has expanded to the present two billion doses. the preparation work and investment for h n are showing rewards with the current pandemic of a/swine h n . most manufacture is still located within the eu, but production is increasing in the usa, korea, japan, china, and most recently, india. the international collaboration in face of the outbreak of a/california/ / (h n ) in mexico around christmas to the present, the exchange of clinical data and viruses enabled vaccine manufacture to start production by may/june . by october , the production of hundreds of millions of doses of a monovalent vaccine containing mg of ha and immunogenic after a single ion dose in the over -year olds is a quite remarkable achievement. many countries have started to immunize at-risk groups, namely younger people < years of age with diabetes, obesity, chronic heart, or lung problems, and the immunosuppressed including pregnant women. it is forecast that up to - % of some countries could volunteer for the vaccine. it is especially important that medical and nursing staff take the vaccine to protect both themselves and their patients. however, such large vaccination campaigns open schisms in modern societies,which on the one hand become very concerned about young persons dying but on the other hand have prejudices about vaccines in general. in the first winter wave the over s, unusually for a pandemic virus, are protected by prior experience of the h n family and hence overall mortality is likely to be less than a seasonal year but the mortality is likely to be in younger persons, thus exposing our achilles heel. additionally nearly half the deaths to date have been in young persons without comorbidities. finally, within a year the virus is likely to mutate to allow it to infect the over group, so, paradoxically, mortality in the second pandemic year could easily exceed the first. twenty-first century vaccines induce protection across the different virus subtypes? there are known subtypes of the ha of influenza a virus. only three subtypes have caused pandemics in humans, h , h , and h , while h , h , and h predominantly circulating in birds have crossed the species barrier into humans and caused human outbreaks. we do not know whether these latter three subtypes could mutate into human-to-human transmitters and thereby acquire pandemic potential. at present, h n is causing considerable concern in se asia. an important question therefore is whether a vaccine could be engineered to give socalled heterotypic or cross-subtype immunity to protect against all these potentially pandemic viruses. it is well known that the internal proteins of influenza a virus such as m , m and np are shared by all influenza a viruses. these internally situated proteins are certainly immunogenic (particular np) but could the immunity induced, either t cell or antibody, be broadly reacting? to back up the central core of this approach, it has been known for years that mice infected with an influenza a (h n ) virus would later resist a lethal challenge from an influenza a (h n ) virus. given the lack of genetic and antigenic relatedness between the h and h proteins, or indeed the corresponding n and n proteins, this strong cross-immunity was attributed to an internal protein such as np or m. however, it has been difficult to construct a solid database and there has been a lingering doubt about this so-called cross-protective immunity. most virologists deduced, virtually by elimination, that a cross-reactive portion of the ha (ha ) could have provided the cross protection. furthermore, this cross protection is particularly seen in the mouse model, leading some to conclude that the mouse recognized cross protection epitopes that perhaps humans did not. fundamental studies to correlate the genetics and immunology of np and m established the cytotoxic t-cell response to portions of these proteins. however, the work clearly showed that m could be a cross-reactive immunogen, although a relatively weak one [ ] . the m protein is an integral membrane protein of influenza a viruses that is expressed at the plasma membrane of virus-infected cells and is also present in small amounts on virions. the important extracellular domain, potentially targeted by antibodies and t cells, is conserved by virtually all influenza a viruses. even the pandemic virus differs only in one amino acid. the first indication that the m was immunologically active was the observation that an anti-m monoclonal antibody reduced the spread of virus cell culture. not unexpectedly, the antibody reacted with the extracellular domain of m . even more excitingly, the antibody reduced the replication of virus in mouse lungs. immunization studies with m constructs, however, have given more mixed results. immunization of mice with dna plasmid of m and m gene gave protection mainly via t-helper cell activity. an alternative approach utilized a hepatitis b core and m fusion protein. the cross protection resided in antibodies, although m specific antibodies did not neutralize the virus in vitro. presumably, protection was mediated by an indirect mechanism such as complement-mediated cytotoxicity or antibody-dependant cytotoxicity. however, the protection induced in the mouse model was considerably less than that induced by a conventional sub unit ha/na vaccine. it could be argued that weak heterotypic immunity may be present already in the community and that this is helping to prevent the emergence of chicken influenza a (h n ) in se asia [ ] . certainly with evidence of tens of millions of domestic birds infected since late in countries in se asia, with only a handful of human infections and only human-to-human transmission in family groups, there is a possibility that the unique cocirculation since of two influenza a viruses (h n and h n ) may have enhanced heterotypic immunity in most communities, which in turn abrogates the emergence of chicken influenza a (h n ) into humans. it would be foolhardy, though, to take this argument to a fuller conclusion and relax preparations for a new pandemic influenza a virus. at present, with the unprecedented research investment into influenza vaccines, there are new discoveries of adjuvants and vaccine formulations to be tested as well as fundamentals of virus transmission, infectiousness, and pathogenicity. the ultimate test is in influenza-infected volunteers. this specialized work was initiated over years ago. during the great pandemic of , when the precise nature of the causative microbe of the spanish influenza had not been established, a group of american scientists asked for young volunteers from the army and navy. the quest was to probe the nature of the microbe that was already causing devastation in their own country and where, by , , young people were to die. however, this was not the first study into the precise nature of the microbe. the infection had first been documented a year earlier as a herald wave in the great city-sized military base and encampment of etaples [ , ] . here the british army constructed the largest establishment [ ] in its history, where , newly recruited soldiers each day intermingled with thousands of wounded soldiers, pigs and, in the nearby villages and markets, with ducks, domestic chickens, and geese. these are now recognized as the necessary biological features of an epicenter for the creation of a pandemic virus. we surmise, in retrospect, that an avian virus from a silently infected goose or duck could have crossed species either to a pig or to a soldier already infected with a human strain of influenza. this is the mixing bowl hypothesis. indeed, common epidemic influenza was known to be circulating in the winter of - in etaples. another factor in etaples could have been the hundreds of tons of gases of varieties contaminating the landscape of the nearby somme battlefield, as well as many of the wounded soldiers brought by the night trains into the hospitals on site and causing respiratory distress. a group of pathologists there and at abbeville, led by g. gibson, raised the question of the nature of the microbe. could it be a gram-negative bacterium such as h. influenzae, already described by pfeiffer as the cause of the previous influenza pandemic of ? or could it be a virus? viruses were rather unknown entities at that time but had been identified by their filter-passing nature. hence, gibson's experiment was quite simply to take sputum from a soldier victim and filter it through a berkefield candle filter, which would hold back any known bacterium but allow the passage of the much smaller ultrafilterable virus. but what then? gibson had not even considered that a human volunteer would receive the filtrate. in fact, he gave it to a series of macaques and, inadvertently, to himself. he died and the macaques became ill. his premature discovery of new virus influenza has lain undiscovered and hitherto unquoted in the archives of the first world war [ ] . meanwhile, in the usa, a more vigorous decision had been taken, and army and navy volunteers were infected intranasally with filtered material from spanish influenza victims. some volunteers were placed . m from dying servicemen, who coughed in their faces. the incredible result of this heroic endeavor is that not a single volunteer became ill, whereas all around the usa their companions were dying. it is more than possible that the volunteers had already been subclinically infected in the early summer outbreak of , which was less virulent than the autumn virus and would be expected to give cross-immunity. unit in salisbury (uk) as soon as the second world war was over, the medical research council in the uk established the common cold unit in salisbury at the harvard hospital. the hospital was a donation from the usa to cope with expected bomb casualties from london. in the event, this fully equipped multibuilding facility was used as an acute surgical hospital for servicemen. with christopher andrewes as its first chief scientist, the unit recruited volunteers to unravel the virological mysteries of respiratory disease. for the next years, a small team of virologists and clinicians infected volunteers and discovered the first human coronavirus, the common cold virus, and were the first to describe the clinical effects of interferons. essentially similar units were set up in the usa and ussr. the considerable difficulties encountered in mounting field trials led to experiments in which immunized volunteers were subjected to deliberate inoculation with live virus in the form of either attenuated strain or modified wild-type strain. this protocol was suggested by henle et al. [ ] , who immunized a group of children with inactivated influenza a (h n ) virus vaccine and then inoculated them with egg-cultured virus of the same subtype but recently isolated, by inhalation of an aerosol. high rates of infection ( %) were produced in unimmunized children of whom became ill. those receiving vaccine either escaped subsequent infection or developed serological changes; only child of the vaccinated children thus challenged became ill. although this study illustrated the outstanding success of the immunized protocol, there are probably few observers today who would be prepared to submit their children to a similar risk of deliberately induced illness. ideally young adults - are used for quarantine experiments. such a risk is, of course, experienced during epidemics and bell et al. [ ] undertook a similar experiment in adult volunteers some of whom were immunized with a single dose of inactivated a/japan/ / (h n ) virus vaccine soon after the a/asian epidemic began. the volunteers were isolated before being given intranasally pooled nasopharyngeal washings from patients with influenza and this caused clinical illness in % of volunteers previously given a placebo. as % of the vaccinated volunteers developed fever after challenge in this experiment, the single injection of inactivated vaccine proved relatively ineffective, presumably because of its inadequate immunogenicity. the information obtained by deliberate challenge of immunized volunteers has been explored in the past using modified attenuated virus strains. beare et al. [ ] did this in their comparison of inactivated or live influenza b vaccines in which a challenge from the live virus b strain was used to assess the comparative efficacy of the two vaccines. reinoculation with live virus was resisted better by those receiving the same material a month previously than by those injected with inactivated vaccine. couch [ ] has reported a number of trials in volunteers after inactivated vaccine using a low dose of an essentially unmodified h n virus that had received one or two passages in human embryonic kidney culture. it was first established by greenberg et al. [ ] that previous infection by homotypic h n virus gave protection against deliberate exposure for up to years after the original infection. comparison of inactivated vaccine a/hongkong/ (h n ) given intranasally or subcutaneously showed that following challenge with live virus only those who had developed a serum antibody response after vaccine by either route resisted infection. in a further trial of an anti-na inactivated vaccine made from an heq n virus, it was shown that a reduced frequency of illness and a reduced titer of virus in nasal wash specimens resulted following live h n virus challenge compared with the findings in control subjects. the number of those who contracted infection was also reduced somewhat by the inactivated na vaccine, thus supporting the suggestion of schulman et al. [ ] that na antibody, although incapable of neutralizing viral infectivity, could limit the extent of viral replication. beutner et al. [ ] also immunized children with an na-specific vaccine and noted that antibody to na had a role protecting against illness rather than against infection. slepushkin et al. [ ] and monto and kendal [ ] came to similar conclusions with regard to na vaccine and the clinical evidence of protection from illness. a series of experiments on volunteers, designed to obtain evidence of protection from vaccines containing viruses that were homotypic or heterologous to the challenge virus, is important in relation to the determination of the best composition of inactivated vaccine. potter et al. [ ] gave one of four inactivated monovalent h n virus vaccines to groups of students, measured their pre-and postimmunization antibodies by hi and ni tests, and later challenged all the groups with a live intranasal h n virus (wrl ). this virus was antigenically nearest to the a/port chalmers/ virus and vaccine from this latter strain and also that containing a/scotland/ virus gave better protection against infection than earlier h n virus vaccines; the result thus correlated with the induced hi antibody titers. larson et al. [ ] also challenged the immunity produced by inactivated vaccine made from a/port chalmers/ (h n ) virus with that from a strain developed by the pasteur institute [ ] . this virus ( c) with an antigen closely similar to a/england/ (h n ) was selected in the laboratory by a method analogous to natural selection by antigenic drift, and thus represents the first human attempt to anticipate antigen variation in nature. challenge of those immunized with one or the other vaccines showed that protection by the heterologous c virus was about one-quarter as effective as that produced by the homologous a/port chalmers/ virus. experiences related by couch also confirm [ ] that antibody effective against the homologous ha of the challenging virus is more protective than that formed by heterologous antigen. protection was also compared after inactivated vaccine by intranasal or subcutaneous routes, which showed that the important mediator of immunity was the serum igg content of anti-ha rather than the respiratory secretion content of specific iga. we have established a new quarantine unit, based in london (http://www.retroscreen. com), but very much centered upon the experience and ethos of the common cold unit of the past [ ] . in a series of experiments over the past years, we have infected over young volunteers with influenza a (h n ), influenza b, and influenza (h n ) virus and more recently respiratory syncytial virus, and we now have fully characterized virus pools [ ] . in the usa, a quarantine unit had already been established in virginia and also at baylor and pioneered work into the new na inhibitors of influenza using an influenza a virus isolated in [ ] . so far our own unit has focused on evaluating new influenza vaccines [ ] . we use groups of young volunteers and quarantine them in a student hostel or hotel or phase i clinical unit along with clinicians and scientists (fig. ) . the mrc common cold unit was rooted strongly in the postwar era with deck chairs, free run rabbits, country walks, afternoon cream teas, and two-course english meals. our new unit reflects a more diverse community, so chicken tikka is as common on the menu as roast lamb and baked potatoes, but the wish of many of the volunteers is the same: to contribute to knowledge. influenza a virus has a proven record as a "bioterrorist" virus but driven not in churchill's words by the "evil forces of perverted science" but by the vast unfathomable laws of nature and emergence, reemergence, and resurgence of natural disease. we are experiencing the attacks on pregnant women and younger persons at the present moment with a/swine h n [ ] [ ] [ ] . information from the human genome project, whereby a significant proportion of the , active genes are already known to be involved in innate and acquired immunity, provides reassurance that the immune system will continue to provide some protection against new fig. a volunteer room at the common cold and influenza unit, harvard hospital, salisbury, in the s. volunteers would stay for weeks in this country-placed unit to be infected and carefully studied for clinical symptoms viruses. this is excellently illustrated with a/swine where most of the population has immune memory to this h n family of viruses. gauguin in his last great painting "who are we, where have we come from, where are we going?" asks crucial questions about the future of humankind. but it was the medieval painter breugel who asked the major question, yet to be answered in the twenty-first century. his medieval painting "the triumph of death" shows a horseman on a white charger scything at random and gathering souls during an outbreak of pasteurella pestis in medieval times. the question haunting the painting is "why do some persons survive while others die." even in in most communities % of persons infected with the virus survived. but why did some die and exactly how were they killed by such a minute and fragile form of life that we know as the orthomyxovirus influenza? was the immune reaction and ensuing cytokine storm overwhelming or was virus replication in the endothelial cells of the air sacs more important? an extraordinary clear message is emerging, which tells us to build our public health infrastructure and continue and expand our epidemiological vigilance and surveillance against all these infectious viruses and bacteria. the virus cannot be permanently dislodged from its avian and swine reservoir. for pandemic influenza, every country needs a detailed and practical plan and a supply of antiviral drugs and new vaccines at hand for an emergence of h n . this virus will be a lot more difficult to deal with than a/swine h n . we would then be "at the end of the beginning" as regards protection of all citizens. influenza was the twentieth century's weapon of mass destruction. nature is the greatest bioterrorist of our world and emerging viruses could do for us all, as easily and as quickly, or even more so, than the great influenza of , except for the fact that we now have the ammunition to fight back: knowledge of virus transmission and how to break it with disinfectants and social distancing, and effective antivirals and vaccines. the current a/swine h n pandemic has exposed flaws in pandemic plans and also has exposed many countries that have no preparation whatsoever. the spanish influenza pandemic of - : new perspectives the great war america's forgotten pandemic studies of influenza in hospitals of the british armies in france reports on the pandemic of influenza - . reports on public health and medical subjects influenza a pandemics of the th century with special reference to : virology, pathology and epidemiology medical services diseases of the war human virology: a text for students of medicine influenza: the viruses and the disease strategies for containing an emerging influenza pandemic in southeast asia the great influenza, the epic story of the deadliest plague in history preparing 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fertilised chicken eggs immunological studies with the virus of influenza influenza: further experiments on the active immunisation of mice commission on influenza, board of influenza and other epidemic diseases in the arm ( ) a clinical evaluation of vaccination against influenza comparisons of serological and febrile responses in humans to vaccination with influenza viruses or their haemagglutinins respiratory virus vaccines. iii. some biological properties of sephadex-purified ether-extracted influenza virus antigens antibody response in humans to deoxycholate-treated influenza virus vaccines experience with vaccination against influenza in the spring of results of vaccination against influenza during the epidemic of future influenza vaccines and use of genetic recombinants purification of large quantities of influenza virus by density-gradient centrifugation a field evaluation of inactivated, zonal-centrifuged influenza vaccines in children in chapel hill influenza in the ussr in : recurrence of influenza virus a subtype h n reactions to concentrated influenza vaccines the antibody response and immunity to challenge infection induced by whole inactivated and tween-ether split influenza vaccines correlation of laboratory studies with clinical responses to a/new jersey influenza vaccines application of an improved singleradial-immunodiffusion technique for the assay of influenza haemagglutinin antigen content of whole virus and subunit vaccines a serological trial of asian influenza vaccine after the autumn epidemic clinical studies of monovalent inactivated whole virus and subunit a/ussr/ (h n ) vaccine serological responses and clinical reactions studies on inactivated influenza vaccines. ii. effect of increasing dosage on antibody with resistance to influenza in man immunity to attenuated influenza virus wrl infection induced by heterologous, inactivated influenza a virus vaccines a surface antigen influenza vaccine. . purification of haemagglutinin and neuraminidase proteins. . pyrogenicity and antigenicity pandemic working group of medical research council's committee on influenza and other respiratory virus vaccines ( ) antibody responses and reactogenicity of graded doses of inactivated influenza a/new jersey/ whole-virus vaccine in humans medical research council committee on influenza vaccine ( ) clinical trials of influenza vaccine clinical trials of influenza vaccine trials of an asian influenza vaccine clinical trials of oiladjuvant influenza vaccine, - dose-response relationship after immunisation of volunteers with a new surface-antigen-adsorbed influenza virus vaccine further studies of the neuraminidase content of inactivated influenza vaccines and the neuraminidase antibody responses after vaccination of immunologically primed and unprimed populations influenza virus subunit vaccines. ii. immunogenicity and original antigenic sin in humans the specificity of the antihaemagluttinin antibody response induced in man by inactivated vaccines and by natural infection strain specificity of serum antibody to the haemagglutinin of influenza a (h n ) viruses in children following immunisation or natural infection immunisation with influenza virus a vaccines: comparison of intradermal and subcutaneous routes immunisation with asian strain influenza vaccineequivalence of the subcutaneous and intradermal routes effect of dosage and route of inoculation upon antigenicity of inactivated influenza virus vaccine (hong kong strain) in man influenza antibody in human respiratory secretions after subcutaneous or respiratory immunisation with inactivated virus specificity of respiratory secretion antibody against influenza virus purified influenza vaccine; clinical and serological response to varying doses and different routes of immunisation antibody in respiratory secretions following immunisation with influenza virus vaccines humoral and secretory antibody responses to immunisation with low and high dosage split 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recombinants derived from parents of known virulence for man gene constellation of live influenza a vaccines recombinant influenza a viruses as live vaccine for man the study of antiviral compounds in volunteers analysis of virion rna segments and polypeptides of influenza a virus recombinants of defined virulence poliomyelitis congresses ( - ) papers and discussions at st, nd, rd, th and th international poliomyelitis congresses the replication of type a influenza viruses in the infant rat: a marker for virus attenuation the basis of attenuation of virulence of influenza virus for man genetic approaches to control of influenza temperature-sensitive mutants of influenza a virus xii. safety, antigenicity, transmissibility and efficacy of influenza a/udorn/ -ts- [e] recombinant viruses in human adults adaptation and growth characteristics of influenza virus at c biological and immunologic characteristics of cold-adapted influenza virus cold adapted variants of influenza a. ii. comparison of the genetic and biological properties of ts mutants and recombinants of the cold-adapted a/ann arbor/ / strain cold-adapted recombinant influenza a virus vaccines in young seronegative children use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza a virus vaccine obtaining of an additionally attenuated vaccinating cryophilic influenza strain studies on some biological properties of vaccinal influenza strains cultivated at low temperatures some problems with modern influenza prophylaxis with live vaccine recombinant wrl strain live attenuated influenza vaccine. immunogenicity, reactivity and transmissibility evidence for host-cell selection of influenza virus antigenic variants a universal influenza a vaccine based on the extra cellular domain of the m protein induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines testament of youth: an autobiographical study of the years - the etiology of influenza: a filterable virus as the cause (with some notes on the culture of the virus by the method of noguchi) demonstration of the efficacy of vaccination against influenza type a by experimental infection of human beings artificially induced asian influenza in vaccinated and unvaccinated volunteers assessment of immunity to influenza virus using artificial challenge of normal volunteers with influenza virus duration of immunity to type a influenza protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice evaluation of a neuraminidase-specific influenza a virus vaccine in children. antibody responses and effects on two successive outbreaks of natural infection neuraminidase and resistance to vaccination with live influenza a hong kong vaccine effect of neuraminidase antibody on hong kong influenza immunity to challenge in volunteers vaccinated with an inactivated current or earlier strain of influenza a (h n ) sélection par pression immunologique de mutants dominants du virus de la grippe a (hong kong) cold wars: the fight against the common cold fluinsure tm , an inactivated trivalent influenza vaccine for intranasal administration, is protective in human challenge with a/panama/ / (h n ) virus. in: kawaoka y (ed) options for the control of influenza volunteer challenge studies dna vaccination protects against an influenza challenge in a phase b double blind randomised placebo controlled clinical trial the emerging influenza pandemic: estimating the case fatality ratio assessing the severity of the novel a/h n pandemic cdc. novel h n influenza vaccine acknowledgments we are pleased to receive grant income from the eu to develop new influenza vaccines. key: cord- -ts g ux authors: katragkou, aspasia; roilides, emmanuel; walsh, thomas j. title: role of immunoglobulin therapy to prevent and treat infections date: - - journal: management of infections in the immunocompromised host doi: . / - - - - _ sha: doc_id: cord_uid: ts g ux immunoglobulins have been used widely in medicine for a variety of diseases including infectious diseases. while the main clinical applications of immunoglobulin therapy concern their use as replacement for patients with primary immunodeficiencies, or as treatment for autoimmune and inflammatory disorders, their role in infectious disease is limited largely to viral and toxin neutralization and replacement therapy in patients with immunoglobulin deficiencies. many aspects of the therapeutic regimen of immunoglobulins even in the established indications remain open. recently, due to the worldwide surge of immunosuppression caused by aids, organ transplantation, cancer, and autoimmune therapies, as well as the emergence of multidrug-resistant bacteria, there has been renewed interest in the use of antibody preparation to prevent infections in high-risk groups. knowing the limitations of the current anti-infective armamentarium, approaches that target the host through manipulations to augment the host immune response provide a helpful aid to conventional treatment options. a substantial body of evidence has demonstrated that strategies aiming to support or stimulate immune response could be feasible approaches that would benefit immunocompromised patients. in the present chapter, we present contemporary indications of immunoglobulin administration for therapy and prophylaxis of infections in the immunocompromised population. salmonella species, no consistently effective sera were produced [ ] . with the discovery of antibiotics in s, the interest in "serum therapy" for the treatment of infectious disease waned. the initial interest in using combination therapies with antibiotics and serum was abandoned, as the potential benefits were marginal. antimicrobial chemotherapy proved to be less toxic and more effective than serum therapy in the treatment of infections. however, dr. cohn's discovery of purified antibodies through cold ethanol fractionation of plasma during the second world war revived the interest in antibody treatment especially for infections not able to be treated with antibiotics. the fractionation procedure stabilizes the product, denatures most viruses, and assures a more uniform antibody content. cohn fraction (igg from plasma after cold alcohol fractionation) was initially used for prophylaxis against prevalent and life-threatening infections, such as measles. it was not until that bruton reported for the first time the use of immunoglobulin preparation injected subcutaneously for the treatment of a young boy with agammaglobulinemia [ ] . thereafter, the use of immunoglobulin injected intramuscularly became established as the standard therapy for primary immunodeficiencies, lasting until the development of purer and safer intravenous immunoglobulin preparation in the early s [ ] . the advent of hybridoma technology, which allows continuous generation of large quantities of monoclonal antibodies specific to antigens of interest and the generation of humanized antibodies, revolutionized antibody therapeutics [ ] . monoclonal antibody technology offers supply advantage, reduces the risks of adverse events, and decreases lot-to-lot variation. in the mid- s a monoclonal antibody (mab) to cd was introduced into clinical practice to prevent organ rejection. almost a decade later, the humanized mab palivizumab (synagis®, a humanized mouse monoclonal antibody to prevent rsv pulmonary infections in high-risk patients, especially infants) was licensed. palivizumab was -fold more potent than the polyclonal product, resulting in reduced volume of administration and intramuscular use [ ] . during the last three decades, therapeutic mabs have been licensed, mainly for treatment of malignancies and rheumatic or autoimmune diseases, but only two were licensed for infectious diseases (palivizumab and raxibacumab: human mab to anthrax toxin). although the use of mabs to treat infectious diseases does not depend on discrimination between self-antigen as there are large antigenic differences between the microorganism and the host, the pace of discovery and development of new mabs against infectious disease is limited. currently, the areas of mabs development have been focused on viral diseases without available vaccines [hiv, ebola, severe acute respiratory syndrome (sars), middle east respiratory syndrome, marburg virus], viral disease with limited effective antiviral drugs (influenza, rabies), and bacterial toxinmediated disease (anthrax, clostridium difficile colitis). in the clinical setting, therapeutic mabs can be used when there are nosocomial/iatrogenic outbreaks. for example, a new approach to the prevention of recurrent c. difficile infection is the administration of mabs against c. difficile toxins (in addition to antibiotic therapy) as a form of passive immunity. actoxumab and bezlotoxumab are fully human monoclonal antibodies that bind and neutralize c. difficile toxins a and b, respectively. a double-blind randomized placebo-controlled phase trial showed that a single intravenous dose of bezlotoxumab when given with standard-of-care antibiotics provided protection against recurrent c. difficile infection for up to weeks that was superior to that provided by treatment with standard-of-care antibiotics alone [ ] . other therapeutic mabs can be applied in drug resistance (staphylococcus aureus, vrsa), pandemic outbreaks (ebola virus), bioterrorism attacks (bacillus anthracis), emerging infectious diseases (nipah or hendra virus), and use in high-risk host groups or in severe diseases (respiratory syncytial virus, cytomegalovirus retinitis in hiv patients, hepatitis c virus, influenza virus). another application of therapeutic mabs concerns their use as adjunct therapies that have anti-inflammatory or immune modulatory roles (mabs against tnf-α and other immune mediators) [ , ] . immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to antigens or immunogens and which function as antibodies. serum contains a heterogeneous immunoglobulin pool that reflects the host response to endogenous microbiota and the immunological memory of the host for a variety of acquired microbial agents [ ] . different immunoglobulins can differ structurally; however, they are built from the same basic units. there are five classes of immunoglobulin, classified according to the type of heavy chain they possess (table . ) [ ] . each class of immunoglobulins has a specific function, and deficiency of each class leads to particular dysfunction of immune system. serum igm predominates in the acute immune response to most antigens and is the most efficient complementfixing immunoglobulin. immunoglobulin class switching subsequently occurs, leading to a predominance of igg, which is responsible for protection during the first infectious attack and long-term protection via memory b cells. secretory iga, due to its abundance in mucosal secretions, provides primary defense mechanism against some mucosal infections. ige primarily defends against parasitic invasion [ ] . immunoglobulins together with t cells are the key mediators of adaptive immunity, and deficiencies in either of these two arms of the adaptive immune system can result in higher host susceptibility to bacterial, fungal, or viral infections [ ] . immunoglobulins interact with the [ , ] . understanding the mechanisms of interactions between immunoglobulins, immunomodulatory molecules, and cells of the immune system, both innate and adaptive, is the basis for understanding the future therapeutic perspectives of immunoglobulins [ ] . immunoglobulins, upon binding of a specific antigen, stimulate significant direct and indirect "effector functions." classically, in bacterial disease, immunoglobulins neutralize toxins, facilitate opsonization, and, with complement, promote bacteriolysis. in viral diseases, immunoglobulins block viral entry into uninfected cells, promote antibody-directed cell-mediated cytotoxicity by natural killer cells, and neutralize virus alone or with the participation with the complement [ ] . furthermore, more recent studies have demonstrated the immunomodulatory functions of antibodies, including the potential for antibody therapy to reduce damage from the host inflammatory response to major infections [ , ] . notably, igg can exert pro-and antiinflammatory activities depending on its concentration. low dose of igg has pro-inflammatory activity and requires complement activation or binding of the fc fragment from igg to iggspecific receptors (fcγr) on innate immune effector cells. this results in receptor clustering, recruitment of secondary effector functions, and subsequent activation of signaling pathways, leading to an increase in intracellular calcium levels and cell activation. by comparison, high concentrations of igg have anti-inflammatory properties. the mechanisms proposed for this mode of action are modulation of the expression and function of fcγrs, interference with activation of the complement cascade and the cytokine network, neutralization of autoantibodies, and regulation of cell proliferation [ ] . the immunoglobulin preparations used in passive immunization are the standard human serum immunoglobulin, which is available in three forms: immune globulin (ig) for intramuscular use (imig), intravenous use (ivig), and subcutaneous use (scig). imig is used primarily for the prevention of certain infections, such as hepatitis a, measles, and rubella, and less commonly for the treatment of antibody immunodeficiencies. ivig is used in the treatment of primary and secondary antibody deficiencies, many immunoregulatory disorders (e.g., immune thrombocytopenic purpura, kawasaki disease), and neurologic disorders (e.g., guillain-barré syndrome, peripheral neuritis). igsc is used exclusively for the antibody deficiencies. ivig preparations comprise the pooled fraction of serum from ~ to , donors, which is generated by a cold ethanol precipitation, providing, thus, a broad spectrum of opsonic and neutralizing igg antibodies. opsonic and neutralizing igg antibody content varies with each product batch, primarily due to differences in the local pathogen ecology of donor exposure. igg and complement proteins are the principal classes of opsonins contributing to bacterial clearance. in addition to igg, varying amounts of immunoglobulin isotypes, especially iga, can be found in the ivig preparation. regarding the different human igg subclasses (igg -igg ), ivig preparations reflect the hierarchy present in the serum, consisting mainly of igg and igg and containing much smaller amounts of the other igg subclasses. only the product pentaglobin® (biotest, germany) is igm-enriched [ ] . the clinical use of ivig can be distinguished by the infused amount [ ] . the principal manufacturing process in all current igg preparations is cold ethanol fractionation with product-specific additional processes for manufacturing. the commonest processes for virus reduction include solvents/detergent, low ph (ph ), incubation, nanofiltration, and chromatography [ ] . other major quality control practices in the production process, besides viral reduction, include the depletion of blood coagulation factors and the removal of igg aggregates, since these aggregates could result in a cytokine release syndrome owing to the ubiquitous activation of innate immune effector cells via activating fcγrs. igg aggregations are absent from the majority of ivig preparation; however, depending on the provider and batch, up to - % of igg can be found in dimeric form in most ivig preparations [ ] . the two major indications for which immunoglobulins are used are igg replacement therapy and anti-inflammatory therapy in a variety of acute and chronic autoimmune diseases. apart from immunoglobulin replacement therapy, currently licensed application of immunoglobulin (ivig) administration includes guillain-barré syndrome, kawasaki disease, and chronic inflammatory demyelinating polyneuropathy. licensed indications, however, only account for approximately - % of the worldwide immunoglobulin sales, as most immunoglobulin administrations are "off-label" [ ] . the use of immunoglobulins for infectious disease can involve the passive transfer of antibodies for pre-/postexposure prophylaxis or for treatment. passive immunization provides temporal immunity to unimmunized individuals either prophylactically or therapeuti-cally. the different forms of passive immunotherapies are shown in table . [ ] . the technology of ethanol fractionation of plasma resulted in products used for the treatment and prophylaxis of infectious diseases (table . ). human immune sera have fewer adverse effects, but there are concerns about availability, potency, and consistency. table . summarizes the adverse reactions of immunoglobulin used in the prevention and treatment of infectious diseases. administration of immunoglobulins is indicated for the majority of patients with primary immune deficiencies and for patients with combined immune deficiencies and for those with secondary immune deficiency with significant antibody deficiency. the benefits of replacement immunoglobulin therapy for the prevention of infections in patients with antibody deficiencies are well established and pertain to the reduction of the incidence and the severity of infections and prevention long-term deterioration in organ function [ , ] . primary immune deficiencies (pidd) are one of the us food and drug administration (fda)approved indications for immunoglobulin therapy. over % of all pidd involve antibody-mediated immunity; however, each individual disorder has a different immunopathogenesis in terms of the number of b cells in the blood and b-cell function. moreover, any persisting endogenous antibody production varies both between specific conditions and within individual disorders [ , , ] . table . describes the pidd for which immunoglobulin replacement is or may be efficacious. the recommendations for immunoglobulin replacement treatment in primary and secondary immune deficiencies are shown in table . [ , , ] . the main indications are primary antibody deficiencies including agammaglobulinemia (autosomal recessive or x-linked) and common variable immunodeficiency disorders. rarely, other antibody deficiencies, such as igg subclass deficiency, may be managed by immunoglobulin replacement. in these immunodeficiencies, a trial of months may be indicated if there is a substantial infection burden. on the contrary, for selective iga deficiency, immunoglobulin replacement is not required or recommended, as anaphylactic reactions may occur during ivig infusions. combined immunodeficiencies with antibody deficiency also benefit from immunoglobulin therapy until the defects in cell-mediated immunity are corrected by hematopoietic stem cell transplantation. however, b-cell function is not restored universally after transplantation, and immunoglobulin therapy may be continued [ ] . it is important that each patient receives a thorough evaluation before starting immunoglobulin therapy especially those with partial antibody defects. the american academy of allergy, asthma, and immunology, based on a review of evidence, recommends for pidd the dose of - mg/kg of ivig every weeks, titrating the dose and interval between infusions to achieve a trough igg level at least greater than mg/dl in agammaglobulinemic patients [ ] . however, recent evidence suggests that the goal of igg replacement therapy should be to reduce or prevent serious or recurrent infections instead of aiming to achieve a specific igg level. the clinicians should identify for each patient with pidd an individual "biological" igg level with which the patient achieves the best clinical outcome instead of trying to reach a specific igg level [ , ] . the two modes of igg replacement (ivig and scig) have significant pharmacokinetic differences, which are important to know when choosing the mode of igg delivery or switching from ivig to scig. scig causes sustained release of igg and thus attains higher igg trough levels; this mode of delivery may benefit the - % of patients who show increased risk of infection during the rd and th weeks after receiving ivig or who experience extreme lethargy during the same period. ivig achieves higher peak levels ( % higher than that obtained by sc infusion), and this mode of delivery is usually initially preferred for patients with pidd who are very symptomatic (present with pneumonia or other serious infectious such as sepsis) and who present with pneumonia or for those with other medical problems such as sepsis [ ] . ivig has also been used in a number of diseases that cause secondary humoral immunodeficiency. while for the majority of secondary immunodeficiencies, the use of ivig was supported only by anecdotal reports, and b-cell chronic lymphocytic leukemia (cll) and pediatric hiv infection are fda-approved indications. for both, cll and hiv, infections are the most common complications. ivig has been shown to be a useful prophylactic therapy against infections in such patients [ , , , , , ] . administration of ivig in cll patients with hypogammaglobulinemia has been shown to decrease the rate of bacterial infections; however, decision analysis modeling showed that this decrease might not improve the length or quality of treated patients' lives, and, furthermore, it is extraordinarily expensive [ ] . the prophylactic administration of ivig in cll patients has not been studied extensively, and, thus, there are no guidelines to define the patient population that would benefit from this treatment; also the optimal dosing and timing of ivig administration remained to be defined. some experts support the use of ivig in selected cases, depending on the history of the patient and especially in patients that ivig has been shown to work in the past. ivig therapy together with antiviral therapy was beneficial in infants and children with aids and hypogammaglobulinemia or two or more bacterial infections in the previous year. other indications for ivig therapy in hiv-infected patients include those with severe parvovirus b or measles infection [ , , ] . however, it is important to note that these studies occurred before the era of highly active antiretroviral treatment for hiv [ ] . ivig has been utilized in allogeneic bone marrow transplantation (bmt) in an attempt to decrease the incidence of cytomegalovirus (cmv) infection, infections due to other pathogens, and graft-versus-host disease (gvhd). immunoglobulin use in the setting of bmt is fda approved. the rationale for using ivig in transplantation is that the administration of passive antibodies may prevent infections in these immunocompromised patients and especially infections caused by cmv [ ] . several randomized controlled trials provided the basis to recommend ivig after allogeneic bmt [ , , , , , , ] . meta-analysis of these trials found significant reduction of fatal cmv infections, cmv pneumonia, non-cmv interstitial pneumonia, and transplant-related mortality among patients receiving prophylactic ivig [ ] . while an improvement in survival was reported in some studies [ , , ] , a more recent meta-analysis showed that ivig or hyperimmune cmv-ivig had no effect on the reduction of allcause mortality [ ] . collectively, the data regarding the benefit of prophylactic administration of ivig after bmt remain controversial and contradictory. in addition, until currently, there is no consensus on the type, schedule, dose, and patients benefiting from ivig. subsequent studies suggested that double prophylaxis consisting of high-dose ivig and ganciclovir was more successful than either treatment alone in reversing cmv pneumonia in patients after bmt [ , ] . the american society for blood and marrow transplantation does not recommend the routine use of ivig to hematopoietic cell transplant recipients for prophylaxis for cmv disease or for bacterial infections within the first days after transplantation. for patients with severe hypogammaglobulinemia (igg < mg/dl), ivig prophylaxis of bacterial infections may be considered. ivig dose and frequency for these patients should be individualized to maintain trough serum igg concentrations > mg/dl [ ] . routine use of ivig appears to offer little benefit to patients with malignancies undergoing hla-identical sibling bmt [ ] . given that the landscape of patients receiving bmt is evolving, it is likely that the available data are outdated, and more updated randomized trials are warranted to inform clinical practice. gvhd and infection are major complications of allogeneic bmt. in vitro and in vivo experimental models showed that the prevention of acute gvhd by ivig is mediated by the induction of apoptosis of activated alloreactive cd + expressing cd + donor t cells and reducing the amount of ifn-γ produced by donor t cells [ ] . ivig was shown to decrease the severity of acute gvhd in recipients of allogeneic bmt [ , , ] . on the contrary, administration of ivig prophylaxis has no effect on the incidence or mortality of chronic gvhd on bmt [ ] . while there is no consensus on the optimal dose of ivig, it appears that the incidence of acute gvhd is less in patients receiving higher doses of ivig. the benefits of ivig appear to correlate with igg trough levels where acute gvhd was less frequent among patients achieving maximum serum igg levels ≥ mg/dl after the administration of ivig. trough serum igg levels > mg/dl were associated with less severe acute gvhd [ , , ] . over the last decade, ivig usage in solid organ transplantation has increased significantly. there are encouraging data on the role of ivig for the treatment of antibody-mediated rejection, desensitization to hla and/or abo antigens, as well as prevention and treatment of infectious complications for patients undergoing solid organ transplantation [ , ] . there is also some evidence that ivig may be useful for the treatment of autoimmune cytopenias after solid organ transplantation [ ] . dosing of ivig is empiric although higher than those for replacement therapy. especially for the treatment of antibodymediated rejection, the dose is - gm/kg [ , , , , , ] . the use of higher doses of ivig is related to higher rates of adverse events. these include aseptic meningitis thrombotic events and bronchospasm [ ] . sepsis is the systemic inflammatory response of the host to an infectious insult. severe sepsis is characterized by acute organ dysfunction, while septic shock is characterized by hypotension, which is refractory to fluid replacement, or by hyperlactatemia [ ] . severe sepsis and septic shock represent one of the oldest and most pressing problems in medicine. care of patients with sepsis has improved over the last decades; however, the incidence of sepsis is increasing along with morbidity and mortality rates especially in critically ill adults. worldwide, the annual incidence of severe sepsis lies between and cases per , population, and mortality for severe sepsis and septic shock reaches % and %, respectively [ , , , ] . while our understanding of the underlying biologic features of sepsis has made significant progress, the clinical assessment of several new strategies for implementation for sepsis treatment has led to disappointing results [ , , , , ] . there have been more than randomized clinical trials of strategies to modify the systemic inflammatory response during sepsis; however, no strategy showed to improve dramatically the survival of patients with sepsis [ ] . the development of highly purified human plasma-derived polyclonal ivigs presented a very compelling therapy for severe infections including sepsis and septic shock. ivigs have broad and potent activity against microorganisms, their extracellular products, and potent immunomodulatory effects [ ] . ivig preparations, in particular igm-enriched preparations, contain antibodies against lipopolysaccharides of escherichia coli, pseudomonas aeruginosa, and klebsiella spp. [ ] . the effects of ivigs on the sepsis-induced host response seem to be pleotropic, not yet completely clarified, and are likely to be secondary to both suppression of synthesis and direct scavenging of upstream and downstream mediators of the host response and complex immunomodulatory effects [ ] . the cellular effects of immunoglobulins are mediated through the igg constant fragment (fc). immunoglobulin acts as an adaptor between the innate and adaptive immune system by interacting with fc, which mediate both pro-and antiinflammatory signals. ivigs have direct antibacterial effects through pathogen recognition and increased clearance. ivigs also have anti-inflammatory properties mediated by the scavenging of bacterial toxins and proinflammatory cytokines, by immune cell depletion, by the blockade of activating receptors, and by modulating fcγr expression, dendritic cell activity, and t-cell expansion [ , , ] . the challenging pathobiology of sepsis is associated with acquired hypogammaglobulinemia, which seems to prevent optimal pathogen clearance and pathogen toxin scavenging [ , , ] . furthermore, sepsis, by causing endothelial dysfunction and capillary leak together with the iatrogenic fluid resuscitationrelated increase in extravascular volume, eventually causes an alteration in the distribution of immunoglobulins [ ] . consequently, it is logical to predict that the administration of ivig during sepsis would be of benefit. in the clinical setting, the role of ivig as an adjunctive treatment in sepsis has been controversial for years. a number of randomized placebo-controlled clinical trials in adult critical care patients evaluating standard polyclonal ivig-or igm-enriched polyclonal adjunctive therapy in severe sepsis as well as the metaanalyses of these trials have been published [ , , , , , ] . positive findings of controlled trials and anecdotal reports have been criticized for methodological weakness including the small number of the patients and adequacy of blinding. the more recent studies, which were more meticulously designed, have shown much less effect of ivig than older, smaller, and less well-designed studies [ ] . of note, the studies that used albumin as control showed less benefit of ivig than those that did not [ ] . the score-based immunoglobulin g treatment in sepsis (sbits) study, one carefully designed, large study representing almost half of all the adults studied to date, showed no reduction in mortality by ivig in patients with score-defined sepsis and sepsis-induced multi-organ failure [ ] . the first clinical trial, which evaluated the effect of igma-enriched immunoglobulin preparation ( . g igm, . g iga, and . g igg), which have shown to contain superior antibody content against bacterial lipopolysaccharides, in an appreciable number of neutropenic patients with hematologic malignancies and sepsis or septic shock, showed that immunoglobulins had no beneficial effects [ ] . however, as the editor comments, the study, with a high evidence level, demonstrates that neutropenic patients with malignancies and low-grade sepsis with no or only one organ failure will not benefit from adjunctive ivig treatment [ ] . the prophylaxis and treatment of neonatal sepsis has been a major global priority, and large international trials have been carried out testing ivig ( [ , [ ] [ ] [ ] [ ] [ ] ; group et al. ). mortality during hospital stay in infants with clinically suspected infection at trial entry was not significantly different after ivig treatment [ ] . the results of the international neonatal immunotherapy study (inis) and recent metaanalyses showed that ivig did not reduce mortality during hospital stay or major disability at years of age in infants with sepsis [ , ] . based on the results of the inis trial ( subjects), routine administration of ivig to prevent mortality in infants with sepsis is not currently recommended [ ] . when considering the administration of ivig during sepsis, important aspects that should be taken into account are the dose, the type, the timing, and pharmacokinetics of ivig [ , ] . while dose-ranging studies have not been completed, studies that used high (> g/kg body weight) doses of ivig demonstrated better effects. this seems plausible given the clinical observations in other inflammatory conditions, such as kawasaki disease, where greater effect was noted with higher doses [ ] . the type of ivig may have an important effect, possibly in favor of a greater pooled effect of igma-enriched compared with standard preparations of ivig. igma-enriched preparations are associated with greater complement inactivation and improvement in microvascular perfusion in experimental models [ ] . however, collectively, the results from animal models and in vitro experiments show contradictory results and do not allow for a definite conclusion regarding the superiority of one specific immunoglobulin preparation in patients with sepsis. in an efficacy study, administration of polyvalent igg versus igma in selected patients at high risk for sepsis was associated with a comparable improvement in disease severity [ ] . regarding the timing of ivig administration during sepsis, there is probably a "window of opportunity" in the first days that follow clinical presentation of sepsis [ ] . if this window is missed, probabilities of success could be greatly diminished [ ] . pharmacokinetic studies of ivig in sepsis have not been performed yet. data for dosage selection in current practice are primarily derived from studies in volunteers and in patients with primary immune deficiencies and other indications for immunomodulation. existing pharmacokinetic studies also do not address immunoglobulin clearance or area under the curve parameters and target serum immunoglobulin concentrations [ ] . in addition, it is still unknown whether the main goal of ivig in sepsis is to refill low levels of endogenous immunoglobulins or alternatively whether ivig could exert a beneficial effect regardless of these levels [ ] . most studies evaluating the use of ivig for sepsis are small; some have methodological flaws and high-quality, large studies showed no effect [ , ] . given immunoglobulin high-cost, limited supply and the lack of strong evidence to support their beneficial effect, widely used guidelines either neglect or grade as a weak recommendation the use of polyclonal ivig in sepsis [ ] . while clinical judgment may guide immunoglobulin use in individual cases, particularly those due to gram-negative etiologies or streptococcal toxic shock syndrome, these practices are based largely on theoretical rationale, anecdotal, and retrospective clinical observations [ , , ] . the effect of monoclonal antibodies against tumor necrosis factor (tnf)-α has been evaluated in a series of trials on different anti-tnf-αdirected therapies [ , , , ] . the long-anticipated sepsis trial (monarcs [monoclonal anti-tnf, a randomized controlled sepsis trial]) reported that afelimomab, which is made up of the fab component of a monoclonal antibody against tnf-α, in patients with severe sepsis and elevated il- levels decreased mortality and had a safety profile similar to placebo [ ] . however, combining the results of these studies, a small improvement in mortality can be detected [ , ] . as sepsis is increasingly being considered as an exaggerated, poorly regulated innate immune response to microbial products, by the time of diagnosis, an entire network of cytokines has already been activated. in this regard, the results of the previous studies would have been anticipated, as it seems unlikely that therapy aimed at only one cytokine would by itself have the highly significantly impact on sepsis mortality [ ] . immunoglobulins have been used widely in medicine for a variety of diseases including infectious diseases. while the two major indications for immunoglobulin use are as replacement and antiinflammatory therapy in a variety of acute and chronic autoimmune diseases, their use in the prevention and treatment of infectious diseases is emerging as an attractive option especially in the era of multi-antibiotic resistance. many aspects of immunoglobulin therapy remain controversial and contradictory. consequently, immunoglobulin use is sometimes determined by clinical judgment or expert opinion, which is based largely on theoretical rationale, anecdotal, and retrospective clinical observations. gaps of knowledge that need to be addressed are certain categories of patient populations that would benefit from immunoglobulin treatment or prophylaxis, the optimal immunoglobulin dosing, and duration, as well as timing of administration. monoclonal antibody technology has opened a new era in antibody therapy. on many occasions, human monoclonal antibodies have better therapeutic properties than immunoglobulins including low toxicity, longer protective immunity, higher than natural protection, and high specificity. several antibodies for the treatment of bacterial and viral infections have been developed [ ] . however, some challenges need to be overcome before they become preferred agents for the treatment and prophylaxis against infectious diseases. biofilms are now acknowledged to contribute to a plethora of chronic and recurrent infections. while treatment or eradication of biofilm-related infections is still challenging, there are sufficient in vitro and preclinical data to support the use of antibodies directed against extracellular dnabinding proteins entrapped into the extracellular biofilm polymeric substance [ , ] . while still an area of ongoing preclinical and clinical research, this use of antibodies constitutes a novel therapeutic approach for treatment of biofilm-related infections. comparison of two doses of intravenous immunoglobulin after allogeneic bone marrow transplants different brands of intravenous immunoglobulin for primary immunodeficiencies: how to choose the best option for the patient? efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. a randomized, controlled, double-blind, multicenter clinical trial. tnf-alpha mab sepsis study group intravenous immunoglobulin for treating sepsis and septic shock the original sins of clinical trials with intravenous immunoglobulins in sepsis e murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. e study investigators epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care severe sepsis and septic shock efficacy of immune globulin in preventing complications of bone marrow transplantation: a meta-analysis goals of therapy in antibody deficiency syndromes principles of and advances in immunoglobulin replacement therapy for primary immunodeficiency relationship between the timing of administration of igm and iga enriched immunoglobulins in patients with severe sepsis and septic shock and the outcome: a retrospective analysis the effects of ibuprofen on the physiology and survival of patients with sepsis. the ibuprofen in sepsis study group using intravenous immunoglobulin (ivig) to treat patients with primary immune deficiency disease biologic igg level in primary immunodeficiency disease: the igg level that protects against recurrent infection a controlled clinical trial of high-dose methylprednisolone in the treatment of severe sepsis and septic shock cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplantation evaluation of the kinetics and mechanism of action of anti-integration host factor-mediated disruption of bacterial biofilms normal igg protects against acute graft-versus-host disease by targeting cd (+)cd (+) donor alloreactive t cells a randomized and prospective study comparing treatment with high-dose intravenous immunoglobulin with monoclonal antibodies for rescue of kidney grafts with steroid-resistant rejection passive antibody therapy for infectious diseases antibody-mediated regulation of cellular immunity and the inflammatory response a reappraisal of humoral immunity based on mechanisms of antibodymediated protection against intracellular pathogens serum therapy revisited: animal models of infection and development of passive antibody therapy return to the past: the case for antibody-based therapies in infectious diseases immunoglobulin replacement in patients with chronic lymphocytic leukaemia: a comparison of two dose regimes intersept: an international, multicenter, placebo-controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. international sepsis trial study group prevention of cytomegalovirus infection by prophylaxis with an intravenous, hyperimmune, native, unmodified cytomegalovirus globulin. randomized trial in bone marrow transplant recipients guidelines for the use of human immunoglobulin therapy in patients with primary immunodeficiencies in latin america some but not all benefits of intravenous immunoglobulin therapy after marrow transplantation appear to correlate with igg trough levels a new paradigm for the treatment of sepsis: is it time to consider combination therapy? key aspects for successful immunoglobulin therapy of primary immunodeficiencies surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock cdp , a humanized antibody to human tumor necrosis factor-alpha: safety, pharmacokinetics, immune response, and influence of the antibody on cytokine concentrations in patients with septic shock. cpd sepsis study group intravenous immunoglobulins -understanding properties and mechanisms cytomegalovirus pneumonia after bone marrow transplantation successfully treated with the combination of ganciclovir and high-dose intravenous immune globulin reduced dose intravenous immunoglobulin does not decrease transplantrelated complications in adults given related donor marrow allografts a comparison of albumin and saline for fluid resuscitation in the intensive care unit influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. the cb sepsis syndrome study group the results of the serum treatment in thirteen hundred cases of epidemic meningitis biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins history of passive antibody administration for prevention and treatment of infectious diseases intravenous immunoglobulin may lessen all forms of infection in patients receiving allogeneic bone marrow transplantation for acute lymphoblastic leukemia: a pediatric oncology group study afelimomab-another therapeutic option in sepsis therapy? treatment of neonatal sepsis with intravenous immune globulin immune globulin therapy in allogeneic bone marrow transplant: a critical review clinical applications of intravenous immunoglobulins (ivig) -beyond immunodeficiencies and neurology igma-enriched immunoglobulin in neutropenic patients with sepsis syndrome and septic shock: a randomized, controlled, multiple-center trial high-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood the national institute of child health and human developments intravenous immunoglobulin study group. intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection cooperative group for the study of immunoglobulin in chronic lymphocytic leukemia. intravenous immunoglobulin for the prevention of infection in chronic lymphocytic leukemia. a randomized, controlled clinical trial molecular properties of human igg subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases longterm cognitive impairment and functional disability among survivors of severe sepsis the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis posttransplant therapy using high-dose human immunoglobulin (intravenous gammaglobulin) to control acute humoral rejection in renal and cardiac allograft recipients and potential mechanism of action clinical aspects of intravenous immunoglobulin use in solid organ transplant recipients intravenous immunoglobulin a natural regulator of immunity and inflammation evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: report of the nih ig trial passive immunity in prevention and treatment of infectious diseases continuous cultures of fused cells secreting antibody of predefined specificity pharmacokinetics of intravenous immunoglobulin: a systematic review use of polyclonal immunoglobulins as adjunctive therapy for sepsis or septic shock intravenous immunoglobulin for severe infections: a survey of canadian specialists polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and metaanalysis origin of the terms 'antibody' and 'antigen' treatment of interstitial pneumonitis due to cytomegalovirus with ganciclovir and intravenous immune globulin: experience of european bone marrow transplant group discovery of antibodies why have clinical trials in sepsis failed? crossover of placebo patients to intravenous immunoglobulin confirms efficacy for prophylaxis of bacterial infections and reduction of hospitalizations in human immunodeficiency virus-infected children. the national institute of child health and human development intravenous immunoglobulin clinical trial study group epidemiology of sepsis: race, sex, and chronic alcohol abuse guidelines for the use of immunoglobulin therapy for primary immune deficiency and solid organ transplantation costeffectiveness of immunoglobulin m-enriched immunoglobulin (pentaglobin) in the treatment of severe sepsis and septic shock anti-inflammatory actions of intravenous immunoglobulin intravenous polyclonal igm-enriched immunoglobulin therapy in sepsis: a review of clinical efficacy in relation to microbiological aetiology and severity of sepsis intravenous immunoglobulin for preventing infection in preterm and/or low-birth-weight infants intravenous immunoglobulin for suspected or subsequently proven infection in neonates intravenous immunoglobulin for preventing infection in preterm and/or low birth weight infants intravenous immunoglobulin for suspected or proven infection in neonates intravenous immunoglobulin for suspected or proven infection in neonates use of intravenous immunoglobulin in human disease: a 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immunoglobulin therapy for sepsis -biological plausibility from a critical care perspective a controlled trial of intravenous immune globulin for the prevention of serious bacterial infections in children receiving zidovudine for advanced human immunodeficiency virus infection. pediatric aids clinical trials group intravenous immunoglobulins as therapeutic agents therapeutic use of immunoglobulins immunomodulatory and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation a controlled trial of long-term administration of intravenous immunoglobulin to prevent late infection and chronic graft-vs.-host disease after marrow transplantation: clinical outcome and effect on subsequent immune recovery gamma-globulin levels in patients with community-acquired septic shock monoclonal antibodies in infectious diseases: clinical pipeline in modulation of the cellular immune system by intravenous immunoglobulin guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective bacterial lipopolysaccharide (lps)-specific antibodies in commercial human immunoglobulin preparations: superior antibody content of an igm-enriched product metaanalysis: intravenous immunoglobulin in critically ill adult patients with sepsis a survey of physician's attitudes regarding management of severe group a streptococcal infections veterans administration systemic sepsis cooperative study, g. effect of high-dose glucocorticoid therapy on mortality in patients with clinical signs of systemic sepsis international study of the prevalence and outcomes of infection in intensive care units cost effectiveness of prophylactic intravenous immune globulin in chronic lymphocytic leukemia immunoglobulin treatment in sepsis -is the answer "no"? ccm. . .bb mirror, mirror on the wall, which is the fairest meta-analysis of all? score-based immunoglobulin g therapy of patients with sepsis: the sbits study bezlotoxumab for prevention of recurrent clostridium difficile infection emil von behring and serum therapy intravenous immunoglobulin and cmvseronegative blood products for prevention of cmv infection and disease in bone marrow transplant recipients intravenous immune globulin for prevention of cytomegalovirus infection and interstitial pneumonia after bone marrow transplantation high-dose weekly intravenous immunoglobulin to prevent infections in patients undergoing autologous bone marrow transplantation or severe myelosuppressive therapy. a study of the american bone marrow transplant group does intravenous immune globulin have a role in hiv-infected patients? textbook of pediatric infectious diseases key: cord- -wfuzk dp authors: meza, diana k.; broos, alice; becker, daniel j.; behdenna, abdelkader; willett, brian j.; viana, mafalda; streicker, daniel g. title: predicting the presence and titer of rabies virus neutralizing antibodies from low-volume serum samples in low-containment facilities date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: wfuzk dp serology is a core component of the surveillance and management of viral zoonoses. virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. here, focusing on rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmrffit) that overcomes these limitations. specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein–tagged murine leukemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. we further used statistical analysis to generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing antibodies from microscopic imaging of neutralization outcomes. using serum samples from domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus neutralization test (favn), pmrffit showed moderate sensitivity ( . %) and high specificity ( . %). despite small conflicts, titer predictions were correlated across tests repeated on different dates both for dog samples (r = . ), and for a second dataset of sera from wild common vampire bats (r = . , n = ), indicating repeatability. our test uses a starting volume of . μl of serum, estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. the pmrffit enables high-throughput detection of rabies virus neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained. serology is a core component of the surveillance and management of viral zoonoses. virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. here, focusing on rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmrffit) that overcomes these limitations. specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein-tagged murine leukemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. we further used statistical analysis to generate rapid, quantitative predictions of the probability and titer of rabies virus neutralizing antibodies from microscopic imaging of neutralization outcomes. using serum samples from domestic dogs with neutralizing antibody titers estimated using the fluorescent antibody virus neutralization test (favn), pmrffit showed moderate sensitivity ( . %) and high specificity ( . %). despite small conflicts, titer predictions were correlated across tests repeated on different dates both for dog samples (r = . ), and for a second dataset of sera from wild common vampire bats (r = . , n = ), indicating repeatability. our test uses a starting volume of . µl of serum, estimates titers from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. the pmrffit enables high-throughput detection of rabies virus neutralizing antibodies in low- biocontainment settings and is suited to studies in wild or captive animals where large serum volumes the last few decades have seen a surge in newly emerging human viruses that originate from wildlife ( of gfp fluorescence. next, the command "analyze particles" was used to count the total number of fluorescent cells per field (i.e. infected cells). this command grouped and counted the white neighboring pixels with a predetermined size area and circularity to be a single cell (size area: - circularity: . - . ), so counts corresponded to the number of infected cells. cell count outputs were converted into a standardized spreadsheet using a python version . . script (python core team, ) (script available in supplementary materials). at the end of the image processing step, each serum sample was described by data points consisting of the number of the fluorescent cells in each of fields (photographs) in the : and : dilutions (figure b) . all statistical analyses were executed in r (r core team, (scaled to improve model convergence) and ) the serum dilution level (two factors: : and : dilution). random slope and intercept terms were also considered for the date the test was run ("test date") to account for observed variation in the relationships between srig titers and infected cell counts across dates (figure ) and for the field number ( to ) within each microscope well ("field") to account for variation in cell counts between fields (the middle field, field , had more agglomerated cells in particular). to evaluate whether a simpler, single dilution test produced comparable results, the full dataset was then subset to the : dilution only. the binomial and log-normal models fit to this data subset included only the fixed effect of the virus-infected n a cell counts, but the random effects were identical to those explained above (i.e. test date and field). models were fit using the 'lme ' package (bates et al., ) . the 'predict' function was used to generate the predicted probability that a srig concentration or serum sample was seropositive (binomial model) and its corresponding rvna titer (log-normal model). predictions per field were averaged to obtain results per sample (figure c) with a threshold of > . iu/ml (positives) were used as the benchmark reference. to understand the variability of the pmrffit, replicate srig titer concentration curves were produced on different dates between / / and / / (hereafter "test " through "test "). as expected, the number of infected cells declined at higher srig titers in all replicates; however, the shape of the antibody decay curve varied across test dates (figure ) . at the . iu/ml srig concentration, infected cell counts were more dispersed in the : dilution than in the : dilution, as indicated by higher interquartile range (iqr) within each of the test dates. across all the srig concentrations in all test dates (n = ), . % of the count comparisons were less dispersed in the : dilution suggesting this dilution could be more precise for downstream statistical analysis (si ). binomial glmms accurately predicted seropositive and seronegative srig concentrations (figure ) . the best random effects for the binomial model included a random slope and intercept for test date ( table ) . the models built with the : dilution data only ("one-dilution model") and from both the : and : dilution data ("two-dilution model") had equivalent specificity ( %), but the one- dilution model was more sensitive ( % versus . %, figure a, b) . furthermore, the two-dilution binomial model failed to correctly predict the seropositive controls on out of the test dates, confirming improved performance of the one-dilution model (figure b) . the log-normal glmms gave repeatable predictions of rvna titers from the datasets generated through our protocol across test dates (figure c) . the best log-normal model included a random intercept and slope for test date. although the most complex model had the lowest aicc, the simpler model (without the random intercept of field) had a Δaicc < ( table ) . observed and predicted srig titers were highly correlated for both the one and two-dilution models (r = . , figure c). when comparing test dates (i.e. one-to-one comparison between correlations of the one-dilution and the two-dilution model from the same test dates), the correlation coefficients were similar, suggesting the simpler one-dilution model is sufficient for titer prediction (figure d) . the most commonly applied serological tests to detect rvna titers challenge a range of serial dilutions of serum with infectious rv. this process is labor-intensive and requires laboratory capacity to grow large quantities of pathogenic rv. here, we provide an alternative serological framework that uses a combination of digital image analysis and statistical analysis to estimate the presence and titer of rvna from a single dilution using only . µl of serum. the pmrffit differs from other lyssavirus neutralization tests in several key aspects. it uses an mlv(rg) pseudotype rather than pathogenic rv, allowing the pmrffit to be performed in any low- containment laboratory with appropriate cell culture and microscopy facilities. the addition of gfp expression is significant, since it removes the need for fitc-conjugated antibody (reducing reagent costs) and the fixation and staining steps used in traditional rffit or favn. one potential drawback of using gfp expression to measure infectivity is the prolonged neutralization period ( h versus h rffit and h favn) required to gain sufficient fluorescence for image processing (aubert, ; smith et al., ) . longer neutralization requirements ( h) were also required in a favn modification using a gfp expressing recombinant cvs- -egfp but did not alter results relative to the test run with cvs- (xue et al., ) . fortunately, extended incubations are unlikely to alter neutralization outcomes since mlv(rg) pseudotype is replication incompetent, preventing infection of additional cells during the incubation (temperton et al., ) . the pmrffit also uses an imaging pipeline that combines systematic photography of microscope fields with automated digital image processing to count infected cells. microscopy in neutralization tests is time consuming and presents challenges for interlaboratory comparisons due to multiple sources of variation, especially those that affect the manual readout (e.g. laboratory user, manual pipetting, uneven cell monolayer) ( the pmrffit standardized approach minimizes these sources of error while potentially reducing microscope operator time. moreover, the imaging process generates traceable and permanent electronic records of the raw data, eliminating the need to manually digitize records of field counts. several investigators have previously incorporated image processing into rv neutralization tests. to count pixels using a microrffit but did not make full use of the quantitative nature of imaging data to obtain rvna titers and used pathogenic rv rather than a viral pseudotype. a final distinction is that instead of scoring microscope field or wells as virus positive or negative, the pmrffit predicts serological status and rvna titer from infected cell counts in a single serum dilution using statistical modelling. the efficacy of this approach highlights the value of historically underutilized quantitative data on cellular infectivity for lyssavirus serology. model selection indicated substantial day-to-day variation in the srig dilution series. this was unsurprising, since virus neutralization tests are biologically dynamic systems that can be influenced by many factors (e.g. variability in the humidity of the incubator, technical manipulation, light condition of the microscope, variability in gfp expression in the cells) (briggs et al., ; hammami et al., ; kostense et al., ) . since our statistical approach handles this variability through the random effect of test date, the pmrffit is best suited for large numbers of serum samples that require testing to be carried out across multiple batches. however, performance is only marginally reduced when running single models for each test date, implying the pmrffit may still be useful when fewer samples are available for testing (see si , ). surprisingly, fitting the glmms to data from a single : dilution of srig predicted both seropositivity and rvna titer more accurately than models fit to both the : and : dilutions. the reduced performance of the two-dilution model reflected higher variability in the : dilution compared to the : dilution, as evidenced by greater iqr values (si ). ultimately, this variability likely reflects both higher stochasticity in infected cell counts at lower serum concentrations and pipetting error. regardless, the ability to detect low titers (< . the authors declare no conflict of interest. perform the cell count of the fluorescent cells to construct a database to fit the statistical models. c) construction of the statistical models with two different types of prediction. srig data to fit models~ hrs fields/well antibodies to rabies virus in terrestrial wild mammals in native rainforest on the north coast of são paulo state, brazil practical significance of rabies antibodies in cats and dogs rabies virus exposure of brazilian free-ranging wildlife from municipalities without clinical cases in humans or in terrestrial wildlife mumin: multi-model inference measurement of rabies-specific antibodies in carnivores by an enzyme-linked immunosorbent assay fitting linear mixed- effects models using lme temporal and spatial limitations in global surveillance for bat filoviruses and henipaviruses the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination resolving the roles of immunity , pathogenesis and immigration for rabies persistence in vampire bats supporting online material contents : s seroprevalence data s estimation of seasonal birth rate s methods for parameter estimation use of a transient assay for studying the genetic determinants of fv restriction estimating time of infection using prior serological and individual information can greatly improve incidence estimation of human and wildlife infections a comparison of two 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detection of rabies antibodies in domestic carnivores an evaluation of two commercially available elisas and one in-house reference laboratory elisa for the determination of human anti-rabies virus antibodies human rabies: updates and call for data who expert consultation on rabies, third report. world health organization technical report series virus neutralising activity of african fruit bat (eidolon helvum) sera against emerging lyssaviruses a robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in africa investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross- species comparison generation of recombinant rabies virus cvs- expressing egfp applied to the rapid virus neutralization test a pneumonia outbreak associated with a new coronavirus of probable bat origin mixed effects models and extensions in ecology with r tables table . random key: cord- -mygj nd authors: nan title: proceedings of the nd annual meeting of the american rheumatism association a section of the arthritis foundation june & , new york city abstracts of papers presented date: - - journal: arthritis rheum doi: . /art. sha: doc_id: cord_uid: mygj nd nan azathioprine has been shown t o be an effective agent in the treatment of active rheumatoid arthritis (ra); however, potential long-term neoplastic effects remain a serious concern. to clarify this relationship, patients with classic or definite ra being treated with azathioprine (group ) were chosen at random and studied by means of clinical, laboratory, and radiologic measures to detect potential early markers of neoplastic disease. nineteen age matched patients with ra were concurrently similarly studied as controls (group ). group had a longer disease duration ( . versus . years). the mean duration of treatment with azathioprine was . months, with a mean dose of . mg/day. histories of cigarette smoking, hormone treatment, and fertility were comparable. chest x-rays revealed comparable numbers of inflammatory and fibrotic abnormalities. there was an increased frequency of tumors in first degree relatives in group i ( versus ). hematologic studies revealed a trend to lower cell counts in group with a striking increase of marrow megaloblastosis in this group ( versus i ) . there were no differences in iron saturation or b levels. serum folate was decreased in group i ( versus ). there were neither liver function test abnormalities in group , nor evidence of malignancy in gastrointestinal x-rays when clinically indicated. no neoplastic lesions were identified on mammography in either group. four patients in group and none in group had atypical or dyskaryotic cells on urine cytology. one patient in group had an abnormal pap smear and was subsequently shown to have endometrial carcinoma, on chromosomal analysis, group i showed a greater percent of cells with aberrations, a higher aberration incidence and index, and more patients with hyperdiploidy. patients in group had a history of benign neoplasms. patients in group i had a history of i benign neoplasm prior to the initiation of azathioprine; subsequently neoplasms were diagnosed, benign and malignant ( endometrial carcinomas and carcinoma-in-situ of the cervix). in summary, group i had an increased number of cellular abnormalities, with cytologic atypia, megaloblastic bone marrows, and increased chromosomal aberrations. clinically this group had more tumors diagnosed while on treatment, particularly in females, and arising from the genitourinary tract. more long-term studies of patients on cytotoxic agents are clearly warranted. solid phase clq (sclq) radioimmunoassays. c was determined by radial immunodiffusion and antibodies to dna by binding of radiolabeled dna by the millipore filter technique. these assays were correlated with each other, general presence or absence of symptomatic disease, active renal disease, and active joint disease. to evaluate predictive capabilities, a change in cic result was correlated with a change in disease activity. change in disease activity was defined as an sle related event requiring hospitalization, a change in symptoms requiring an increase in or addition of steroids or immunosuppressive agents, or a marked improvement in symptoms such that drugs were tapered or discontinued. the fclq results did not correlate with any parameters of disease activity. a fall in c or an increase in antibodies to dna usually signaled a change in disease activity; however, many patients experienced a change in disease activity without any change in c or dna binding. the sclq assay correlated with symptoms of sle in general (p < . ), with active renal disease (p < . ), and with active arthritis (p < . ). an appropriate change in sclq determination correlated best with a significant change in disease activity (p < . ). we conclude that the sclq method of determining cic correlates best with active clinical manifestations of sle and is a useful predictor of disease activity. we have recently found that certain rheumatoid factors (rfs) cross react with dna-protein (dnp) . this type of cross reacting r f is frequently present in seropositive rheumatoid arthritis (ra), of patients, and also in order r f positive patients: rheumatoid overlap syndrome / , mixed connective tissue disease (mctd) / , other diseases / , thus far rfs reactive with dnp have not been found in systemic lupus erythernatosus (sle) / or essential mixed cryoglobulinemia / . rfs which react with dnp can give positive antinuclear antibody (ana) and le tests which can be blocked by aggregated igg or dnp. in contrast, positive ana and le tests in sle are not blocked by aggregated igg. hence rf induced ana and le tests may be considered "false" positives when these tests are used in the diagnosis of sle. in some sera these rfs could be detected only following isolation with insoluble igg or dnp, suggesting the presence of rfs complexed with antigen. by use of a monoclonal r f which cross reacts with dnf in a sensitive competitive inhibition radioimmunoassay designed for detection of immune complexes (jci : , ) , complexes larger than s by ultracentrifugation studies could be detected in several ra sera and synovial fluid and mctd sera. in some studies dnase treatment of these complexes resulted in a decrease in size. these studies suggest that complexes consisting of dnp antigen and r f may be present in the circulation in ra and mctd. the relationship, if any, of these complexes to the immunopathology in these diseases is currently under study. these studies also suggest that not all rfs may be induced by immune aggregated igg and that a re-exploration of rfs in various diseases for cross reactions with non-igc antigens is indicated, such antigen may provide clues to etiologic factors in these diseases. previous studies demonstrated that patients with rheumatoid arthritis (ra) frequently have an antibody referred to as rheumatoid arthritis precipitin (rap). this antibody was shown by the indirect immunofluorescent technique to react with a nuclear protein in human, b lymphocytes from continuous cell culture (wil,) and the antigen was termed ra associated nuclear antigen (rana) . infectivity experiments with epstein-barr virus (ebv) or other herpes viruses have suggested that rana may be associated with ebv infection; although rana shares some characteristics with eb nuclear antigen, they are probably two different antigens. in this study we have expanded our work on rana. in addition to its presence in wil, cells, it has now been found in other human, b lvmphocyte lines but was not present in t lymphocyte lines. rana was not found in extracts from normal spleen, kidney, heart, or lungs, or a spleen extract from a patient with felty's ra. this antigen was not present in normal lymphocytes stimulated with b or t cell mitogens. rana was not found in peripheral leukocytes from patients with ra. however, it was found in of extracts made from pannus layers and an extract made from nodules taken from patients with seropositive ra but was not present in extracts made from pannus layers taken from seronegative ra patients and synovium from a traumatized joint. initial physicobiochemical characterization of rana demonstrated that it was stable to heat ( oc or oc) or cold ( o to - ooc) treatments or at ph values of ph . - . . studies performed by immunoelectrophoresis indicated that rana was an anion at ph . - . . will extracts were separated on a calibrated % agarose column and the molecular weight estimated at , d. in other studies the antigen could not be detected on the surface of wil, cells. immunodiffusion studies using burkitt's lymphoma sera ( ) or nasopharyngeal carcinoma sera ( ) containing high titers of ebv antibodies to: viral capsid antigen or early antigens d or r do not react with wil, extract, suggesting these ebv antigens are not present in the extract and therefore cannot be rana. the data suggest that rana may be a new, heat stable, acidic, nuclear protein associated with ebv infection which could be important in the pathogenesis of ra. after medical workup, arthroscopy was performed on patients ( : , men: women; ages - ) with painful knees of unknown cause. arthroscopy of the knee was achieved on ambulant patients under sterile technique in an outpatient minor surgery suite utilizing a needlescope or watanabe arthroscope. biopsies were guided by visualization. postarthroscopy diagnoses are reflected in the table. in most cases the procedure allowed adequate visualization of the suprapatellar sac, articular cartilage, menisci, anterior cruciate, and synovial lining. no arthroscopic abnormality was visualized in %. nearly half of the patients had osteoarthritis including patients with chondromalacia patella. the group with osteoarthritis also included patients who had at least one torn meniscus. septic arthritis was confirmed in % by synovial biopsy including one case of tuberculosis. previous undiagnosed synovitis was confirmed but not further defined in patients. four biopsies suggested rheumatoid synovitis; the diagnosis was confirmed by subsequent clinical and laboratory course. results of arthrography ( ) were confirmed in , contradicted in , and provided additional findings in i. patients were ambulant after the procedure with minimal morbidity- hemarthroses and an acute pseudogout, all treated by arthrocentesis. the gainful knee often presents a diagnostic enigma particularly when degree of disability is a question. ar-throscopy is a safe minor surgery procedure that may reveal the underlying cause of pain and preclude the need for arthrotomy. diagnosis computer assisted tomography of the spine and/or roentgenographic transaxial lumbar tomography were performed on patients (age iz sd years) with back pain and concomitant paget's disease. clinical assessment with routine lumbosacral x-rays was performed by a rheumatologist and an orthopedic surgeon. both tomographic methods are xray techniques that transect the spinal canal and the intervertebral foramina producing a computer printout or laminogram along the axial plane of the spine. the following was found: . spinal stenosis: spondylotic osteophytes encroaching on the spinal canal in patients. three had a history of pseudointermittent claudication. lumbar x-rays supported a diagnosis of paget's disease at the involved site in . . lateral recess syndrome: spondylotic osteophytes encroaching on the intervertebral foramina in patients. all had sclerotogenous pain (nonspecific pain referred to the buttocks, groin, and/or thighs). lumbar x-rays supported a diagnosis of paget's disease at the involved site in . . pagetic pain: increased thickness of the laminae, pedicles, or bodies of the vertebrae secondary to paget's disease in subjects without alteration of the spinal canal. . spondylotic changes of articular facets without stenosis or lateral recess in ; had adjacent pagetic changes. in the assessment of back pain, axial tomography proved to be a useful tool in defining anatomic spinal lesions that were not appreciated by routine x-ray techniques. the methods were particularly helpful in outlining the zygapophyseal articulation for definition of radiographic joint space and hypertrophic changes. neural entrapment by bone due to hy-pertrophic changes was similarly defined. in several cases the procedure obviated the need for myelography. in paget's disease, determining the degree of spondj osis could guide therapy since it would predict those that might be expected to respond to suppressive therapy of their paget's disease. hla-b is a potent genetic marker for ankylosing spondylitis (as), reiter's syndrome (rs), and other spondylitic variants. this cell surface antigen is determined by the hla-b locus, one of a linear array of genes clustered on the sixth chromosome. whether hla-b is the disease-conferring gene or only linked to another nearby gene remains unclear. studies of hla-d on one side of hla-b ( map unit) have shown no associations with as or b . the hla-c locus lines . map units to the opposite side of hla-b. it was our purpose to study c locus antigens in b positive and negative patients in order to more precisely localize the disease promoting gene. eighty-eight patients ( as, rs, sacroiliitis, psoriatic spondylitis, and colitic spondylitis) were hla cwl, p < . for cw , and p < . for either cwl and cw . similar significant differences were found when both the b + patients and the b + controls were compared with the matched controls. there were no significant differences noted between b + patients and b +controls or between b patients and matched controls. the occurrence of cw and cw was similar within all groups. thus, these data confirm strong linkage disequilibrium between b , cwl, and cw which does not relate to disease. most importantly, the absence of cwl and cw in b patients provides further evidence that recombination between b and c loci is unlikely, and that disease susceptibility is intimately related to or inseparable from the b locus. control groups (n= ), % (n= ), % (n= ), % (n= ),% typed for a, b, and the c locus antigens, cwl, cw , cw , and cw . in addition, age, sex, and race matched normal controls and b positive normal controls were similarly studied and compared to those with disease. hla-b was present results of c locus typing are shown in the table. an immunofluorescent complement fixation (ifcf) test was used to determine if nuclear antigen-antibody complexes of different specificities possessed different capacities to fix complement. sera from patients with rheumatic diseases were specially selected and demonstrated to contain only antibodies to sm antigen ( patients), nuclear rnp ( ), ss-b antigen ( ), or nuclear histones ( ). column chromatography isolated-igg from these sera were reacted with sections of mouse liver to form nuclear ag-ab complexes of the different specificities described above. binding of complement components to the nuclear ag-ab complexes was determined after washing away excess serum and incubating with different sources of complement: ) normal human serum (nhs); ) nhs-egta mg, conditions which are known to block activation of the classic pathway; ) immunologically clq-depleted sera in which other complement proteins were demonstrated to be % hemolytically active. fixation of complement was determined by using fluorescein-conjugated antiserum to clq, c , properdin, factor b, c , c , and c . all sera with antibodies to sm antigen, nuclear rnp, and ss-b nuclear antigens showed fixation of all complement proteins when nhs was used as the source of complement. when the source of complement was egta-mg or clq-depleted serum, clq and c were negative by ifcf. however, complement proteins properdin, factor b, c , c , and c were positive, showing that these nuclear antigen-antibody complexes were able to fix complement proteins of the alternative pathway, without concomitant fixation of the classic pathway. the sera with antibodies to nuclear histones did not fix any complement component of either the classic or alternative pathways. these studies demonstrate two important findings. bodies of certain specificities to activate complement may have certain nuclear ag-ab complexes are capable of activating the relationship to their capacities to cause tissue damage by realternative complement pathway, independent of classic path-lease of inflammatory peptides mediated by complement actiway activation. reactions between nuclear histones and anti-vation. our results may explain why drug-induced lupus erbody, as demonstrated by the immunofluorescent technique, ythematosus, characterized by anti-histone ab, is rarely appear not to be complement-fixing reactions or at least not associated with inflammatory kidney disease. detectable by this technique. the capacity of antinuclear anti- it has been suggested that renal involvement in sle patients may depend upon qualitative immunochemical characteristics of antibodies to dna, e.g., precipitating ability, affinity, ig class or subclass, and complement fixing (cf) capacity. we used the immunofluorescent crithidia luciliae method [a-dna(cl)] to delineate some of these characteristics in sera from patients with active sle. using fitclabeled polyspecific antiserum to igg, iga, and igm, a-dna(cl) was found in of patients with active lupus nephritis (gp-i) and of patients with lupus activity without nephritis . median titer in gp-i was : and in gp-i : ; the difference in titers between these groups was significant (p < . ). c f antibodies to dna were found in patients in g p i and patients in gp-i using a double sandwich technique to detect c binding. when titer of c f activity was compared with a-dna(cl) titer, a strong correlation was found (r = . ; p < . ). igg, iga, and igm a-dna(cl) were detected with high frequency in both groups. in addition, we compared titers of a-dna(cl), which detects antibodies primarily directed against dsdna determi-nants, with dna binding capacity detected by the millipore filter (mf) method of ginsberg and keiser, in each of these groups. as antigen we employed * -labeled calf thymus dna (type i, sigma) passed through an . p, type ha millipore filter. in contrast to the cl method, levels of a-dna(mf) did not differ significantly in gps i and . when we compared a-dna(cl) titer in gp-i sera with quantitative a-dna(mf), a highly significant correlation was found (r = . , p < . ), while no correlation between these methods was evident in sera from gp-i patients. these findings suggest that: ) the presence of high titered a-dna(cl) is strongly correlated with active renal lupus; ) detection of c-fixing a-dna(cl) may reflect total amount of antibody; demonstration of c-fixing ability may generally be expected in high titered sera; ) antibodies to at least sets of constituents of the dna preparation employed in the mf method can be detected, one occurring in active lupus nephritis and correlated with antibodies detected by cl, and one in patients with active lupus without nephritis, detected minimally or not at all by the cl method. cell mediated cytotoxicity is an important pathogenic mechanism in certain rheumatic diseases. recent studies from this laboratory have demonstrated that adherent monocytes are potent killers of antibody coated (adcc) nonerythroid target cells and also kill uncoated target cells to a lesser extent. although immune complexes can inhibit lymphocyte cytotoxicity, their effect on monocyte cytotoxicity is unknown. this study compares human monocytes adcc and natural killer (nk) activities with that of lymphocytes. the effects of heat aggregated igg ( . - mg per ml) and preformed soluble keyhole limpet hemocyanin (klh)/anti-klh complexes were evaluated. monocytes and lymphocytes were separated by their differential adherence properties in plastic microtiter wells and were at least % pure. cytotoxicity was measured by "chromium released from antibody coated (adcc) and nonsensitized (nk) k cells, an established myeloid cell line. precipitated klh/anti-klh complexes formed at equivalence were the strongest inhibitors of cell mediated cytotoxicity (suppression: monocyte nk / , monocyte adcc %, lymphocyte nk o%, lymphocyte adcc %). aggregated igg also strongly inhibited both monocyte and lymphocyte nk ( % and % respectively) and also suppressed adcc to a lesser extent (monocyte %, lymphocyte %). klh/anti-klh in antigen excess had no effect on monocyte nk and suppressed lymphocyte nk by only %. although soluble complexes did not block monocyte nk, they did significantly block monocyte adcc ( %). by use of serial dilutions of complexes in aggregated igg, a dose dependent relationship was established between complexes and percent suppression of cytotoxicity. these studies reveal that both klh/anti-klh immune complexes and aggregated igg can inhibit monocyte cytotoxic activity and that, in general, these effects correlated with lymphocyte nk and adcc. significant differences between soluble immune complexes and aggregated igg were demonstrated. finally, the finding that certain immune complexes inhibit both nk and adcc would imply that fc receptors are required for both cytotoxic responses. the relatively greater inhibitory effect on nk would imply that antibody fixed to target cells potentiates attachment by the effector cell and renders it less susceptible to inhibition by immune complexes. gout is easily diagnosed from a classic clinical history and the demonstration of urate crystals in synovial fluid and/ or tophi; however the diagnosis may not be suspected in patients with an atypical history or physical examination. to ascertain the prevalence of atypical findings we are conducting a prospective study in our gouty population randomly admitted to the clinical research unit. we report the following findings in the first patients with crystal proved gout: ) % had saturnine gout " to moonshine consumption, % gave a history of heavy ethanol consumption but did not have lead intoxication, and % were obese. all patients were male, % black, % white and % american indian. the average age at the time of study was . years with a mean duration of gout of years. seventythree percent had evidence of liver disease (elevated sgot or hepatomegaly), % were hypertensive, % had elevated triglycerides, and % had abnormal glucose tolerance. ) twenty-seven percent had a polyarticular onset of gout (> joint); % subsequently had polyarticular attacks. seventythree percent had tophi at the time of the study. chronic synovitis was present in % of patients. in the patients examined the following joints of the upper extremity were involved; % m p and/or ip, % wrist, ' elbow, % shoulder. in the lower extremity the knee was involved in %, m p joints in %, and ankles in %. ) a positive latex fixation test was observed in % of patients; being > : and > : . ra latex was present in % of patients with tophi and % of patients without tophaceous deposits. in the patients with chronic synovitis % had a +ra latex, and % free of chronic synovitis were also positive. the mean age in patients with a positive test was . years and . years for those who were negative. in contrast to these negative associations, evidence of liver disease was correlated with the finding of a +ra latex. fifty percent of patients with liver disease had a +ra latex whereas it was absent in all without liver disease. we conclude that chronic synovitis and polyarticular arthritis are common in our gouty patients. in addition, rheumatoid factor is found in a significant proportion of gouty patients and it does not seem to correlate with age, tophi, or chronic synovitis. ra latex seems to correlate best with the presence of liver disease which in most instances was a concomitant of chronic ethanol abuse. a polysaccharide iden tical to a proprionibacterium acnes polysaccharide (pps) has been isolated from hyaluronidase treated rheumatoid synovial fluids and synovial leukocyte pellets using a phenol-water (westphal) extraction and sepharose b column chromatography. pps was identified by counterimmunoelectrophoresis (cie) by rabbit antibody to sonicated p acnes organisms. sensitivity by this method is approximately . ng of pps and identity of synovial and bacterial pps was shown by cie. of common aerobic and anaerobic bacteria tested, only ps aeruginosa contained a similar antigen. pps is not present in e coli or s typhosa lipopolysaccharide. pps could not be identified in synovial fluid without extraction procedures even when a more sensitive radioimmunoassay with lzsi labeled rabbit antibody was used. sixty percent of rheumatoid fluids and % of rheuma-toid synovial leukocyte pellets contained pps. only of ( %) nonrheumatoid fluids was positive. two of ( %) nonrheumatoid inflammatory synovial leukocyte pellets contained pps. antibody to pps by cie was found in % of rheumatoid sera ( p < . ), % of nonrheumatoid inflammatory arthritic sera ( p < . ), and % of normal control sera. characteristics of pps (bacterial and synovial) include sensitivity to mm periodate oxidation, resistance to rnase, dnase, pronase, and boiling. uronic acid and hexosamine account for approximately % by weight. less than . % protein is present. it appears polydisperse on sephadex g with a m w of approximately x ' to >lp. electrophoresis in . % polyacrylamide showed a single pas positive band which formed a preciptin line with anti-p acnes antibody by crossed cie. no lipid staining bands were noted. these findings are consistent with a polysaccharide antigen. the isolation of a bound antigenic bacterial polysaccharide primarily from rheumatoid effusions associated with diminished antibody suggests a pathogenetic role for pps in rheumatoid arthritis possibly as an immune complex type. an in vitro system for examining the cellular requirements necessary for initiating and regulating antinucleic acid antibody responses has been developed to facilitate a better understanding of the mechanisms responsible for triggering antibody responses to this group of antigens in murine and human sle. it has been found that spontaneously appearing anti-ssdna hemolytic plaque forming cells (pfc) can be detected when spleen cells from normal mice of different hz types (cba, dba, bdf , cfw, c /b ) or young ( weeks) autoimmune (nzb x nzw f (b/w), nzb, nzw) strains of mice are cultured under standard mishell dutton conditions in the absence of added antigen. such pfc( - /culture) which peak on day and cannot be detected prior to culture consist entirely of specific igm anti-ssdna antibody. the response, which appears to be dependent on active antibody synthesis, can be selectively abolished by specific removal of ssdna antigen binding cells, by rosette depletion. while low responses can be augmented by pokeweed mitogen, there is no obligatory requirement of serum, thymic (t) cells, or macrophages in the culture system. this in vitro response can blocked by: ) adding small quantities of ssdna (lo- opg) but not similar concentrations of dna or rna directly to culture on day ; ) pre-incubation of spleen cells with ssdna ( hour) and washing prior to culture. unblocking can be demonstrated by subsequently treating these cells with trypsin-dnase, suggesting that this inhibition may be due to a membrane receptor blockade mechanism; ) the addition to young b/w spleen cells of t cells from young ( weeks) or old (> months) syngeneic mice similarly abrogates this in vitro anti-ssdna response without effecting cell viability or the anti-srbc antibody response. on the other hand syngeneic bone marrow cells which markedly suppress the anti-srbc antibody response have no effect on the anti-ssdna response of these spleen cells. these in vitro studies suggest mechanisms that may be relevant to the normal regulation of anti-nucleic acid antibody and other autoantibody responses in vivo. the ability of old b/w t cells to exhibit inhibitory effects similar to those of young preautoimmune b/w mice on the in vitro antinucleic acid antibody response suggests that the suppressor potential of these old b/w t cells may be blocked in vivo. the ehlers-danlos syndrome (ed-s) is an uncommon, genetically determined disorder of connective tissue. of the seven clinically distinct types, a basic molecular defect has been reported in types iv, vi, and vii, all of which are inherited by a recessive mode, and appear to be related to deficiencies in the activity of specific enzymes involved in postribosomal changes. types i to i are inherited by a dominant mode and might be expected to have defects at the transcriptional level. type v is sex-linked and has been reported to result from a cross-link deficiency. we have studied patients with ed-s type i, patients with type , and with the x-linked type v. the results show that the reducible crosslinks are present and undergo the same maturation process to nonreducible cross-links as in normal skin. we were unable to confirm the absence of reducible cross-links in the x-linked type ed-s. the ability of the ed-s skin to produce apparently normal cross-linked collagen was supported by cell and tissue culture studies. transmission electron micr~scopy and optical birefringence revealed a normal ultrastructure of the collagen fibrils. at a higher morphological level of organization scanning electron microscopy demonstrated a gradual increase in fiber bundle disorder from the x-linked to the myasthenia gravis, where the fibers making up the large fiber bundles demonstrated a considerable inability to aggregate. this disorder could account for the prolonged lag phase in the stressstrain curves, while the presence of cross-links ensures that once stress is on the individual fibers the elasting modulus is normal and the skin can return to normal after hyperextension. the absence of cross-links would lead to an irreversible elongation of the fiber. the low breaking point of the fiber is therefore due to the lack of integrity of the fiber bundles and suggests that the defect lies at a higher order of organization. the cryoprecipitates (cryos) from many sle sera contain lymphocytotoxic antibody and, when used to immunize rabbits, induce antibodies to human lymphocytes, suggesting that they contain membrane fragments complexed to antibody. lymphocyte-reactive antibodies in sle sera also recognized antigens on neuronal cells. to determine whether the membrane fragments in the cryos have the antigenic determinants shared with neurons, antisera generated against sle cryos were tested in an antibody-dependent, cell-mediated cytotoxicity assay by use of cr-labeled human neuronal cells, sk-n-sh, as targets and normal human peripheral blood lymphocytes (pbl) as effectors. the anti-cryos produced . f . mean % "cr-release. two of the antisera were strongly positive at and % cytotoxicity, and others were modestly cytotoxic in the range of . to . %. the remaining antisera were not significantly different from the controls; . f . % ' -release from normal rabbit sera (nrs), and . f . % cytotoxicity from a control group consisting of non-sle anti-cryos. the antibody activity was not linked to sk-n-s h cells. the same sera reacted with human neuronal line, la-n- , and human glial lines, a-i and u-l mg. unexpectedly there was no correlation between reactivity against the neuronal cells and antibody activity against normal human pbl. antibody t o pbl was not detected in / anti-cryos with neuronal antibody, and of the antisera unreactive with sk-n-sh had anti-pbl activity. absorption of the anti-cryo reactive against both sk-n-sh and pbl with the human lymphoblast line wll removed all anti-pbl activity but not the neuronal reactivity. absorption with sk-n-sh eliminated the neuronal reactivity and also diminished the antibody to pbl. the antineuronal activity was not removed by absorptions with human platelets and human rbc. passage of the anticryos over immunoglobulin containing immunoabsorbents removed the detectable anti-immunoglobulin activity without diminishing the neuronal reactivity. thus, sle cryoproteins contain antigenic determinants shared by neuronal and glial cells but not by the major cellular elements in blood. it is not yet clear whether those antigens are present on fragments of neuronal membranes or on antigenic structures eliciting cross reactive antibodies. it is generally accepted that antibodies to double stranded (ds) dna are a feature of active sle, while antibodies to single stranded (ss) dna occur in many rheumatic diseases. the natural occurrence of antibodies to dsdna presumes an interaction with the sugar-phosphate backbone of dna, the immunogenicity of which is unproved. we have attempted t o define the preferential reaction site of dsdna antibodies on a molecular species of dsdna containing the multiple ss regions, herewith designated ds/ssdna. calf thymus dna was sheared to a molecular weight of x iw daltons. characterization by hydroxyapatite and benzyolated naphthoylated deae cellulose chromatography (j immunol : , ) indicated the dna was predominantly ds with numerous ss regions. antibodies to chromatographically pure dsdna (> % binding in a standard farr assay) were partially purified by ammonium sulfate precipitation, iodinated with lz i, and reacted with ds/ssdna. the resulting complexes were precipitated with . % polyethylene glycol (mw, ) and redissolved in . m tris-hci, ph . , . m mgci , . m nacl (the endonuclease buffer). the solubilized complexes were reacted with ss specific endonuclease from neurospora crassa ( . pg enzyme/ pg dna) and compared t o undigested complexes by velocity sedimentation in a % to % sucrose gradient run at ,ooog for i hours. undigested complexes sedimented at a position of s; after endonuclease digestion there was a dramatic decrease in the sedimentation velocity to a s position. these findings could be explained by: ) a disintegration of the dna into many smaller ds molecules still attached to labeled antibodies, ) a liberation of antibodies formerly attached to ss regions of the ds/ssdna, ) a displacement, by the endonuclease, of antibodies reactive with ss regions. to test these possibilities the procedure was repeated without labeling of the immunoglobulins, the resulting complexes were reacted with lz l dsdna in a standard farr assay. after endonuclease digestion free dna antibodies were readily detected, as shown by a -fold increase in dna binding, % to %. these findings indicate that dsdna antibodies preferentially react with ss portions of dsdna molecules. whether dsdna antibodies ever react with determinants unique to dsdna, or are merely a manifestation of high avidity binding to a very short ss region of the dsdna molecule, remains to be determined. t cell cytotoxic function in nzb mice has previously been tested utilizing target cells differing from nzb at the major histocompatibility complex (mhc) of mice, h- . in the experiments reported here, t cell cytotoxic functions of nzb mice against targets identical to nzb at the mhc were investigated. normal mice do not generate cytotoxicity under these circumstances. spleen cell suspensions of nzb mice (h- d) were cultivated for days in the presence of irradiated stimulator cells of other h- d strains (b .d , balb/c, dba/ , hw ). on the fifth day labeled target cells identical to the stimulating cells were added and the 'cr release from these targets was measured after hours. nzb effector cells exerted a highly significant specific lysis in all four of the h- d strains tested; none of these reacted against nzb. in experiments in which nzb cells were sensitized against one h- d strain (balb/c) target cells from all four h- d strains were lysed, demonstrating crossreactivity of the lytic reaction. the amount of lysis observed was dependent on the ratio of effector to target cells. the action of the effector cells was abolished by treatment with anti-thy- serum, indicating their t cell character. the capacity to respond against balb/c could be demonstrated in nzb mice as early as weeks and as late as months of age. the capacity of nzb mice to generate cytotoxic t-cells following primary in vitro sensitization against targets carrying the same major transplantation antigens as nzb establishes a qualitative difference between nzb mice and all normal strains since generation of cytotoxicity is restricted under the applied conditions to targets differing at the h- in all normal strains heretofore tested. it is suggested that the observed lack of restrictioq of the cytotoxic reaction in nzb mice is related to the autoimmune reactivity of this strain. immune deposits in the choroid plexus of patients with neuropsychiatric manifestations of systemic lupus erythematosus (sle) and nzb/nzw f, hybrid mice are thought to relate to the pathogenesis of central nervous system (cns) lupus. since previous studies did not include in their control groups patients with sle who had no neuropsychiatric manifestations, we looked for immune deposits in the choroid tissue of patients with and without cns lupus. fixed brain tissue containing choroid plexus was retrieved from sle patients and cont.rols. ependymal lining cells, choroid stroma, and cortical vessels were reviewed for deposits of igg, igm, iga, igd, ige, and kappa and lambda light chains by an immunoperoxidase technique (rabbit antihuman immunoglobulin and peroxidase-antiperoxidase). complement components were destroyed by fixation. immunoglobulins and light chains were found in the ependymal cells and/or choroid stroma of each sle patient. the pattern or intensity of immunoglobulin deposition did not distinguish those patients with and without neuropsychiatric manifestations. no staining was seen in tissue from the controls. though the choroid plexus serves a filtering function, the finding of immunoglobulin deposits in the choroid plexus cannot be correlated specifically with any of the diverse manifestations of cns lupus. other clinicopathologic correlations must be sought. multinucleate cells (heterokaryons) are frequently found in rheumatoid synovium as well as in cultures of isolated, adherent cells from this tissue. using an experimental polyethylene glycol (peg) was used to fuse cells in monolayer cultures of rabbit synovial fibroblasts. fusion began within minutes of peg treatment and continued for about hours; approximately % of the cells developed or more nuclei. in some experiments, up to % of fused cells contained or more nuclei, indicating true giant cell formation. measurement of intracelluar ( rubidium) to indicate the presence of a leaky cell membrane showed that immediately after peg treatment, intracellular ( rb+) dropped to % of control levels. it began to approach normal by minutes but did not recover fully for about hours, at completion of fusion. during the first hours after peg treatment, the treated cultures incorporated / as much ( h) thymidine as did control cultures, but incorporation of ( h) leucine into tca-precipitable radioactivity was unaffected. autoradiographic studies using ( h) thymidine revealed that peg depressed incorporation of the label into dna for at least days. secretion of proteinases from peg-treated and control cultures in serum-free dulbecco's modified eagle's medium was compared. peg-treated cultures containing multinucleate cells secreted u (cumulative) latent collagenase into medium changed every hours over days, compared with u produced by control cells during the same period ( u collagenase degraded kg collagen/hour at °c. collagenase was activated from its latent precursor form by tpck trypsin-i wg/ml, minutes, oc, followed by addition of excess soybean trypsin inhibitor.) neither peg-treated nor control cells released substantial or significantly different amounts of neutral or acid proteinases into medium during the same period. our data show that compared to controls, cultures of multinucleated cells have a decreased rate of cell replication and increased rate of collagenase production. multinucleate cells in rheumatoid synovium may amplify mechanisms for active collagenolysis. fourteen previously untreated polymyositis (pm) patients were treated with prednisone mg/day until cpk values normalized and then with mg/day for a total of weeks. they were also randomly placed on either azathioprine (a) mg/kg/day or placebo (p) in double-blinded manner. manual muscle testing, total prednisone dose required over weeks, and muscle biopsy changes were used to detect differences in the two groups. the a group ( patients) turned out to be weaker at onset (total manual muscle testing score - ) than the p group ( patients with score of - ) but improved more ( points to - ) than the p group ( points to - ). however, the difference in improvement was not statistically significant. cpks in the two groups normalized a t about the same rate and each group therefore received comparable doses of prednisone over the weeks. all followup muscle biopsies at the end of weeks showed improvement in the majority of the eight microscopic features reviewed and in total numerical score. the trend was toward greater improvement in the a group but not significantly so. changes consistent with steroid myopathy were noted in of women but only of men ( in a and in p) and of these patients were weaker at weeks. of special note is the observation that of patients with normal cpk at i weeks, still had significant inflammatory infiltrates. therefore, azathioprine plus prednisone is not dramatically better than prednisone alone in polymyositis over the initial weeks of treatment. the development of steroid myopathy in over one half the patients is not influenced by a or p and at the prednisone dosages used complicates the evaluation of changes in muscle strength; nevertheless, the trend is toward greater improvement in the azathioprine group in muscle strength and also follow-up muscle biopsy scores. surprisingly, a normal cpk does not indicate resolution of inflammation in the muscle and cannot be used reliably to indicate full control of the disease. followup is continuing. with the appreciation of the relationship between immunogenetics and infection in the pathogenesis of rs, interest in this condition is increasing. in order to determine more precisely the natural history of rs, data are presented from two distinct california communities: university (pa) and community (sb). to date, consecutive patients have been studied: (pa) and (sb). rs is defined as an asymmetric oligoarthropathy (predominantly lower extremity) accom-panied by one or more of the following symptoms: ) urethritis, ) diarrhea at onset, ) inflammatory eye disease, ) mucocutaneous manifestations consisting of balanitis, buccal ulceration, or keratodermia blennorrhagica. patients with ankylosing spondylitis, psoriatic arthropathy, or other rheumatic syndromes were excluded. nineteen ( %) of the presented with diarrhea and ( %) had at least a triad of the above features. there were more similarities than differences between sb and pa. for example, females represented % (sb) and % (pa). the mean duration of disease to date is . months (sb) and . months (pa), disease activity continuing (albeit, sometimes, episodically) in % of the sb practice and % of the pa patients. hla-b was present in % (sb) and % (pa). only % had a persistent monoarthropathy. twenty-four percent of patients had a positive family history for an inflammatory polyarthropathy. other similarities included keratodermia blennorrhagica in % (sb) and % (pa), tendinitis in % (sb) and % (pa) and heel disease in % (sb) and % (pa). sacroiliitis occurred in %, % of whom had asymmetric change. only patient with sacroiliitis was hla-b negative. the esr ( - , mean ) appeared unrelated to disease activity. aortitis was present in only (pa) patients. differences between pa and sb patients were minimal; balanitis and uveitis appearing more frequently in the former group. buccal ulceration was seen in % of the pa patients and % of the sb group. from this study we conclude: ) rs is a major chronic rheumatic disease; at least two-thirds of patients have active disease at an month followup. the prognosis for occupation requiring significant exertion should remain guarded. ) there are minimal differences between an academic and community population of rs patients. ) there were no discernible differences between disease severity in hla-b positive and negative patients. the activities of the major fragment of the third component of complement, c b, are controlled by at least two proteins, c b inactivator and b h globulin. using in vitro model systems, we have previously demonstrated that h physically binds to c b and blocks its activity. in the present report immunofluorescent examination of renal biopsies has been used to demonstrate that similar c b-blh interaction occurs in vivo. twelve biopsies from patients with systemic lupus erythematosus or acute poststreptococcal glomerulonephritis were examined. igg and complement components were found by immunofluorescence to be deposited in a granular fashion in the glomeruli of these specimens. by electron microscopy, mesangial, subendothelial, intramembranous and/or subepithelial electron dense deposits were found. a single biospy in which only c was found to be deposited, consistent with activation via the alternative pathway, was also studied, as was one patient with goodpasture's syndrome, whose biopsy contained linear deposits of proteins along the glomerular basement membrane. six specimens from patients with such nonimmune renal diseases as arteriolar nephrosclerosis and acute tubular necrosis were also examined. by use of direct immunofluorescence with anti-blh specifically shown to be free of any contaminating anti-c , deposits of b h were found in every instance ( of exams) in which c deposits were observed. this was true regardless of the underlying disease which resulted in the c deposits. the spatial distribution of p l h within the glomerulus was often congruent with the distribution of c in the same tissue, suggesting that these two proteins were intimately associated. n o deposition of b h was observed in those with nonimmune renal disease. we conclude that binding of / h to fragments of c , presumably c b, occurs in vivo during immunologic activation of the complement system, just as we had previously demonstrated it to occur in vitro. hahn et al. (ann int med , ) have shown a relationship between anti-hdz and anti-dna in patients with hdz-sle, and yamauchi et al. (j clin invest : , ) have shown immunologic cross reactivity between dna and hdz with rabbit antisera to hdz-albumin conjugates. to find if the development of antibodies to dna or dnp in patients taking hdz represents the production of anti-hdz which cross reacts with dna/dnp determinants, we followed prospectively hypertensive patients taking this drug (average dose mg/day) for one year. antibodies to hdz were measured by passive hemagglutination, to native dna by in the prospective group the major finding was development of increased levels of anti-dnp in patients, of whom also made anti-hdz. nine other patients made anti-hdz without anti-dnp. none made anti-ndna. one developed a mild hdz-sle syndrome with anti-hdz and anti-dnp. examination of sera of other patients with hdz-sle syndrome by radioimmunoassay failed to show an immunologic cross reaction between hdz and dnp although such a reaction was found in guinea pig anti-hdz-bsa serum. the results show a relationship between production of antibody to hdz and to dnp in patients taking hdz but do not support the hypothesis that anti-dnp in such patients is antibody cross reacting with hdz. they are consistent with the findings of fritzler and tan (clin res :a , ) that anti-dnp in drug-related sle reacts with the histone moiety. the relationship of the immune response to hdz to that to dnp remains unexplained. anti-idiotypic antibodies recognize antigenic determinants (idiotypes) uniquely associated with a given antibody molecule, or closely related molecules. the in vivo interaction between idiotypes and anti-idiotypic antibodies has been postulated to play a central role in the regulation of the immune system. the purpose of this study was to characterize the idiotypic antigens on three monoclonal igm anti-y-globulins and to determine if cross-reacting idiotypes were present in the sera of patients with rheumatoid arthritis (ra). anti-idiotypic antibodies against the purified igm ( k ) anti-y-globulins lay and si, and the igm (a) anti-y-globulin koh, were raised in rabbits. when analyzed by solid phase radioimmunoassay (ria), each antibody reacted only with the immunizing antigen, and not with pooled igg or other igm paraproteins lacking anti-y-globulin activity. studies with recombinant molecules reconstructed from lay or si light or heavy chains and light or heavy chains from heterologous proteins demonstrated that the idiotypic antigens were formed from a specific heavy-light chain interaction. the idiotypes were closely related to the antibody combining site, as judged by the inhibition of idiotype-anti-idiotype binding by antigen, i.e., igg, and of idiotype-antigen binding by anti-idiotype. idiotypes cross-reacting with the monoclonal igm ( k ) anti-yglobulin si, but not with the igm (a) anti-y-globulin koh, were found in increased concentration in the sera of patients with seropositive ra. the idiotype positive material, as analyzed by gel filtration, was found in the s but not the s fraction of serum. from these experiments we conclude: ) that the idiotypes on igm anti-y-globulins are associated with the antibody binding site and are the product of a specific light-heavy chain combination; ) igm rheumatoid factors in ra patients, although polyclonal, may have idiotypic antigens cross-reacting with those on monoclonal igm ( k ) anti-y-globulins; ) igg rheumatoid factors probably have different idiotypic antigens than igm rheumatoid factors and may therefore have different active sites and antibody specificities; ) anti-idiotypic antibodies may be useful for the selective suppression of anti-yglobulin production without a general depression of immune responsiveness. connective tissue activating peptides from lymphocytes (ctap-i) and platelets (ctap- ) are known to stimulate glycosaminoglycan synthesis, glycolysis, and mitogenesis in connective tissue cell cultures. direct evidence suggested that increased accumulation of cyclic amp was involved in the mechanism of action of these peptide agonists, and increased prostaglandin e synthesis was postulated on the basis of indirect evidence. in the present experiments, ctap-i and - were incubated with human and murine cells in culture, and prostaglandin e was measured by radioimmunoassay using antibody directed primarily to prostaglandin e,. both ctap-i and - markedly stimulated the elabo-ration of prostaglandin e, into culture medium, the earliest evidence of increased synthesis occurring at hours with maximal concentration found at hours. in a typical experiment, after hours incubation, buffer, ctap-i and ctap-i treated cells produced, respectively, , , and pg of prostaglandin e,/ . x cp normal human synovial cells. substantial residual stimulation persisted at least through hours. the lymphocyte factor (ctap-i) appeared to be more potent than ctap-i in stimulating prostaglandin synthesis. indomethacin these studies substantiate the postulated increase in synthesis of e series prostaglandins by human connective tissue cells on exposure of ctap-i and - , and clarify the mechanism of action of these agonists on "activated" target cells. the importance of elevated extracellular concentrations of prostaglandins is uncertain, although they may potentiate the actions of ctap-i and - . in order to examine the repair collagens produced by cells present in injured cartilage, the femoral articular surfaces of three groups of new zealand white rabbits were scarred in the following manner: superficial and deep lacerations, and drilled holes. eight weeks after surgery the rabbits were sacrificed and slices of injured articular cartilage harvested. the types of collagen produced at the site of these lesions were identified by labeling the recovered specimens with *h proline and characterized by sds gel electrophoresis, cmc chromatography, and cnbr peptide analysis. in all cases, tissue-specific type i ([ai( ) ) cartilage collagen was synthesized. histologic examination revealed the chondrocytes bordering the cartilage injured by deep laceration and drill holes responded by increased cellular activity. grossly, the drilled holes were completely filled with tissue possessing staining and morphologic characteristics similar to that of hyaline cartilage. these data strongly suggest that in the lacerative type of injury, the surrounding tissue will produce enough matrix for repair only when the subchondral bone is violated. both repaired and normal cartilage produce tissue-specific type i collagen. we recently demonstrated that, from birth on, nzb splenic b cells spontaneously secrete to times as much pentameric igm as normal b cells (j immunol : (j immunol : , . spleen cells from to week old mice have been examined in further study of this abnormality. nzb spleen suspensions contain to times as many spontaneous plaques to sheep erythrocytes and to tnp conjugated sheep erythrocytes as normal mice. there is a similar increase in the number of cells secreting igm as detected by a reverse plaque assay. after cold ethanol-acetic acid fixation, it can be shown that nzb spleen contains - cells with cytoplasmic igm compared to . - . % in normals. nzb spleen cells were sorted by flow microfluorometry (fmf) into surface ig negative cells and pools with increasing levels of surface igm or total ig. the surface ig negative fraction produced no igm but the positive pools each had the same level of igm production and cytoplasmic staining. when sorted into pools on the basis of size, however, all the igm production was found in the largest fraction. nzb spleen suspensions contain more large cells than normal spleens by coulter volume measurements and fmf reveals more large surface ig positive cells in nzb than normal spleens. when surface ig was enzymatically removed before fixation, it was found that the nzb cells with cytoplasmic ig were to s times brighter than those from normal mice. thus nzb spleen contains approximately times as many cells which synthesize and secrete igm as normal mice. each such nzb cell appears to contain and presumably secrete about times as much igm as do cytoplasmic ig positive cells in normal mouse spleen. because nzb mice spontaneously produce antibody to thymocytes, we utilized fmf at high gain to search for immunoglobulin on peripheral t cells. none could be demonstrated. after fixation, however, immunoglobulin was readily detected in nzb splenic t cells. whether this immunoglobulin is passively acquired and rapidly pinocytosed and thus not available for staining on the cell surface or endogenously synthesized by the t cell is under investigation. in an effort to define the cellular basis of abnormalities of polyclonal b cell activation previously noted in nzb mice, the surface immunoglobulin (sig) isotypes of spleen cells from young and old nzb mice were examined. after lactoperoxidase-catalyzed radioiodination, cells were lysed, the immunoglobulins bound to rabbit anti-mouse immunoglobulin and the immune complexes absorbed to s aureus. the complexes were solubilized and the cell surface immunoglobulins analyzed by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. cell surface immunoglobulin isotypes were also analyzed by staining spleen cells with fluorescein-conjugated rabbit anti-p, anti- , and anti-ig sera and examining stained cells by fluorescence microscopy or by automated cytofluorometry on a becton-dickinson facs- cell sorter. spleen cells from both young ( weeks old) and old (one year old) nzb mice were found to have markedly increased ratios of cell surface igm/igd compared to cells from balb/c control mice. the altered ratio of sig isotypes was not a consequence of increased proteolytic activity present in nzb cell suspensions since the addition of nzb cells to iodinated balb/c cells did not alter the balb/c ratio of sigm/sigd. it was not due to the presence of cytophilic antibody or autoanti-body since the sigm was found to have a sedimentation coefficient of s and was thus not of serum origin. when examined by fluorescence microscopy or by cytofluorometry, nzb spleen cells were found to have normal numbers of sigm+ and sigdf cells, indicating that the altered sigm/sigd ratio observed in the radioiodination experiments was due to abnormal densities of these isotypes on b cells rather than to the absence of an igd-bearing b cell subset. the increased sigm/sigd ratio may be a consequence of in vivo polyclonal b cell activation, since in vitro polyclonal activation of m o p e spleen cells has been shown to alter this ratio in a similar manner. since the increased ratio was noted as early as weeks, these data provide support for the concept that polyclonal b cell activation precedes the onset of autoimmune disease in nzb mice. sera from patients with systemic lupus erythematosus (sle) will bind native dna (n-dna). poly dat, a synthetic n-dna, consists of regularly recurring base pairs. this results in a restricted antigenicity but ensures that the molecules do not contain single-stranded (ss) regions. many laboratory preparations of n-dna will be contaminated with either ss dna or structures with ss regions in predominantly duplex molecules. the aim of this study was to determine a relationship between antibodies to poly dat and native dna in individual sle sera. one hundred sera from patients with sle were used in this study. antibodies to poly dat and n-dna were measured in duplicate in each serum using a millipore filter technique. poly dat was prepared from datp and *h labeled dttp in the presence of e coli dna polymerase. native ah labeled dna was extracted from hae cells after incubation with 'h thymidine. optimal concentrations of each antigen were calculated for use in the assay. each preparation was structurally analyzed by both hydroxyapatite (hap) elution studies and ethidium bromide fluorescence (ebr) to determine the presence of ss dna or ss regions within a predominantly duplex molecule. the specificity of antibodies to poly dat and n-dna was assessed by passing sera over agarose columns bound to either circular pm dna or poly dat and measuring both n-dna and poly dat antibodies in serum and eluate. neither preparation contained ss dna assessed by hap elution. poly dat was entirely duplex as assessed by ebr fluorescence but our n-dna contained % ss regions within the predominantly duplex molecules. under optimal conditions each serum showed consistently less binding to poly dat than to n-dna. a significant correlation was seen between the binding to poly dat and n-dna (r = . , p < . ). this correlation was consistent in all sera tested and no serum showed exclusive binding to only one preparation. binding to both antigens could be removed by passing a serum over an agarose column carrying either poly dat or n-dna. these results suggest that in clinical practice diagnosis and management of sle will be provided by measurement of antibodies to either poly dat or n-dna provided that the n-dna preparation is well characterized and does not contain ss dna or significant ss regions. despite its limited potential antigenic sites, poly dat provides a useful alternative to n-dna and does not possess ss regions to which other antibodies may bind with less diagnostic specificity. cultured adherent rheumatoid synovial cells (asc) produce collagenase and prostaglandins, particularly pge;. with age in culture and passage, collagenase and pge, production decrease; however, release of these substances can be stimulated up to > one hundredfold by a factor (apparent mol wt of - , ) released by cultured human peripheral blood mononuclear cells (mcf). mcf stimulation of collagenase production by asc is enhanced by addition of low concentrations of indomethacin (indo), nm; such concentrations of indo block pge, synthesis > wo even in the presence of mcf. at higher indo concentrations ( - fim) which inhibit pge, synthesis - , collagenase stimulation is usually inhibited by - %. in some asc stimulated with mcf, this inhibition by indo ( pm) is reversed by addition of small amounts of pge, ( ng/ml). addition of these small amounts of pge, results in augmentation of collagenase stimulation to levels greater than those seen when cells are stimulated with mcf alone. addition of larger amounts of pge, ( pg/ml) under these conditions tends to inhibit collagenase. asc respond to exogenous pge, with increased levels of camp. mcf also modulates this camp response: preincubation of asc with either indo or mcf plus indo augments camp response to exogenous p g b . it is possible that some of the effects of pge, on collagenase in asc are mediated through adenylate cyclase. pge, levels thus have profound effects on collagenase production by asc and modulate collagenase response to stimulation with mcf. therefore, pge, inhibition with pharmacologic agents in vivo could have beneficial or deleterious effects with regard to connective tissue destruction depending upon the type, sensitivity, and prior environment of the responding cell. ty cells, a subset of t lymphocytes, bear fc receptors for igg and have suppressor activity for antibody production after activation with igg immune complexes. levels of ty cells as well as total t lymphocytes were measured in patients with systemic lupus erythematosus (sle), with active and with inactive disease, and in normal subjects. mononuclear cells were separated on a ficoll-hypaque gradient, depleted of adherent and phagocytic cells, and pelleted with neuraminidase-treated sheep erythrocytes. rosette-forming cells were counted to determine the percentage of total t lymphocytes. rosettes were then mechanically dissociated and t cells separated from sheep erythrocytes through a ficoll-hypaque gradient at °c. purified t cells were pelleted with ox erythrocytes sensitized with rabbit igg and rosette-forming cells recorded as ty cells. a total of cells in each of three replicate assays was counted. active sle patients showed a significant decrease in total t lymphocyte percentages compared to normal subjects (p < . ), confirming previous reports. in addition, markedly depressed percentages of ty cells (p < . ) were found in patients with active disease. patients with inactive sle were not significantly different from normal subjects, both with regard to total t lymphocytes as well as ty cells. two acute sle patients who went into remission showed significant increases in both total t lymphocytes and ty cells with clinical and laboratory improvement. active sle patients show decreases both in total t lymphocytes and the ty subset of lymphocytes associated with suppressor activity. low levels of detectable ty cells in sle may reflect the presence of high levels of circulating immune complexes and/or the defect in suppressor activity reported in these patients. the - fold increase in red cell turnover in patients with sickle cell anemia results in uric acid overproduction. since uric acid overproduction in these patients begins within the first years of life, we studied patients with sickle cell anemia and normal gfr ranging in age from to to determine the natural history of serum uric acid and urate excretion in sickle cell disease. hyperuricemia (plasma urate . mg/dl) was observed in only of patients younger than age . however, urinary uric acid/creatinine ratios and hour urinary uric acid levels were elevated in these patients, indicating uric acid over-production and hyperuricosuria. thirty-seven percent of adults ( / ) were hyperuricemic (mean plasma urate . f . mg/dl); were normouricemic. of normouricemic adults studied while on a purine-free diet, of ( %) had hour urine uric acid excretion greater than mg. urate clearance in these patients was increased (cur . f . ml/min), indicating that normouricemia was maintained by hyperuricosuria. urate clearance in the hyperuricemic subjects was decreased (cur . f . ml/min) as compared to both normal subjects (cur . f . ml/min) and normouricemic adults with sickle cell disease (cur . f . ml/min). urate clear-ance appears to be the major determinant of serum uric acid concentration even in sickle cell patients with urate overproduction. responses of urate clearance to probenecid and pyrazinamide were exaggerated in the normouricemic overexcretors [pza suppressible urate clearance (psur):ss, . f . ml/min; control . f . ml/min; probenecid (pb) response; ss, . f ml/min; control, . f . mi/min] and were diminished in the hyperuricemic subjects with diminished urate clearance (psur, . f . ml/min; pb response, f . ml/min). urate clearance was correlated pah clearance. these results suggest that changes in urate clearance were secondary to changes in tubular secretion in urate. the initial response to urate overproduction in ss disease is hyperuricosuria with increased urate clearance and maintenance of normal serum urate concentration. hyperuricemia is found in adults with diminished urate clearance and is associated with other evidence of impaired renal tubular function. nephropathy is a little known complication of polymyositis. thus, we describe the features of glomerulonephritis in patients with polymyositis. the patients ( men and woman) aged - years each presented with symmetrical limb girdle weakness, widespread polyarthritis, myalgias, recurrent fever, and proteinuria. cpk, ldh, and sgot levels were elevated in all cases, and the diagnosis of polymyositis was confirmed by electromyography and muscle biopsy. although raynaud's phenomenon and sacroiliitis were each present in one case, no patient had the skin changes of dermatomyositis, and no evidence of other diffuse connective tissue disease or underlying neoplasm. ana was negative in all patients; cryoglobulins, le preparations, and asot were absent in patients tested. c complement level was decreased in one case. rheumatoid factor was negative in and latex positive ]:i in one case. blood urea nitrogen and serum creatinine were normal in each patient; however, the hour urine protein excretion ranged from . to . gm. three patients had an abnormal urine sediment and one had myoglobinuria. renal biopsies ( patients) showed a mild focal segmental mesangial increase. immunofluorescence was negative in one biopsy and mildly positive in a granular pattern for igg and igm in others. treatment of all patients with high-dose corticosteroids led to rapid clearing of the proteinuria and slower improvement of the polymyositis. a -year review in our center of polymyositis cases showed proteinuria exceeding mg/ hours in patients and abnormal urinary sediment in . none of these latter patients had underlying malignancy. a focal glomerulonephritis associated with polymyositis may be more common than is generally appreciated. although the pathogenesis of the renal lesion in these cases is unknown, it may be related to myoglobin. two enzymes of the purine nucleotide degradation pathway, adenosine deaminase and purine nucleoside phosphorylase, are associated with specific immunodeficiency syndromes. a third catabolic enzyme, '-nucleotidase, was measured on the external surface of peripheral circulating lymphocytes from patients with immunoglobulin deficiencies. all male patients with congenital agammaglobulinemia demonstrate reduced activity of lymphocyte ecto- '-nucleotidase to to % of the normal value. the mean activity is . f .o nm/hr/lob cells for congenital agammaglobulinemia as compared to the mean normal value of . * . . patients with common, variable hypogammaglobulinemia or selective iga deficiency have values within the normal range. two other lymphocyte plasma membrane ectoenzymes, atp'ase and nonspecific phosphatase, have similar values in lymphocytes from normal subjects or from subjects with congenital agammaglobulinemia. the activity of '-nucleotidase in lymphocyte lysates has similar values in normal and enzyme deficient subjects. ecto- '-nucleotidase has similar activity in both t and non-t lymphocytes in all subjects and the deficiency occurs in both these categories of cells in the affected patients. ecto- '-nucleotidase from normal and enzyme deficient subjects has a similar p h optimum'of . , is similarly inhibited by adenosine- ' , p-methylene diphosphonate, has hyperbolic kinetics and a similar michaelis constant of pm for amp. these data suggest that the reduction of lymphocyte '-nucleotidase activity is an abnormality localized to the plasma membrane in this x-linked, b-cell immunodeficiency syndrome. in vivo clearance of exogenous single-stranded dna (ssdna) is extremely rapid and occurs primarily through the liver. with higher doses of ssdna, however, both liver uptake and blood clearance approach a maximum, enabling large molecular weight ssdna to persist in the circulation (emlen, mannik; j exp med, march, ) . in the present study, we examined the effects of prior saturation of the liver with immune complexes on the clearance kinetics of ssdna. heat denatured calf thymus '*'i ssdna was chromatographed on hydroxyapatite and a defined molecular weight was obtained by gel filtration over sepharose b. immune complexes were prepared at -fold antigen excess with human serum albumin and purified rabbit anti-human serum albumin. female c bl/ j mice were injected at time with immune complexes containing mg antibodies or with buffer. at , , , , or hours, animals were given pg of ' ssdna; serial blood samples were assayed for radioactivity and clearance velocities were calculated by linear regression analysis. clearance of ssdna was slowed in the mice pretreated with immune complexes. control mice cleared ssdna at a rate of . f . pg/ml/min, while animals pretreated with immune complexes cleared the ssdna at a rate of . f . at hours (p < . ), . f . at hours (p < . ) and . f . at hours (p < . ). clearance returned to normal at and hours. the degree of suppression of ssdna clearance velocity at different times after administration of immune complexes correlated with the previously established values for the amount of immune complexes present in the liver at a given time. to examine the effects of pretreatment with immune complexes on the saturability of ssdna clearance mechanisms, , , or pg of ssdna were given hours after administration of immune complexes. with the pg dose the clearance velocity approached a maximum, but this value was less than half of the maximum clearance velocity in normal mice. these observations indicate that immune complexes alter ssdna clearance by decreasing the number of sites in the liver to which dna can bind, or by decreasing access of dna to the liver. by altering clearance kinetics, immune complexes may contribute to the persistence of dna in the circulation of patients with sle. (supported by nih grant am .) rheumatoid arthritis (ra) frequently affects the wrist, leading to pain, loss of function, and destruction of normal anatomy. surgical treatment by synovectomy alone does not correct carpal deformities and ulnar deviation; fusion of the wrist, although effective in reducing pain, severely limits function. to prevent loss of upper extremity function while simultaneously correcting deformities and relieving pain, we combined synovectomy with distal ulnar excision, correction of carpal supination-subluxation, and repair of damaged extensor tendons. this surgical stabilization approach was performed times in patients with ra. all had definite or classic ra and had not responded to aggressive physiotherapy, including splints and local and systemic medications. all had synovitis with deformity and instability. the deformity most commonly seen was carpal supination-subluxation with relative prominence of the distal ulna. the primary reasons for operation were frank ( ) or potential tendon rupture ( ), persistent synovitis with pain at rest ( ), and pain with significant deformity ( ). the mean age was years (range: - ). the wrist was exposed with preservation of the exten-sor retinaculum. following excision of the distal ulna, a synovectomy of the radiocarpal and intercarpal joints was carried out. any existing carpal dissociations were realigned, and the carpal supination-subluxation deformity was corrected by placing the wrist in pronation and reconstructing the triangular ligament using the volar capsule. extensor tendon repair and grafting were then carried out. length of followup was months with a range of - years. results were classified as good if the patient was satisfied, had only occasional pain, a stable wrist, and no recurrence of synovitis. three patients were lost to followup and of the remaining , good results were obtained in . an additional had objective improvement but experienced recurrence of synovitis and pain and an extensor lag of up to o . less satisfactory improvement was found in of the . the average loss of motion was ' , leaving patients with a combined palmar and dorsiflexion range of ". although this stabilization procedure is not considered appropriate for the severely destroyed wrist, the overall / improvement (fair and good results) justifies its consideration in preference to either synovectomy or wrist fusion alone. the destructive inflammation observed in synovia of rheumatoid joints may be largely due to autoimmune processes. antibodies to types i, , collagens have previously been detected in joint fluids of rheumatoid patients and cellassociated antibodies which react only with cartilage type i collagen can be locally detected in inflamed rheumatoid synovia. we are interested in finding evidence for cell-mediated immunity to collagen. immune lymphocytes challenged with antigen produce substances (lymphokines) that can grossly stimulate the secretion of the enzyme collagenase from macrophages. collagenase, which can degrade cartilage collagen producing cartilage destruction, is also secreted by macrophage-like cells in rheumatoid synovia and its secretion can be stimulated from synovial cells by a factor(s) produced by lymphocytes, unstimulated or stimulated with phytohemagglutinin. in the present study inflamed synovia were removed at surgery from patients with classic rheumatoid arthritis. synovial fragments were maintained in a viable state in organ culture for up t o weeks. culture media were changed every days and the collagenase secreted into the culture medium was assayed. to these cultures were added type i or type collagen in the form of diffusible immunologically active fragments prepared by cyanogen bromide cleavage of native collagen. in of rheumatoid patients, the addition of type i collagen fragments resulted in a significant increase in collagenase secretion compared with explants which had not been stimulated: artificial stimulation of lymphocytes in these explants with phytohemagglutinin also resulted in a similar increase in collagenase secretion. type i collagen peptides had no stimulatory effect. these results support the thesis that in some patients rheumatoid synovia contain immune lymphocytes which can respond t o type i collagen peptides produced by degradation of hyaline cartilage collagen. when challenged with antigen, these cells may produce factors that stimulate the secretion of collagenase from synovial cells leading to further cartilage destruction. although intraarticular corticosteroid injection has been considered a therapeutic adjunct in rheumatology for the last years, scientific proof of efficacy is still lacking. the present study was undertaken, therefore, to determine the effect of intraarticular steroids on pain in the osteoarthritic knee by use of a controlled, double-blind protocol. thirty-four patients with symptomatic osteoarthritis of the knee were included in the study. patients previously injected with local steroids were excluded. half the patients were treated with mg of triamcinolone hexacetonide injected into the knee, the other half with the same volume of placebo (vehicle without steroid). the two physician-experimenters performed the arthrocentesis and withdrew any synovial fluid, if possible. a nurse-assistant then injected either steroid or placebo through the same needle according to a predetermined, random schedule. thus, the physicians who conducted the study did not know the nature of the material each patient received. knee pain was quantified prior to, and at , , , and weeks after injection. at week the steroid group had significantly reduced knee pain compared with the pretreatment assessment. this reduction persisted through the week evaluation period. the placebo group also exhibited a significant amount of pain relief at week, but this was significantly less than that experienced by the steroid group. from through weeks there was no statistically significant difference between the groups. whether or not synovial fluid was initially aspirated did not affect results. postinjection flares occurred as often with placebo as with steroids and did not affect the subsequent course. it is concluded that intraarticular steroids reduce the pain of osteoarthritis, but such a response cannot be distinguished from a placebo effect by weeks postinjection. it has been reported previously that antibodies to histones can be demonstrated by immunofluorescence. when tissue sections are extracted with . n hci, histones are eluted from nuclei, and the extracted tissue contains only d n a devoid of nuclear proteins. histones can be reconstituted to nuclear d n a by incubation of acid-extracted tissues with purified calf thymus histones. sera containing antibodies to histones show positive antinuclear antibody (ana) staining on untreated tissue, become ana negative on acid-extracted tissue and regain ana positivity on histone-reconstituted tissue. sera from patients with drug-induced lupus erythematosus (procainamide , isoniazid , nitrofurantoin ) and patients with idiopathic (not drug-induced) systemic lupus erythematosus (sle) were studied. all drug-le patients had positive ana on control tissues but ana became completely negative on acid-extracted tissues. on histone-reconstituted tissues, / again became ana positive. in contrast, of idiopathic sle sera which were positive for ana on control tissues, only became negative and of these, again became ana positive on histone-reconstituted tissues while the other remained negative. three other sle sera showed partial reduction in ana titer but increased in titer on histone-reconstituted tissue. thus, / drug-le patients ( %) had antibodies to histones compared to / sle patients ( %). to determine the specificity of anti-histone antibodies, the h , h a-h b and h -h fractions of calf thymus histone were prepared by differential salt extraction and sephadex gel filtration and used in reconstitution experiments. it was shown that in / of the drug-induced sera antibodies were primarily against h a-h , while in the idiopathic sle sera studied, antibodies to all classes of histone were found. a further difference was that idiopathic sle sera had antibodies to native dna and to the nonhistone proteins sm and nuclear rnp whereas drug-induced le sera did not. the heterogeneity of anas in idiopathic sle and the striking prevalence of anti-histone antibodies in drug-induced le, as demonstrated by immunofluorescence, have been valuable clinically in differentiating between these two syndromes. the etiology of pss is unknown and progress toward its understanding has been hampered by the lack of a model for the disease. it has recently been reported that skin changes similar to those of scleroderma develop in some long-term survivors of bone marrow transplantation (bmt). five such subjects, who lived an average of months post-bmt, developed sclerodermatous cutaneous involvement and also underwent examination for visceral complications resembling pss. at present, are alive and have died. we compared their findings with the findings in well-characterized patients with scleroderma to ascertain the similarities and differences between these two groups. taut hidebound skin was found in all patients in both populations and selected skin biopsies were also similar. restrictive lung disease, characterized by decreased vital capacities and diffusing capacities, was found in % of the pss patients and in / ( wo) of the bmt group. cardiac disease and raynaud's phenomenon, similar to that found in the pss group, were noted in one post-bmt patient, and pathologic evidence of pss-like esophageal involvement was found in another. in both the bmt and pss groups, the responses of lymphocytes to suboptimal concentrations of pha were d e pressed, while complement levels, screening tests for autoantibodies and circulating levels of iga and igm were essentially normal. the frequency of patients with abnormal ana and dna-bindings was higher in the pss group than in the bmt group. the incidence of abnormal levels of immune complexes, igg levels, anergy, and decreased percentages of b-and t-lymphocytes was higher in the bmt patients than in those with pss. even though only months have elapsed since transplantation, these post-bmt patients have begun to develop signs and symptoms of a pss-like disease. we suggest that bmt bears closer examination as a model for pss. this study was initiated to further investigate the inhibitory effect of igm rheumatoid factor (rf) in an antibodydependent cell-mediated cytotoxicity (adcc) assay. this assay employs human peripheral blood lymphocytes as effector cells and igg or igm sensitized, cr labeled ox erythrocytes as target cells. serum and synovial fluid were obtained from patients with seropositive classic rheumatoid arthritis (ra) and heat inactivated. rf was prepared by dialyzing the sera and synovial fluid against sodium acetate buffer (ph . ), fractionating on sephadex g- with the same buffer and pooling the fractions constituting the leading side of the s peak, dialyzing against pbs and further purifying by passage over an igg-coupled sepharose b column and eluting with acid. this igm-rf was tryspin digested (enzymexubstrate ratio = : ) at °c for minutes and the reaction stopped by adding soybean trypsin inhibitor. the fragments were fractionated on biogel a m to separate undigested igm, fc,p and fab. the igm-rf, fc& and fab fragments were tested for inhibitory capacity in igm and igg induced adcc at both the effector and target cell levels. it was found that igm and igg induced adcc was inhibited by igm-rf and its tryspin digest fragments. inhibition at the effector cell level was achieved by rf fc& and fab whereas inhibition of the target cell level was achieved only by rf fab. these results indicate that r f may inhibit adcc at the effector or target cell level and thus modulate an immune response that is of potential importance in immune-complex disease. patients with seropositive rheumatoid arthritis (ra) of adult-onset were compared to control groups by utilizing two primary histocompatibility procedures, b-cell (ia) alloantigen typing and mixed lymphocyte culture (mlc) reactivity. by use of a two-stage microcytotoxicity assay with a panel of alloantisera, the frequencies of various b-cell alloantigens in patients with ra were compared to those of control populations, including patients with multiple sclerosis. three b-cell alloantisera were identified that gave particularly high frequencies of reactivity with ra patients. delineation of the specificity of these alloantisera on a panel of b-cell lines derived from individuals homozygous for mlc alleles revealed that reactions were obtained only with those lines from individuals that were positive for hla-dw , dw , or dwlo. absorption of the alloantisera with b-cell lines from dw , dw , or dwlo positive individuals eliminated the seroreactivity with all b-cell lines having any of these three de-terminants, thus demonstrating that the alloantisera react with a b-cell antigen common to cells with either of the three mlc specificities. mlc testing of the ra patients with a panel of normal homozygous cells defined in the seventh international histocompatibility workshop revealed an increase in the frequencies of hla-dw and dwlo as compared to controls ( % versus . % and % versus . %, respectively), while the frequency of hla-dw was not significantly different ( % versus . %). the individuals who were negative for the hla-d alleles dw , dw , and dwlo by conventional mlc testing yet positive for the shared b-cell alloantigenic specificity were of particular interest. the results indicate that a shared antigenic specificity exists among alleles of b-cell alloantigens that is, in turn, partially related to particular mlc alleles. thus susceptibility to ra may be a function of the inheritance of the molecules bearing this antigenic specificity. activation of mediator cells such as platelets and neutrophils plays an important role in the pathogenesis of gout. monosodium urate crystals (msu), the probable initiating agents of gouty inflammation, have been shown to stimulate suspensions of washed platelets or neutrophils. when msu crystals are coated with igg (as occurs in plasma), stimulation is markedly enhanced. these studies, using msu induced human platelet serotonin secretion as a model, examined the nature of cellular recognition mechanisms for the igg-coated msu crystal and the uncoated crystal. f(ab') fragments of specific anti fc antibody blocked and the lipopolysaccharide of s minnesota r enhanced human platelet secretion induced by igg-coated urate crystals. these agents had little effect on stimulation by uncoated crystals. this indicated that urate crystals stimulate platelets independently of fluid phase igg. urate crystals directly stimulated suspensions of washed rabbit platelets which lack fc receptors. in contrast to human cells, stimulation was blocked by igg. this again demonstrated igg-independent cell stimulation by urate crystals. calcium pyrophosphate dihydrate crystals could trigger human platelet secretion only when coated with igg. this suggests that when crystals were coated with igg, the surfacebound igg alone may be the stimulus to the cell. this was confirmed by the finding that polyvinyl pyridine-n-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated but not igg-coated urate crystals. in contrast to igg, f(ab') fragments, igm, or iga did not enhance human platelet stimulation by urate crystals. this indicates that the effect of igg on urate crystal stimulation of platelets depends on the fc region of igg. these data demonstrate that urate crystals (and potentially other surfaces or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of absorbed igg or when coated with igg, by triggering the cell via fc receptors. the correlation of serologic abnormalities, especially low serum complement and high levels of anti-dna antibody, with disease activity has been established in sle. the significance of abnormal serologic tests in the absence of active clinical disease is still unclear. this report describes a group of patients seen in the course of a prospective study of sle in whom a discordance between clinical and serologic features was apparent. these patients had persistently positive le preps and antinuclear antibody tests, low serum complements, and high levels of dna binding. the mean lymphocyte response to con a mitogen was suppressed in these patients as compared to age and sex matched controls done on the same day. these patients did not differ significantly from other patients with sle, seen during the same period, in the frequency of skin manifestations, raynaud's phenomenon, and arthritis; they showed trends toward a lower frequency of photosensitivity, serositis, mucous membrane ulcers, and neu-ropsychiatric involvement and had a statistically significant lower frequency of alopecia and nephritis. they also resembled the larger group in the presence of most laboratory abnormalities with only hypergammaglobulinemia occurring less frequently. these patients have been followed untreated for a mean of % years without evidence of clinical exacerbations of disease. thus in individual patients with sle it would seem advisable to determine whether their clinical course and laboratory abnormalities are concordant. some patients will illustrate this pattern and thus therapeutic manipulation according to changes in laboratory variables may be indicated. some patients will display clinical-laboratory discordance and could then be treated for clinical exacerbations only, irrespective of laboratory changes. followup in our patients would indicate that patients with such humoral and cellular immune abnormalities may safely be followed untreated for prolonged periods. independently and in two separate laboratories, cocultivation techniques have demonstrated the presence of viruslike particles (vlps) in rheumatoid synovial membrane cells (rsc). one technique consisted of cocultivation of long-term rsc cultures with human lung fibroblasts (wi ); the other system utilized cocultivation of freshly dissaggregated rsc with fetal rabbit synovialcell cultures. controls in both laboratories consisted of similar cocultivations using cultures derived from nonrheumatoid membranes. all cocultivations were maintained for - months without subculture, during which time small foci of heaped-up cells were observed. by electron microscopy, all the rheumatoid cultures ( ) but none of the control cultures ( ) showed vlps. these appeared as spherical bodies with a spike-like external array, seen both in sonicated negatively stained (pta) preparations, and in thin sections of harvested rsc cocultivations. these vlps were found budding from the cell membranes, and also within cytoplasmic vacuoles. their morphology resembled that of budding rna viruses, although coronaviruses and myxoparamyxoviruses may also show these characteristics in certain situations. similar particles have not been previously demonstrated in rsc. serial passage of cocultivation extracts containing vlps onto other cell types resulted in repeated transient cytopathic effects. extracts of the rsc cocultivations were injected into suckling mouse brains, resulting in a reproducible illness not observed when control extracts were similarly injected. additional studies are under way to identify the agent and to explain its presence in the rsc cocultivation systems. the mechanism responsible for leukopenia in felty's syndrome (fs) is unknown. both increased peripheral destruction of leukocytes and decreased marrow production may play a role in this process. the present study was designed t o determine whether factors in the sera of patients with fs inhibit the maturation of granulocyte precursors from human bone marrow. sera were obtained from patients with fs; patients with rheumatoid arthritis, splenomegaly, and normal white cell counts; patients with active ra, and patients with osteoarthritis. nucleated cells were isolated from human marrow, and single cell suspensions were cultured in mccoy's medium and fetal calf serum. the serum to be tested, . ml, was incubated with . ml of the cell suspension (containing x ' cells/ml) and . ml rabbit serum, and the mixture was placed in % agar. at the end of days, the number of cells giving rise to granulocyte-macrophage colonies (cfu-c) were counted. serum from a healthy adult was run simultaneously as a control. inhibition of maturation was considered to be present if the number of cfu-c in the agar incubated with the test serum was reduced by at least % as compared to healthy control serum. sera from of patients with fs caused a significant reduction in the number of cfu-c. seven of the patients with serum inhibitors had undergone splenectomy; in of these subjects the inhibitor was absent prior to splenectomy, but present after splenectomy. none of the sera from the control subjects gave a positive response. these data indicate that the sera of certain patients with fs contain a factor that inhibits human granulocyte precursors in vitro. this factor may play a role in the induction of leukopenia in fs. the presence of a serum inhibitor of granulocyte maturation may also explain the failure of some patients with fs to respond to splenectomy. this study was designed to determine-whether lymphocytotoxic antibodies (lca) in patients with sle had specificity for the ii antigen system; the ii antigens are known to be present on the surface of all formed elements in blood. sera from patients with sle were tested for lca using the standard microdroplet assay. following determination of the lca in whole sera, each serum was serially diluted twofold in saline and retested for lca. the dilution of the serum which gave a % decrease in cytotoxicity was used in the following absorption experiments. each diluted serum was absorbed threefold with either adult (rich in i antigen) or cord (rich in i antigen) red cells a t "c, and then retested for lca activity. eluates were prepared from sera incubated with either adult or cord cells a t "c, and the eluates assayed for lca. the cytotoxicity of the whole sera was % or greater in all but one serum. seven of the diluted sera showed an obvious reduction in cytotoxicity following absorption with cord cells, but minimal or n o reduction after absorption with adult erythrocytes. of the remaining sera, the lca were equally reduced by adult and cord cells in , and were unaffected by absorption in . in the sera studied, eluates prepared from cord cells showed greater lca activity than eluates from adult cells. statistical analysis confirmed that the mean cytotoxicity values in the sera after absorption with cord cells were significantly different from those obtained after absorption with adult cells (p < . ). additional studies showed that there was a strong correlation between the level of lca and the titers of cold agglutinins against cord red cells. these data indicate that the lca in most sle sera react with i antigen. cold-reactive antibodies with i specificity have been associated with hemolytic anemia and thrombocytopenia in other disease, thus, the capability of lca to react with i antigen may explain the observation that lca were found in higher frequency and greater titer in sle patients who have hematologic abnormalities. clinical and experimental observations indicate that estrogens may play a part in rheumatoid arthritis (ra) and in the regulation of immune responses. in order to delineate this relationship, the effect of oophorectomy in an experimental immune synovitis resembling ra was studied. nineteen adult female rabbits were sensitized to homologous igg and then randomly divided into groups. group i ( ) underwent oorphorectomy. group i ( ) had the same but were treated with mg of estradiol intramuscularly every other week. group i ( ) had a sham procedure; group iv ( ) were unoperated controls. an immune synovitis was then induced in the right knee by the biweekly intraarticular injection of mg of igg and the animal sacrificed after weeks. skin testing to igg was done at week intervals. serum cortisol levels were performed prior to sacrifice by use of a radioimmunoassay technique. gross and histologic assessment of synovial inflammation and cartilage changes was recorded using a "blind" grading previously reported from for no changes to + for severe involvement. synovial cathepsin d activity was analyzed by the anson technique. there were no changes in the initial positive skin tests to igg in any groups. cortisol levels in groups , , and iv were . , . , . hg% without significant differences, while in group i, the . was significantly depressed. synovial inflamation in the oophorectomy rabbits was +, compared to the unoperated and sham groups of +. the reduced synovitis in group i was reflected by a significantly lower cathepsin d level of . p moles/hour/l mg of protein compared to . and . for the other groups. however, when oophorectomy rabbits were given estrogen replacement, there was an increased synovitis rated + with a significantly elevated cathepsin d level of . . cartilage changes were rated + and appeared uniform throughout the groups. the results of this study indicate that in this model of immune synovitis, oophorectomy appeared to inhibit synovial inflammation while estrogen replacement seemed to enhance this response. whether this inhibition is by direct endocrine mechanisms or through the immune system remains to be studied. finally, since ra has a significant female predilection, these findings may be important in the pathophysiology of this disease. we investigated the role of suppressor cells in the depressed cellular immunity of patients with sarcoidosis. we have recently described a glass adherent prostaglandin producing suppressor cell that inhibits t-cell mitogenesis in phytohemagglutinin (pha) stimulated cultures of human peripheral blood mononuclear cells (pbmc) by secreting pge, (j exp med : , . increased activity of this cell is responsible for the hyporesponsiveness to pha seen in pbmc from patients with hodgkin's disease (n engl j med : , the mean response of patients with active sarcoidosis to three concentrations of pha was significantly (p < . ) less than controls. passage of the cells over glass wool resulted in a % increase in the mean response to pha in the sarcoidosis patients and a % decrease in controls. the pha response of the active sarcoidosis patients went from % of control before passage over glass wool to % of control after. addition of indomethacin, a prostaglandin synthetase inhibitor, to pha cultures increased the response of the pbmc from patients with sarcoidosis f % versus a f % ). increase in controls (mean f sem, p < . ). the sarcoidosis patients had an increased percentage of. monocytes in the peripheral blood mononucleat cell preparations ( f % monocytes for patients, f % for controls, mean f sem, p < . ) and the percent monocytes in a pbmc preparation from patients with active sarcoidosis correlated with the percent increase in pha response after glass wool passage (r = . , p < . ). thus, there appear to be at least three mechanisms operating in the depressed pha response of pbmc from patients with active sarcoidosis. first, there appears to be increased activity of the prostaglandin producing suppressor cell. second, the increased proportion of monocytes correlates with increased suppression, either via a diluting effect or perhaps through another active suppressive effect. blockade of these first two mechanisms by indomethacin and glass wool passage does not result in total restoration of the pha response of patients with active sarcoidosis, and therefore, a third, as yet unknown, factor must also contribute to the depressed pha response. the rheumatologist is aware of hand deformities in the adult. very little is written concerning the hand of the child with juvenile rheumatoid arthritis (jra). roentgenographic and clinical analysis was done, review of surgery and outline of treatment is given. five-hundred patients are under treatment. there are three basic types of onset in jra. systemic onset is similar to that described by still; polyarticular closely resembles the disease in the adult; pauciarticular involves four or less joints. jra has a much better prognosis than the adult type but tends to produce more stiffness. examination of successive children showed % had radial deviation of the rnetacarpophalangeal (mcp) joint and % bad decreased flexion. seven percent had boutonniere deformity and % had a swan-neck contracture. there was decreased flexion of the pip in % and decreased extension in %. the wrist showed % decreased extension and only % decreased flexion. a statistical review of the records and x-rays of children showed finger involvement in approximately %. wrist involvement in approximately %, and very few cases of boutonniere or swan-neck. ulnar shortening in joints measured on roentgenogram showed a range of - mm with an average of . mm. ulnar wrist deviation was seen in joints with an average of degrees and radial deviation in joints with an average of degrees. metacarpophalangeal ulnar deviation was seen in with a range of to degrees and an average of , and a mcp radial deviation also of between and degrees with an average of . degrees. there was n o ara abstracts statistical correlation between ulnar shortening, ulnar deviation, and metacarpophalangeal radial deviation, as reported in previous literature. surgery between and was rare and included collateral ligament release in patients, tenolysis on occasions, carpal tunnel release, and reconstruction of thumb mcp. terminal vasospasm required finger amputation. synovectomy of mcp and pipjoints was done and studied between and . n o recurrences were seen in late followup, but stiffness precluded continuing this procedure. the common problem of wrist flexion contracture is best managed by physical therapy and extension orthosis, and the loss of finger flexion by active exercises, occasional injection, and active splinting. we have previously demonstrated that the translational movement of glucose in pathologic synovial fluids is too rapid to be accounted for by bulk diffusion (fed proc : , . such enhanced diffusivity is a property of a n isolated . % matrix of ha as well, wherein both diffusivity and matrix structure are critically dependent on ca++ concentration (see semin arthritis rheum : - , for review). the current study examines translation movement of glucose in a i% matrix. human umbilical h a and agarose were prepared as % matrices in phosphate buffered saline at ph . . the bulk diffusion coefficients (d) for glucose and sucrose within each matrix were determined in codiffusion experiments employing a capillary method. the d for both solutes in h a was indistinguishable from that in agarose. if the matrix was prepared in % horse serum, glucose diffusivity was enhanced twofold in h a while slightly diminished in agarose. a % dialysate of serum as a solvent enhanced glucose diffusivity in ha threefold; the dialyzed protein had little effect. the en-hanced glucose diffusivity in the presence of a % dialysate was inversely dependent on solute concentration. the diffusivity of sucrose in h a was little altered by these solvent changes. in order t o gain insights into matrix structure that might underlie the enhanced glucose diffusivity, darcy numbers were determined. permeability is dependent on h a concentration, has a ph optima of . in a % matrix, and is critically dependent on ca+ + concentration rising rapidly below mm. matrix structure is highly dependent on the solvent environment particularly in the physiological range. constituents in the serum dialysate may induce a matrix configuration that supports the enhanced glucose diffusivity. the matrix configuration that exists in the presence of the dialysate is likely to be present in synovial fluid as well. this configuration can facilitate the translational movement of a nutrient such as glucose. facilitated movement may be homeostatic in inflammatory states such as rheumatoid arthritis where low synovial fluid glucose concentrations are well described. sera from patients with mctd were examined for the presence of immune complexes (ic). the diagnosis was based on serologic and clinical criteria. all patients had antibodies to extractable nuclear antigen by passive hemagglutination. titers of : , , or greater were found in patients and : ,ooo and : , in the remaining two. treatment of the antigen with rnase resulted in a , -fold or greater decrease in titer in patients and a -fold decrease in one. rheumatoid factors were present in patients and antinuclear antibodies in . all patients had a mixed rheumatic syndrome. clinical features included: raynaud's phenomenon- , swollen hands and/or sclerodactyly- , myopathy- i , pleuritis-- , malar or diffuse rash- , and arthritis- . one patient had a stable iga membranous glomerulopathy. ic were measured in sera by a raji cell radioassay (rc-ra), monoclonal rheumatoid factor radioimmunoassay (mrf-ria), and clq binding assay (clq-ba). ic were detected by at least one method in ( %) sera. the single negative serum was from a patient with clinically inactive disease. ic were found by the rc-ra in ( %) sera, by mrf-ria in ( %), and by clq-ba in ( %). mean values for each assay compared to normal controls were: . versus . pg agg.-igg equiv/ml for the rc-ra (p < . ); . versus . pg agg.-igg equiv/ml for the mrf-ria (p < . ); . % versus . % la -clq bound for the clq-ba (p < . ). ic were detected by all three assays in ( %) sera and by two methods in ( %). thus, ic were found by two or more methods in ( ) sera. only three sera failed to react in more than one assay. a different pattern of reactivity was found in rheumatoid arthritis sera where ic were detected at a higher frequency by mrf-ria ( %) and clq-ba ( %) while the frequency by rc-ra ( %) was lower. sufficient clinical data were available for of the mctd patients to permit preliminary evaluation of the relationship of disease activity and ic. changes in levels of ic appeared to parallel clinical activity in patients by rc-ra, in by mrf-ria, and in by clq-ba. these data indicating a high incidence of ic in mctd and a different pattern of reactivity from ra emphasize the wide spectrum of immune complex disease. hydroxyapatite (ha) has been found by others in synovial fluid and fluid leukocytes by electron microscopy and electron probe. we have developed a method to quantify ha in synovial fluid which avoids possible em artifacts. ("c) ehdp, (sa . pci/omole) was used in a final concentraion of nm/ml in normal serum or phosphate buffered saline (pbs) containing standard ha or to joint fluid samples; serum or joint fluids were processed under oil. after rotation x h "c, radioactivity/o.l ml was determined before and after centrifugation ( x g), and expressed as percent loss of nuclide from the supernatant. standard ha binding in serum monsodium urate or calcium pyrophosphate dihydrate (cppd) crystals failed to bind in concentrations up to . mg/ml and .o mg/ml respectively. the centrifuged pellets were washed in distilled water and dried to a spot which showed ha by x-ray diffraction. this method was sensitive to - pg ha standard. we confirm the finding of ha crystals in joint fluids, handled physiologically, in amounts equal to - pg ha standard/ml. such small amounts and the low wbc in these fluids fail to support a phlogistic role for ha crystals. patients with systemic lupus erythematosus (sle) have circulating immune complexes (ic) which are thought to be involved in disease pathogenesis. using a newly described method for in vivo evaluation of res igg fc membrane receptor function, we previously demonstrated a defect in untreated patients with active sle and related this res defect to the presence of circulating immune complexes. a serial study of the correlation of defective res fc receptor function, immune complexes, and disease activity was undertaken to further investigate the interaction of these factors. isologous yr-labeled erythrocytes were sensitized with human anti-rh(d) and their intravenous clearance determined. clearance rates (t- / ) c' levels and ic levels by z -clq precipitation were determined prior to therapy in sle patients, of whom had active disease. fifteen of had markedly prolonged clearance rates (range = to > ,ooo minutes; average clearance t- / in normal volunteers = minutes, range to minutes). the correlations of clinical activity with ) clq binding and ) prolonged clearance were statistically sig-nificant (p < . and p < . , respectively; spearman rank coefficient). in addition, elevated clq binding and depressed c levels were significantly correlated (p < . ). at the end of from to months, patients who initially had prolonged clearance were restudied. patients had been treated with either corticosteroids ( of ) or nonsteroidal, antiinflammatory drugs ( of ). clinically, of were improved; the remaining continued to do well. although a clearance defect persisted in all (t- / from to minutes), in of t- / was markedly improved, suggesting improved fc receptor function. in patients, there was an apparent correlation between increased clearance and clinical improvement. there was also a significant correlation between clinical improvement and a decrease in clq binding (p < . ). these studies underscore the interrelationships between the presence of circulating immune complexes, defects in membrane fc receptor function which might lead to continued circulation of these complexes, and disease activity in sle. we have found immune complexes in patients with lyme arthritis, an apparently tick-transmitted illness that typically begins with the skin lesion, erythema chronicum migrans (ecm). many patients subsequently develop clinical attacks of synovitis histologically like that of rheumatoid arthritis; some also develop other systemic complications (aseptic meningitis, peripheral neuropathy, or carditis). we measured circulating immune complexes (cic) serially in patients with ecm, by the l*si-clq binding assay. the table shows the first test result on each patient's serum in each of the designated categories. circulating immune complexes-an antigenic component of which may be related to the causative agent-were almost always present very early in the illness and in patients with systemic complications. in contrast, they were found less often during attacks of arthritis and still less often during remissions. quantitative levels tended to diminish with time; in individual patients they fluctuated in a dampened hemi-sinewave pattern in parallel with recurrent bouts of active disease. synovial fluid from all of patients contained immune complexes, compared to only of concomitant sera. in the positive sera the concentrations were much less than in the corresponding joint fluids. we suggest that the causative agent is introduced into the skin, where ecm is the initial clinical manifestation of a systemic, immune-mediated inflammatory disorder that often becomes localized to and propagated in synovium. thus, lyme arthritis can be considered a human model for an infectious etiology of rheumatoid arthritis. little attention has been given to analgesia, divorced from suppression of inflammation, in rheumatoid arthritis (ra), despite its importance to patients. this study was designed to determine the comparative efficacy of five often used analgetics for the pain of active ra synovitis. single doses of mg aspirin (asa), mg acetaminophen (ac), mg codeine (co), mg pentazocine (pe), mg propoxyphene (pro), and placebo (pi) were given in a randomized double blind fashion, and on a prn basis, to each of carefully selected hospitalized r a patients with painful synovitis of at least joints. all other drugs and distracting activities were prohibited during the study period and at least hours of observation required between doses of test agents. subjects recorded time of onset and duration of maximal relief, percent of relief achieved from each agent and all possible side effects, and, after taking all agents, ranked their order of preference, with as most effective. onset and duration of action of all agents were similar except ac and pi, which required longer to act and lasted a shorter time. side effects occurred significantly more frequently with pe, somewhat more often with co, but were otherwise insignificant. effectiveness was analyzed according to some of all ranks, mean percent relief and percent of subjects achieving % or greater relief for each agent. as expected, by parameters measured in this study and by cost to the patient, asa is a superior analgetic in ra. however, ac, by cost and effectiveness, is an acceptable alternative, as codeine would be except for its narcotic properties and cost. cost, side effects, and limited effectiveness restrict the usefulness of pe. pro seems no more effective than pi and of questionable usefulness in ra, except perhaps as a placebo. sum there is evidence that the inflammatory properties of crystalline material may be modified by their interaction with serum proteins. in this study, the activation of c by crystals is quantitated by two dimensional crossed immunoelectrophoresis (tdiep). in polypropylene tubes . ml aliquots of fresh human serum were mixed with . ml of buffer containing . mg of washed crystals. the tubes were incubated with agitation of oc for hour and then centrifuged at ,ooog for minutes. aliquots of the supernatants were diluted appropriately in edta-containing buffer to inhibit further conversion of c . by use of standard techniques, tdiep was performed using rabbit antisera to human c . a method was devised whereby samples could be compared simultaneously on a single plate. after staining, the combined area under the pic and bia peaks was measured using planimetry. the numerical results below are percentage points of total area under the p a peak in excess of that seen in saline controls. the coefficient of variation of the ratio of the two peaks was . for a single specimen on the same plate. the areas could be corrected to indicate milligrams of c converted. sodium urate was highly active in the electrophoretic conversion of c with a dose and time related response. activation was prominent a t concentrations of crystals seen in gouty effusions. heating the crystals at ' c for hours reduced c conversion by nine-tenths, but also altered morphologic properties of the crystal preparations. conversion was inhibited by increasing concentrations of edta and eliminated by . m edta. the edta inhibition was not seen in the presence of excess calcium. with the conditions described above, potassium urate crystals were much less active ( %) than sodium urate ( %). eight different commercially available preparations of steroids gave varying but small amounts of c activation (- t o %). it is demonstrated that different crystals and altered forms of the same crystals vary in their interaction with serum and their ability to activate complement. it is not known what properties are responsible for these phenomena. the calcium requirement suggests that the interaction of sodium urate with complement occurs at or before c . it is possible that immunoglobulin, which has a high affinity for sodium urate and is found in sodium urate crystals in vivo, is altered and initiates complement activation by the classic pathway. temporal artery pain, polymyalgia rheumatica, and blindness are well recognized manifestations of giant cell arteritis. however, the disease may often present in a less evident fashion. this is demonstrated by ( %) of our series of patients. of patients with biopsy-proved giant cell arteritis, the chief complaint was polymyalgia rheurnatica in ; temporal pain in , and loss of vision in . in the remaining patients, the predominant complaint that brought them to the physician did not suggest the diagnosis of giant cell arteritis, since headache and polymyalgia were either absent or so minimal as to escape notice. these complaints included fever of unknown origin in patients; malaise, anorexia, weight loss, and abnormal liver function tests suggesting occult malignancy in ; and unexplained anemia in . four patients presented with a neurologic syndrome; had diplopia and acute weakness of one arm. claudication was the chief complaint of patients, involving the leg in one, the arm in one, and the jaw in . all patients responded well t o steroid therapy. the diagnosis of giant cell arteritis in patients such as this who do not present in typical fashion depends on detection of a very rapid erythrocyte sedimentation rate, biopsy of a temporal artery, and the awareness that the disease may manifest itself in a variety of ways. the administration of certain drugs induces clinical and laboratory features of systemic lupus erythematosus (sle) in man but few studies of this human disease model have been undertaken. hydralazine, a useful antihypertensive agent, is one of these drugs and has acquired a "bad press" because of such observations. a prospective study of hypertensive patients with age, sex, and race matched controls was begun years ago. all subjects had complete clinical examinations; anf, d n a binding; immunoglobulin and complement levels; skin tests to a battery of antigens and lymphoprolifera-tive responses t o mitogens and antigens obtained prior t o starting hydralazine and serially throughout the study. all subjects were a n f negative at the start of treatment. fifteen of the have shown varying a n f response (in undiluted serum) but only had a titer of :lo or above, one becoming positive at months and at months. one of these subjects had clinical symptoms suggestive of sle and the drug was stopped. five subjects have shown a proliferative lymphocyte response to hydralazine and/or its metabolites (jci : , ) including the with positive anf. acetylation rates were obtained in of the hypertensive patients. thirteen were slow and fast acetylators. the a n f positive subjects were all slow acetylators as were of the with proliferative lymphocyte responses to hydralazine and its metabolites. this study t o date suggests that hydralazine in its present form may be less likely t o cause serologic and clinical sle than previously reported and that screening by acetylation rates may define a susceptible population. this and other lupus related drugs continue t o be a rich source of data relevant to lupus in humans. (supported by an niamdd grant). normal neutrophils (pmns) develop intracytoplasmic inclusions after incubation with sle sera. the resulting inclusions stain for igg, igm, and c and are believed to be immune complexes removed from the sera in vitro. such inclusions have also been identified in the circulating pmns from sle patients. the present study examined the relationship between pmn inclusions and clinical and laboratory features of sle. sera were carefully collected at " from sle patients. normal buffy coat cells were incubated in the sle sera for minutes a t ' and then centrifuged and washed. slides of the washed cells were prepared in the cytocentrifuge, stained with fitc goat anti-human igg, igm, iga, and c , and examined under ultraviolet light. the composition of these complexes was also examined with a fluorescent probe for double-stranded polynucleotides with the aid of ethidium bromide (eb). eb was added t o sera known to contain high concentrations of anti-dna antibodies and then incubated with normal pmns as before. preparations included ) no added eb, ) eb only, ) fitc anti-human igg (fianti-igg) or ) f -anti-igg and eb. inclusions containing both igg and igm correlated with clinical activity (p < . ), depressed serum complement (p = . ), cryoglobulinemia (p = . ), and anti-ndna antibodies (p < . ). igg inclusions alone were not significantly correlated with any of the parameters. c and igm appeared to be mutually exclusive, i.e. when one was present, the other was never present. eb staining inclusions (red fluorescence) suggested that polynucleotides were present. these findings suggest: ) the pmn inclusions consist of immune complexes which contain double-stranded polynucleotides, ) these complexes correlate with disease activity when igg and igm are both present, and ) such complexes may contribute to a number of granulocyte disturbances seen in patients with sle. in previous studies on preservation of articular carti-( , dpm/mg protein) permitted quantitation of the neulage performed in our laboratories, it was found that addition tral protease activity released into the media of the stored of a-tocopherol ( pg/ml) to the culture media of stored cartilage explants. employing this assay we found that acartilage resulted in improved preservation of its biochemical tocopherol resulted in significant inhibition of this activity as and biomechanical properties when compared to cartilage shown in the table. stored in absence of the vitamin. although it has been shown that a-tocopherol stabilizes lysosomal membranes in other lage were due to stabilization of lysosomal membranes, we no. control + a-tocopherol control + a-tocopherol developed a highly sensitive assay for neutral protease activity employing a biosynthetically prepared protein substrate. this five further patients with sle have been treated with plasmapheresis. six to liters of plasma per week were removed, using a haemonetics model hood cell separator. from to liters were removed in all. levels of complement components were measured by immunodiffusion and by a hemolytic assay. dna antibodies were measured by passive hemagglutination of dna coated red-cells. immune complexes were measured in vitro by the raji-cell assay. two patients were critically ill. one had seizures, pericarditis, pleurisy, and severe edema, despite treatment with mg prednisone daily for one month. the raji assay was pg/ml (normal < pg/ml). following the removal of liters of plasma her condition improved dramatically, complement levels rose to normal, and raji assay fell to - pg/ml. the second acutely ill patient had lupus cerebritis, with a level of only pg/ml on raji assay. after the removal of . liters of plasma there was no significant improvement in her clinical condition. the remaining three cases were less severely ill and had raji assays of , , and pg/ml. they showed a reduction of circulating immune complexes after each plasmapheresis, and an overall, variable fall after to weeks of treatment, but no striking clinical benefit. those cases with a high raji-assay also showed increased binding of doublestranded dna. the raji assay appears to be of value in predicting the response to plasmapheresis: patients with a very high level may show a massive and sustained reduction, with a good clinical response. in those with lower levels, the response is less predictable. plasmapheresis appears to be a valuable accessory form of therapy in the severely ill patient with acute sle. to investigate the vascular lesion in scleroderma (sd), the effect on human endothelial cell (ec, umbilical cord) growth of sera from patients with early sd was compared with healthy control sera. ec growth was monitored by tritiated thymidine chtdr) uptake and by direct cell counts. shtdr uptake of ec was inhibited up to % by of sd sera compared to the remaining sd and control sera. mean htdr uptake was cpm for control sera (range: - l), cpm for the noninhibitory sd sera ( - ). and cpm for the i i inhibitory sd sera ( - ). the sd patients with inhibitory serum activity tended toward shorter disease and symptom duration and were more likely to have peripheral edema and hypergammaglobulinemia than sd patients without inhibitory activity. furthermore, the inhibitory effect was no longer present in one patient after therapy with glucocorticoids which was associated with reduction of peripheral edema and improvement of myositis. the activity was further characterized in selected sera. sd serum completely inhibited the increase in ec htdr uptake seen with increasing normal serum concentration from to %. htdr uptake correlated with reduction in ec number ( % reduction at % sd serum concentration versus % increase with control serum) and with cytotoxicity ( % cell death versus % in control serum). these effects were not observed when the target cells were mouse t or human fibroblasts, smooth muscle cells, or peripheral monocytes, suggesting specificity of the inhibitory effect of sd sera for ec. the inhibitory effect of sd serum was heat stable ( "c, minutes), nondialyzable, present in both plasma and serum, and unaffected by mixing with control serum. when serum was fractionated on sephadex g- column, the inhibitory activity was found to elute in the position of serum albumin. sera from patients with non-sd active rheumatic diseases (ra, sle, pan, dm) showed no inhibitory effect on human ec, fibroblasts or smooth muscle cells or mouse t cells. these preliminary observations describe an ec-specific cytotoxic serum factor(s) in patients with sd. the in vivo role of this factor(s) in the pathogenesis of the proliferative vascular lesions in scleroderma is unknown. activation? activation of the complement (c) system in ra has been amply evidenced by a) detection of c and immune complexes in ra synovial leukocyte inclusions; b) depressed ch , c , and c levels as well as split products of c , c , and fb in ra synovial fluids; c) split products of c and c in ra plasma samples; and d ) hypercatabolism of c in meta-bolic turnover studies. we measured the fractional catabolic rates (fcr) of radioactive labeled c and fb in ra patients and normal controls to assess the relative importance of classical and alternative pathways of complement activation in ra. our results demonstrated: ) hypercatabolism of c in of ra subjects and inverse variation of c fcr with plasma c levels; ) hypercatabolism of f c in of ra patients; ) c fcrs disproportionately higher than fb fcrs in of ra subjects undergoing simultaneous studies; ) correlation of c and fb catabolism and homologous igg catabolism; and ) occurrence of hypercatabolism of c mostly in the extravascular space, a phenomenon previously observed by us also with the igg hypercatabolism of ra. patients with high fb fcrs had higher titers for rheumatoid factors. patients with high c fcrs had higher total joint counts and a greater number of manifestations of extraarticular disease. c and fb fcrs did not correlate with circulating immune complexes by the raji cell assay or with the sedimentation rate. the results suggest that complement activation in ra occurs primarily through the classical pathway and in the extravascular compartment, probably principally by immune complexes not detected by the raji cell assay, and with participation of the alternative pathway in some of the patients. studies from this laboratory have demonstrated that the pha and con-a responses of splenic lymphocytes from rats with adjuvant-induced disease are diminished. these diminished responses return to normal with spontaneous, corticosteroid (cs), or methotrexate (mtxtinduced remissions. the diminished pha and con-a responses are due to two types of suppressor cell: one which adheres to plastic ar glass, and another which adheres only to glass. the latter cell, which accounts for the bulk of the suppression, is more sensitive to the inhibitory effects of cs and mtx in vitro than are the mitogen-responsive lymphocytes. virazole (vzl), a synthetic nucleoside, is a noninterferon inducing antiviral agent which inhibits aid. four groups of highly inbred fisher rats were studied as follows: ) untreated rats; ) rats treated with mg of vzl given days prior to sacrifice; ) rats treated with freund's adjuvant given days prior to sacrifice; ) rats treated with freund's adjuvant followed by vzl days later. whole spleen cell suspensions were studied, as well as suspensions after removal of plastic-adherent cells by incubation in petri dishes or after removal of glass-adherent cells on columns of glass wool. each of these three types of suspension was also incubated with a range of concentrations of vzl and with either pha or con a. ah-rdr incorporation into nucleic acid was determined at hours. spleen cells from untreated rats, rats given mg of vzl days prior to sacrifice, and adjuvant-treated rats given vzl had normal pha and con a responses. the spleen cells from rats given adjuvant alone had markedly diminished pha and con-a responses due to the two types of supprcssbr cells, both of which were more sensitive to vzl in vitro than were the mitogen responsive cells. this preferential sensitivity of suppressor cells to vzl was greater than that noted with cs or mtx. two possible interpretations are: ) vzl has a more specific immunosuppressive effect on suppressor cells than cs or mtx; ) suppressor cell function is due to a virus which is inhibited by vzl. twenty-four cerebrospinal fluid (csf) samples from patients with systemic lupus erythematosus (sle) were evaluated for cyclic-gmp (c-gmp) concentration by radioimmunoassay. for comparison studies, the patients were divided into three groups based on their clinical status at the time of each lumbar puncture: those with ) active neurologic and psychologic abnormalities (group i), ) active neurologic abnormalities only (group ii), and ) psychologic abnormalities only (group ). groups i and i had mean csf c-gmp values of . nm f . (se) and . nm f . respectively, which were both significantly higher than the mean for group i ( . nm f . ) (p < . ) as well as for the mean of a previously studied group of patients with lumbosacral pain syndromes ( . nm f . ) (p < . ). other csf findings did not display this close correlation with activity of neurologic disease. the number of samples with abnormal csf leukocyte counts was significantly greater for group i ( / ) compared with that found for group ( / ) (p < o.ol).however, no significant difference was found between group i and group , for this abnormality. in sle patients significantly higher levels of csf c-gmp were found on serial sampling during times when neurologic abnormaltiies were active. neither prednisone nor psychoactive drug therapy had any demonstrable effect on csf c-gmp levels in these pa-tients. comparison of clinical markers of sle disease activity among the three groups revealed hematologic and serologic abnormalities not to be of significant value in distinguishing sle patients with active neurolgic involvement (groups i and ) from those without (group ). however, fever and, to a lesser degree, rash, proteinuria, and abnormal urinary sediment were present more frequently in groups i and i than in group . thus elevated csf c-gmp concentration may be a marker of active neurologic disease in sle. assay of csf c-g m p may therefore be helpful in the clinical assessment of sle patients with neurologic and/or psychologic abnormalities both with regard to their diagnosis and therapy. this study reports the first serial observations of c-gmp in a group of sle patients with correlation of manifestations of disease activity and extends our previous studies of cyclic nucleotides in sle. mp produces a chronic arthritis in mice with histopathologic features resembling rheumatoid arthritis. since the chronicity of arthritis appears to result in part from persistence of mp within the joints, elucidation of host mechanisms responsible for elimination of mp is of considerable importance. the role of complement in the pathogenesis of mp-induced arthritis was studied in mice congenitally deficient in the fifth component of complement (c ). in all experiments, at least . -week-old male mice per strain free of detectable mycoplasmas were inoculated intravenously with x p color change units of mp. subjective assessment of the arthritis was carried out by scoring ankles, wrists, qetacarpal, metatarsal, and digital joints on a scale of to . in a preliminary study c deficient dba/ mice and strains of normal mice: t.o., c h, cba, and c bl/ were used. in the normal strains the .arthritis peaked in severity within days and declined thereafter. in contrast the arthritis in the dba/ mice reached a plateau at days and gradually progressed in severity for as long as months postinoculation. a second study was carried out with strains of c deficient mice, i.e. dba/ , akr and a strain and normal strains: c h and c bl/ . the arthritis in the c deficient strains persisted at a high level months after inoculation despite considerable variability in the severity of the acute arthritis among the strains. the arthritis in the normal strains rapidly declined as before. at months postinoculation, mp was isolated from the joints, lungs, and spleens of c deficient mice more frequently and in larger numbers than from normal mice, consistent with the failure of cs deficient mice to eliminate mp. complement-fixing antibody to mp was detected in higher titers in c deficient strains. the results demonstrate the importance of c in the elimination of mp from the joints and organs of infected mice. moreover, the study may have clinical relevance since secondary complement deficiencies exist in a number of rheumatic diseases. it supports the concept that complement deficiency might contribute to the persistence of as yet undefined etiologic agents in human arthritidies. we have studied american black patients with primary ankylosing spondylitis (as) unassociated with ulcerative colitis, crohn's disease, psoriasis, or reiter's syndrome. all the patients were unrelated and met the rome criteria for as. sixteen ( %) of them possessed hla-b . we further studied the remaining hla-b -negative patients to see if there was an association with other b locus antigens belonging to the hla-b cross-reactive group (hla-b , b , bw and bw ). hla-b was present in of these patients ( . %) compared to of ( . %) b -negative black controls (x' with yates correction = . , p < . , relative risk = ). bw was found in one patient who also had b . bw was not tested for. antisera used to detect b , b , and bw were free of noteworthy cross-reactivity. duration of disease, age at onset of as, skeletal manifestations, prevalence of acute anterior uveitis and frequency of a positive family history for as were compared between the b -positive (gp-i) and the b -positive (gp- ) patients. duration of disease at the time of study was not significantly different in the two groups. however, mean age at onset of as differed significantly: . years in gp-i and . years in gp-i (p < . ). skeletal manifestations of the disease did not differ significantly in these groups. uveitis occurred in patients ( %) in gp-i and patients ( %) in gp-i (p = . ). a family history of as could be obtained in none of gp-i patients and ( . %) of the gp-i patients (p = . , using fisher's exact test). these data indicate that hla-b may be associated with the development of as in the b -negative black popu-lation. the b -positive black as patients are older at onset of their disease than the b -positive patients and tend to lack obvious familial aggregation of the disease. the findings suggest that american black as patients lacking b but possessing b represent a subgroup of patients with this disease. prostaglandin synthesis inhibitors (pgsi) may affect renal function in humans, especially under conditions of renal injury such as lupus nephritis. to explore this preferential effect in systemic lupus erythematosus (sle), we measured basal excretion of urinary prostaglandin e (ipge) by radioimmunoassay in female sle patients and normal women on a constant meq sodium diet. no patient or normal control received nonsteroidal antiinflammatory drugs during the study period but of patients were on maintenance low dose prednisone ( i mg/day). six of patients had biopsy-proved nephritis; had insufficient urinary findings to prompt a diagnostic biopsy. all ipge values were determined in triplicate on at least three separate -hour collections except in one patient with two such collections. sle patients had a significantly higher mean ipge excretion than normals ( . f . versus . f . ng/hour; mean f sem; p < . ). sle patients with biopsy-proved nephritis had a higher mean excretion than those without biopsy ( . f . versus . f . ng/hour) although the difference was not statistically significant. urinary sediment and general disease activity did not correlate with urinary ipge. all patients showed a decrease in creatinine clearance with pgsi administration but the percent change did not correlate with basal urinary ipge excretion. elevated urinary ipge excretion in sle may reflect renal inflammation due to immune complex nephritis and may explain the greater sensitivity of renal autoregulation to pgsi in this setting. the therapy of lupus glomerulonephritis has been evaluated in a randomized study of corticosteroid treated patients comparing bolus ivcy ( . - . mg/kg, every months), combined oral cy + az ( mg of each, daily), and prednisone alone (pr). five years after the start of the trial, patients have been randomized: ivcy ( ), cy + az ( ), and pr ( ). renal involvement at study entry has been characterized by urine sediment abnormalities ( ), impaired function (crci < ml/min) ( ), nephrotic proteinuria (lo), glomerular proliferation on biopsy ( ), and an estimated disease duration of less than one year ( ). the assigned drugs have been continued without complications in patients with a median course of months. toxicity has necessitated discontinuation of the drugs in courses; cy + az (pulmonary infection, hemorrhagic cystitis, amenorrhea) and ivcy (hepatitis). death has occurred in patients; ivcy (epiglottitis, sudden death), and pr (pulmonary embolism). the changes of renal function of the patients receiving a minimum of months of continuous therapy have been assessed by evaluating the number of patients demonstrating a reduction of function (crci) by at least % (alo) or % (a ) from baseline determinations at study entry. this interim analysis of an ongoing trial is encouraging and suggests that ivcy and cy + az may be more effective than pr in the maintenance of renal function of patients with lupus glomerulonephritis. extended follow-up will be required to determine the duration of these effects and the development of drug related complications. the mechanism(s) underlying the destruction of bone observed in chronic inflammatory diseases such as rheumatoid arthritis or osteomyelitis remain(s) to be elucidated. soluble mediators derived from immunologically activated cells may play an important role in such phenomena. we have observed that alloantigen-stimulated human mononuclear leukocytes (mnl) release a factor(s) capable of resorbing fetal rat bones in vitro. we have investigated procedures for the large-scale generation and partial characterization of this bone resorbing factor(s). human mnl obtained by leukophoresis (ibm cell separator) of two nonrelated healthy individuals (yield = - x lolo cells/donor) were cultured together. ( . x v cells/ml, hours, "c, mishell-dutton atmosphere). supernatant fluids were removed and assayed for bone resorbing activity by measurement of ca release from fetal rat bones in vitro. these supernatants effected significant bone resorption when compared to either control supernatants prepared from a single donor's cells or medium alone. indomethacin ( x to x wm) did not inhibit either the production or biological activity of this factor(s). bone resorbing activity eluted with molecules of - ,ooo daltons on sephadex g- and appeared in the breakthrough when applied to deae cellulose ( . m phosphate, . m naci, ph . ). eluate containing bone resorbing activity after sephadex g- chromatography x and deae cellulose chromatography was applied to amionic disc gels ( "c, ph . , trisglycine). a single peak of bone resorbing activity was observed, although this activity could not be attributed to a stainable protein bond. the results indicate that alloantigen stimulated human mnl elaborate a low molecular weight bone resorbing factor(s) resembling the previously described osteoclast activating factor (oaf). methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this factor in immune tissue destruction. inflammatory cells may play a role in modulating connective tissue growth and function in a number of rheumatic diseases. the interaction of lymphoid and connective tissue cells was studied in vitro by exposing human dermal fibroblasts (fb) to supernatants (sn) of peripheral blood mononuclear cells (pbmc). proliferation was assessed by bh-thymidine incorporation and direct cdl counts. protein synthesis was determined by ah-proline incorporation and collagen synthesis by protease-free collagenase releasable counts. sn of both unstimulated and pha-stimulated pbmc suppressed fb growth in a dose-dependent fashion. sn suppressive activity was non-dialyzable, heat-stable, not cytotoxic, and removed by absorption of sn with fb but not with pbmc. there was suppression of protein synthesis by pbmc-sn in direct proportion to the suppression of cell number ( %), but disproportionately greater suppression of collagen synthesis ( %). culture sn of t-cell depleted pbmc were as active in suppressing fb growth as were sn of unfractionated mc ( - % suppression, mean f % for t-depleted versus - %, mean f % for unfractionated). supernatants of t-cell enriched populations had decreased activity ( - %, mean f % suppression). the growth suppression seen was due, at least in part, to stimulation of fb prostaglandin (pg) synthesis. immunoreactive pge, production by fb was increased -fold ( . versus . ng/ cells) by pbmc-sn. when inhibitors of pg synthesis (indomethacin, sodium meclofenamate) were added to fb cultures just prior to addition of pbmc-sn, the growth suppressive effect was abrogated ( - %). furthermore, exogenously added pge, (final concentration = ng/ml) resulted in suppression of fb growth comparable to that seen with pbmc-sn. thus, lymphoid cells produce soluble factors which induce fb to modulate their own growth in vitro and which alter collagen synthesis. the effector of the growth autoregulation appears to be a pg. this system provides an in vitro model for the study of lymphoid and connective tissue cell interaction which may improve the understanding of proliferative and fibrotic human disease. although there is compelling evidence to support the view that altered endocytic-lysosomal functions of synovial tissue may play a role in the joint tissue injury seen in rheuma-toid arthritis (ra), there are no data which quantitate these functions in human synovial cells (sc). therefore, we investigated the rates of pinocytosis and intracellular digestion of a soluble protein, horseradish peroxidase (hrp), in monolayers ( - days in primary culture) of sc from ra and non-ra patients. pinocytosis was linear with increasing h r p concentrations ( . - mg/ml), time ( - min), cell numbers ( - x ) and cell protein ( -lo pg; x i@ cells equivalent to loopg protein). specific activity (sa; ng h r p taken up/ pg cell protein/ hr) increased in direct proportion to cell concentration. sa of r a cultures ( - pg protein) after exposure to mg/ml h r p was f (mean f sd; n = ); an equivalent concentration of non-ra sc had a sa of f (n = ). uptake in both r a and non-ra sc was inhibited most effectively by - m potassium fluoride ( %) and °c ( %). r a sc digested % of cell bound h r p (t%) in hours, whereas t% for non-ra sc was hours. uptake and digestion of immune complexes by ra sc were also studied. insoluble hrp-anti h r p complexes formed at equivalence were added to sc at . pg hrp/ml (total protein concentration . pg/ml). uptake of complexed h r p was at a rate ( . % of this load/ pg cell protein/hr.) approximately times that for soluble h r p (at a load of mg/ ml). only % of complexed h r p was digested by hours. these studies indicate: ) human sc have a relatively high rate of pinocytosis and r a sc demonstrate the greatest rates of uptake; ) kinetics of pinocytosis in sc are primarily those of fluid phase uptake; ) the rate of pinocytosis increases with increased cell density, a relation which may be important in ra synovial tissue; ) r a sc can readily accumulate immune complexes, but their ability to digest them is much more limited than it is for soluble proteins. further quantitative studies of the mechanisms whereby sc process joint constituents, soluble antigens and immune complexes should lead to a more precise definition of the contribution of sc endocyticlysosomal functions to tissue injury in ra. this study was designed t o evaluate the effects of several nonsteroidal antiinflammatory drugs commonly used in arthritis therapy on gastric mucosa, and to evaluate the effects of agents, tolectin and motrin, in subjects with demonstrated clinical intolerance to aspirin. gastroscopy was carried out before and after all treatment schedules, and gastric mucosa was graded by the endoscopist (fl) on a scale of - +. photographs were taken and subsequently reviewed in a randomized double-blind manner by both the endoscopist (fl) and an impartial gastroenterologist (rn). remarkably consistent results were obtained: part i. forty volunteers were randomly divided into groups with subjects in each group and treated for week with either motrin ( mg/day), motrin ( mg/day), indocin ( mg/day), indocin ( mg/day), naprosyn ( mg/day), naprosyn ( mg/day), aspirin ( . gm/day), and placebo. part . five normal volunteers who had developed + hemorrhagic gastritis during a previous study after week of aspirin ( . gm/day) were given motrin ( gm/day), tolectin ( mg/day), and placebo for week in a randomized crossover manner with at least a -week washout between medications. results. part i. placebo subjects always showed the least pathology followed by motrin ( me), naprosyn ( mg), motrin ( mg), indocin (i mg), indocin ( mg), naprosyn ( mg), and aspirin ( . gm). in patients receiving naprosyn ( mg) and indocin ( mg) frank gastric ulcer was produced. differences between motrin or mg and aspirin were highly significant (p < . ) as were differences between aspirin and placebo (p < , ). differences between motrin ( mg) and indocin ( mg), and between motrin ( mg) and naprosyn ( mg) approached significance ( . < p < . ). part . of the subjects taking tolectin, had hemorrhagic gastritis of a degree comparable to that with aspirin, and had similar but less extensive changes. one subject on motrin had extensive hemorrhagic gastritis as with aspirin, and had minimal or no changes. in both parts i and , gastric mucosal biopsies, blood levels, and photographs confirmed endoscopic pathology. this study demonstrated that severe gastric mucosal hemorrhagic and ulcerative changes occur in subjects using nonsteroidal antiinflammatory drugs, and that significant differences exist between various drugs. seven patients with active gonococcal (gc) arthritis immunoelectrophoresis (cie). the diagnosis of gc arthritis were studied for the presence of gc antigen (ag) and anti-in all seven patients was made by typical clinical presentation, body (ab) in serum and synovial fluid by counter-positive local culture for ngonorrhoeae (ng) , and response to treatment. gram stain and culture of synovial fluid, as well as blood cultures, were negative in all patients. specimens were run against rabbit antisera to n g (difco) to detect gc a g and against a turbid solution of fresh isolates of n g to detect g c ab. glass slides were coated with ml of . m agarose in . m barbital buffer p h . . pairs of wells mm apart and mm in diameter were cut in the agar and filled with lop of samples-ag in the cathode well and ab in the anode well. slides were electrophoresed in barbital buffer for minutes at room temperature at mamps/slide, and examined for the presence of precipitin lines. results are shown in the table. g c a g was found in the serum of patient and in the synovial fluid of others. gc ab was detected in the serum of the remaining patients and in simultaneous synovial fluid of of these. g c ab was absent in convalescent serum in patients # and # at months. only l of patients with initial or recurrent active gc urethritis, cervicitis, or salpingitis had serum g c ab detected by cie; none had g c ag. these synovial fluid patient no. results suggest that the finding of gc a g or ab in synovial fluid o r serum by cie provides evidence for gc arthritis and may be positive when gram stain and culture are negative. cie therefore appears to be a useful test in the diagnosis of this condition. f m f frequently presents a diagnostic dilemma in childhood. its musculoskeletal manifestations may be confused with septic arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, or other rheumatic disease. although % of patients have symptom onset in childhood, diagnosis is usually delayed. we have followed patients who presented before age years. there were male and female children. the mean age of onset was . years (range - ); mean age at diagnosis was . years (range - ). there were armenians, sephardic jews, and of other ethnic origins. all had recurrent fever and peritonitis, pleuritis, or arthritis, without evidence of other rheumatic or nonrheumatic disease. all were of mediterranean ancestry. presenting complaints included fever in loo%, abdominal pain in %, chest pain in %,joint pain in %, and skin rash in %. musculoskeletal manifestations ranged from arthralgias to arthritis. the knees were involved in children, ankles in , shoulders in , sacroiliac joint in , and wrists in . both children with sacroiliac joint involvement had marked erosive changes and were initially thought to have ankylosing spondylitis. four patients had splenomegaly. n o patient had proteinuria o r other evidence of amyloidosis. all of children tested were negative for hla-b . fifteen children with frequent and severe attacks, mean age . years (range - ), were selected for colchicine therapy. patients were begun on the nearest whole tablet equivalent of .o mg/meter squared. in nonresponders, the dosage was raised to a maximum of . mg/day. six patients were noncompliant, but the remaining children have been followed for - months (mean ) on continuous colchicine therapy. the mean number of attacks for the month period prior t o initiation of therapy was . (range - ), but only . (range - ) for the most recent months of colchicine therapy (p < . ). the only side effect was transient diarrhea, which usually resolved without reduction in dosage. although the long term safety of colchicine in childhood has not been established, n o deleterious side effects have been observed. it appears that children tolerate colchicine well. colchicine therapy may allow children incapacitated by severe attacks of f m f t o assume a nearly normal life. a new amino acid, y-carboxyglutamic acid (gla), originally discovered in prothrombin where gla residues are sites for specific binding of ca++ ions, more recently has been identified in osteocalcin, a non-collagenous protein from bone matrix. based on amino acid composition and sequence of bovine bone gla-protein, osteocalcin is distinct from all other vitamin k dependent clotting proteins (factors , vii, ix, and x) synthesized in the liver. the posttranslational formation of gla, studied extensively in liver microsomes from warfarin treated rats, occurs in a vitamin k dependent enzymatic oxidative carboxylation of selected glutamic acid residues in prothrombin requiring bicarbonate ion and nadh or chemical reducing agent. to show that gla synthesis occurs in bone tissue and that the carboxylase enzyme has similar requirements in bone, microsomes were prepared from the long bones of day old chick embryos. the eggs were injected at , , and days with sodium warfarin, an inhibitor of gla synthesis, to allow for the accumulation of endogenous substrate available for specific labeling with nah cob. the microsomes separated from bone homogenates by differential centrifugation incorporated c into gla only in the presence of vitamin k . "c-gla was identified after alkaline hydrolysis of the microsomes and separation on an amino acid analyzer. further confirmation of "c-gla was obtained by acid hydrol-ysis of the putative gla component resulting in decarboxylation to glutamic acid and recovery of approximately % of the incorporated radioactivity in glutamic acid. the posttranslational enzymatic generation of specific calcium binding sites in bone suggests that the vitamin k dependent carboxylation reaction may have a possible role in regulating mineral deposition in calcified tissues. since the formation of a specific calcium binding amino acid in bone is vitamin k dependent, anomalies might be expected to occur in mineralized tissues under conditions of vitamin k deficiency or anticoagulant therapy. of significance is the finding of gla-containing proteins in ectopic calcifications (subcutaneous scleroderma and dermatomyositis plaques, atheromatous plaque) which suggests gla may be important in the formation and resorption of bone in a wide variety of disorders. a retrospective study was instituted on patients in the ucla lupus nephritis clinic in an attempt to determine the ability of three serologic indicators-specifically immune complexes (ic), anti-dna antibodies (dna-ab), and c -to predict the activity of sle renal disease as indicated by changes in hour proteinuria, serum creatinine, and creatinine clearance. we chose not to assess urinary sediment because of difficulty in quantitating terms such as "rare," "occasional,'' and "full field" as used by different observers. patients' records and serum samples from the period - were employed. the mean number of matched clinical samples per patient was with a range of - . similarly, the mean number of matched serologic samples per patient was with a range of - . "normal" daily or short term variation for each clinical and serologic parameter was assessed from paired samples taken within - days of each other. these "normal" values were then used as a baseline, variations from which could be tested for abnormality by statistical techniques. by use of simple regression analysis it was noted that ic as determined by % polyethylene glycol precipitation correlated better than either dna or c with clinical parameters. intriguingly, ic correlated with neither dna-ab or c . more importantly, when attempts were made to predict deterioration of clinical parameters - weeks after either a single abnormal serologic determination or after paired determinations of serologic parameters demonstrating significant deterioration, no test or combination of tests produced less than a % false positive rate. however, all tests were capable of predicting stability or improvement of clinical parameters with a true negative rate that averaged . % for ic, . % for c , . % for dna-ab. no combination of tests improved these rates. pyogenic infection originating within the intervertebral disc space, once thought to be uncommon, is becoming increasingly recognized. we report the clinical findings in adult patients with non-tuberculous pyogenic infection of the intervertebral disc occurring in two teaching hospitals over an month period. chills, rigors, and low grade fever were common prodromal complaints ( :ls). the acute onset of back and neck pain ( :ls) with point tenderness over the involved spinous process ( : ) most accurately indicated the diagnosis. referred abdominal pain or sciatica were less commonly seen. acute paraparesis or quadaparesis occurred in patients with epidural abscess. the sedimentation rate reflected disease activity in all patients. plain radiographs were positive in only patients. tomography was helpful in another . ""'tc pyrophosphate bone scan was positive in : cases and responsible for diagnosis in of patients with normal radiographs. overall the bone scan was positive in of cases studied. responsible organisms were cultured usually from the involved disc space ( : ). staphlococcus aureus ( ) and e. coli ( ) were the most common pathogens, although other microorganisms were responsible. no pathogen was isolated in cases. delay in diagnosis averaged weeks. previous spinal roentgen abnormalities ( ), disc surgery ( : ) , another site of active infection ( : ) , and alcoholism ( : ) were predisposing factors. intensive intravenous antibiotics followed by two months oral antibiotic therapy proved curative. ten of patients could be followed one year after therapy and were asymptomatic except for patients with pre-existing mecha-nial low back pain. none had evidence of active septic discitis. one patient died of pulmonary embolism post-discectomy. epidural abscess was the usual indication for surgery. the sedimentation rate returned to normal in all survivors. septic discitis is a common disease, but delay in diagnosis is usual despite a characteristic clinical syndrome. the sedimentation rate and bone scan are the most useful adjuncts to diagnosis. aspiration of the suspected disc space for responsible organisms is advocated. most patients are afforded excellent prognosis following judicious rest and prolonged antibiotic administration. musculoskeletal complaints of uncertain etiology have been noted in up to % of patients receiving a kidney transplant. we have studied prospectively consecutive renal transplant recipients. joint pain, stiffness, and swelling were assessed - days in every week of the postoperative period. six patients experienced joint pain affecting chiefly knees but also shoulder, ankle, and temporomandibular joints. arthralgias were variable in severity and duration, and in time of onset after transplantation. in patients pain coincided with reduction of steroid dosage and was abolished by modifying the treatment regimen. a third patient, who developed transient joint pain days after appearance of a painless effusion, had hyperparathyroidism. joint effusions, confirmed by arthrocentesis, appeared in knees of patients. mean duration from transplantation to first joint aspiration was days. effusions were generally unaccompanied by pain, except on extreme flexion, and persisted for hours to weeks. twenty-five synovial fluid samples were obtained from the knees. the mean synovial fluid volume was ml (range, - ml) and the mean cell count was /mma (range - /mms). in every case viscosity and mucin test were normal and crystals were not found on polarization microscopy of the centrifuged fluids. joint x-rays showed only soft tissue swelling. electron microscopy of three fluids revealed no hydroxyapatite-like material. synovial biopsies in patients were normal by light microscopy. no cases of aseptic necrosis have been identified. tests for serum ana, immune complexes, and rheumatoid factors were all negative. neither the arthralgias nor effusions bore any apparent relationship to the source of the donor kidney, renal function, fever, or use of anti-thymocyte globulin. no major transplant rejection episodes occurred in any of the patients with effusions or arthralgias, whereas no joint problems arose in any of patients who rejected donor kidneys in the postoperative period. thus, benign effusions are common after renal transplantation. they appear unrelated to immunologic factors or crystal deposition and may be due to transudation associated with high-dose steroid treatment. to investigate the hypothesis that infection may precipitate certain arthritis syndromes, we performed a battery of infectious agent serologic titrations on early-diagnosed arthritis patients and normal controls. forty-five antigens were employed: for common bacteria, for mycoplasma, for respiratory viruses, for other viruses, and for y enterocolitica. the arthritis patients included with sle, with ra, with jra, and with arthritis of undetermined diagnosis (aud). data were analyzed by diagnostic groups and b status. no significant difference was found among the arthritis and control groups in antibody titers to any of the common bacterial antigens or the mycoplasma species, of the respi-ratory viruses, and of the other viruses. in jra patients, adenovirus (group) and herpes simplex (types and ) virus titers were somewhat low, and in the ra and aud groups cytomegalovirus titers were low. in sle, mumps virus titers were slightly higher than in the other groups. however, in contrast to the occasional difference serologic titrations with previously mentioned antigens, highly significant differences in antibody titers to all domestic y enterocolitica antigens ( serotypes) were found among the groups. arthritis patients had consistently lower median levels than normal controls with the lowest titers found in sle, followed by jra, aud, and finally the ra group. the finnish y enterocolitica antigens (serotypes and ) showed no significant difference among groups but yielded the lowest median titers of all yersinia antigens. evidence of yersinia reactive arthritis was not found in this patient sample, and median antibody titers of the b positive and b negative arthritis patients without either rheumatoid factor positivity or the diagnosis of sle were highly comparable. the results provide no serologic evidence of infectious agent association with early-diagnosed arthritis but reveal impressive hyporeactivity to domestic y enierocoliticu serotypes which suggests decreased natural igm agglutinating antibodies to gram negative organisms as previously reported in sle patients (baum j, ziff m: j clin invest : , ) . among the various microvascular abnormalities present in the skin of patients with connective tissue diseases and demonstrable by in vivo microscopy, the most characteristic pattern of capillary abnormalities is found in patients with scleroderma (sd) (maricq hr, spencer-green g, leroy ec: skin capillary abnormalities as indicators of organ involvement in scleroderma (systemic sclerosis), raynaud's syndrome, and dermatomyositis. amer j med : - , ) . the prevalence of this sd pattern in a larger sample of patients with sd and related connective tissue diseases was studied in three separate arthritis centers and in patients with the following diagnoses: sf (so), systemic lupus erythematosus (sle, a), mixed connective tissue disease (mctd, ), and raynaud disease (rd, ) . results from widefield microscopic observations and photomicrography show sd-type capillary abnormalities present in the following numbers of patients in each diagnostic category: sd, ( %); sle, ( %); mctd, ( %); and rd, (%). tortuous capillaries were noticed in patients with sle ( %), with sd ( %). with mctd ( %), and with r d ( %). only patients (with mctd) had both a sd pattern and tortuous capillaries ( % of mctd or % of all patients). non-specific microvascular changes were present in patients with sle ( %), with sd ( %), with mctd ( %), and with rd ( %). many sle ( patients, %) and r d ( patients, %) patients showed no capillary abnormalities, as did patients with mctd ( %) but only with sd ( %). these results confirm the high frequency of sd-type capillary pattern in sd patients and separate them from patients with sle, who do not show a pathognomonic pattern by the present techniques. tortuous capillaries, frequent in sle, occur in other diseases and in normal subjects, although to a lesser degree. it is interesting to note that patients with mctd, an overlap syndrome having clinical features of both sd and sle, exhibited no specific pattern of capillary abnormalities. about half of the patients showed sd pattern, and the other half demonstrated tortuous capillaries, other non-specific changes, or no observable microvascular abnormalities. clinical criteria for classification of systemic sclerosis (ss) were derived from prospectively entered data on early-diagnosed rheumatic patients contributed by centers. cases included patients with definite or probable ss, or scleroderma in overlap. controls were patients with systemic lupus erythematosus (sle), polydermatomyositis, or raynaud's phenomenon alone. computer assisted and multivariate analysis techniques were used to construct criteria with minimal redundancy. careful definition of cutaneous involvement contributed the most powerful criteria. sclerodermatous involvement proximal to the metacarpal phalangeal or metatarsal phala-ngeal joints (ie, "proximal" scleroderma), whether in an acrosclerotic distribution (acrosclerosis), on the face or neck (scleroderma facies), or on the trunk or abdomen, was present in % of definite ss cases and in only % of combined controls. if the major criterion of proximal scleroderma was absent, ss cases could be distinguished by having at least of minor criteria, ie, sclerodactyly, digital pitting scars, pulmonary fibrosis, or large bowel sacculations. employing the major with minor criteria yielded % sensitivity in definite ss patients and % specificity in total controls, without using exclusions. when applied to an independent rheumatic patient the course of articular involvement in early rheumatoid arthritis (ra) is variable and has not been quantitatively characterized. in a prospective study of younger adult, early ra patients (negative for hla-b ), detailed articular, systemic, laboratory, and therapeutic data were collected twice yearly for a mean followup of over years. based upon the number of joints involved during followup, each patient was assigned to separate pain/tender (p/t) and swelling (sw) articular course patterns. monocyclic pattern was defined as one cycle of documented arthritis with articular remission observed for at least year; intermittent as two or more arthritis cycles, each separated by joint remission of at least months; continuing as continuous joint involvement but without persistent progression; and progressive as continued progressive involvement. the table shows numbers of cases in each pattern, mean numbers of joints involved at entry, and the mean annual change (a) in numbers of joints involved during followup, determined by regression analysis. monocyclic groups included significantly more males (p/t, p < . ; sw, p < . ), and seronegative cases (p/t, p < . ; sw, p < . ). only one woman experienced complete articular remission while progressive swelling occurred in only cases. entry joint involvement correlated with subsequent p/t and sw articular patterns (p < . and p < . , respectively). cross-sectional analysis of the numbers of joints involved at each protocol exam and longitudinal analysis of regression slopes of individual patient joint involvement both indicated trimodal distributions: monocyclic; chronic (ie, combined intermittent and continuing groups), and progressive patterns, which were independent of therapy. the data provide quantitative evidence for monocyclic, chronic, and progressive articular course patterns in early ra, with identification of significant sex and clinical correlates. in some postmortem cases of systemic lupus erythematosus (sle) associated with diffuse proliferative glomerulonephritis (dpgn), an antigen related to mammalian type c rna viral core (p ) protein is deposited in the glomerular lesions in an immune complex pattern (proc natl acad sci : , ) . now an attempt is made to support this finding by examining the glomerular immune deposits in sle-dpgn for evidence of type c virus antibody. human immunoglobulins (igs) were eluted from the glomerular immune deposits in two fractions by sequential treatment with dnase to elute anti-dna antibodies, followed by acid buffer to elute remaining antibodies. the eluted igs were then assayed by a sensitive, specific, and quantitative enzymoimmunoassay, which compares favorably with radioimmunoassay, for the detection and measurement of anti-p antibody against purified p proteins of the four chief groups of mammalian type c viruses: murine, feline, endogenous feline rd-i /endogenous primate, and simian sarcoma virus type i (ssv-i)/infectious primate virus groups. human igs which showed specific anti-p antibody activity, particularly against p antigen of the rd-i virus group and to lesser extent against p antigen of the murine and ssv-i virus groups, was eluted by acid buffer from the glomerular immune deposits in two cases of sle-dpgn that were known from previous study to contain deposits of viral p -related antigen in the same tissue lesions. these findings support the hypothesis, stemming from studies of the new zealand mouse model of sle, that subinfectious antigenic expression of a type c virus is involved in the pathogenesis of dpgn associated with sle. (this work was supported by nci, dhew grant no. male b/w mice generally die after one year of age with lupus nephritis and/or lymphoid malignancy. these mice gradually develop a severe depression of cell-mediated immunity which may contribute to the subsequent fatal autoimmune and lymphoproliferative disease. we studied t lymphocyte function in young ( - month) and old ( - month) male b/w mice in separate paired experiments. the spleen cell response to phytohemagglutinin was , cpm (geometric mean) for young mice compared with cpm for old mice (p < .oool). when spleen cells from young and old mice were mixed together, there was significant suppression of young cells by old splenocytes in all experiments ( - % suppression). this suppression is mediated by a radio-resistant, nonadherent, mononuclear leukocyte, probably a small lymphocyte. although this suppressor cell is eluted in the "t cell fraction" of a nylon wool column, it cannot be identified as a t cell because it is resistant to anti and complement treatment . in a typical experiment, unfractionated old spleen cells incorporated cpm compared t o , cpm for young spleen cells. a mixture of old and young cells incorporated , cpm, whereas the predicted incorporation for this / mixture was , cpm ( % suppression). old spleen cells from the t cell fraction of the nylon wool column were enriched for suppressor cells ( % suppression in the mixing experiment). in contrast, the b cell fraction was completely depleted of suppressor activity (actually % greater than predicted incorporation). anti and complement treatment of spleen cells from a n month old b/w mouse demonstrated % suppression by the untreated cells and % suppression by the preparation depleted of &bearing cells. these results have been consistently reproduced. there is no evidence of a suppressor cell in old mice of four normal strains (dba/ , c b / , balb/c, and nzw). we conclude that a mononuclear suppressor cell, probably a lymphocyte, contributes significantly to the severely depressed t lymphocyte function of aging b/w mice. this suppressor mechanism may participate in the pathogenesis of the lymphoproliferative and autoimmune disorder characteristic of the strain. none of the clinical and laboratory parameters presently used for the assessment of sle kidney disease is completely reliable. immunofluorescence of the urinary sediment (ifus) has been recently reported to accurately predict the rejection of renal transplants, and the purpose of this study was to evaluate its usefulness in renal sle. thirty consecutive patients who met the ara preliminary criteria for sle were included in the study. all had recent kidney biopsies and were treated with prednisone and immunosuppressors at usual doses with urinalysis, urinary light chains, c , anti-dna binding, npn, and creatinine as parameters of activity. random urine specimens were collected weekly for months and the urinary sediment was studied by direct immunofluorescence with antisera anti iga, igg, ig m, and fibrinogen degradation products. clinicai grading and fluorescence readings were done by independent observers. nine patients had normal kidney biopsies and ifus was always negative. nine patients had abnormal biopsies, were classified as inactive, and ifus was always negative. in patients with abnormal biopsies and active renal disease, ifus was always positive, even before the appearence of proteinuria, hemoglobinuria, casts, and light chains. ifus was positive to months before c dropped in patients and became negative to months before c returned to normal in patients. in the presence of residual proteinuria, ifus became negative in patients. there was no correlation be-tween the histological type and the immunofluorescence findings in the kidney biopsy with the type of immunoglobulin found in the urine. this procedure is easy to perform, not costly, and the results are available within hour. our data suggest that it reflects the changes in renal status months before the other parameters and therefore it may be a valuable method for guiding a more flexible treatment of patients with sle kidney disease. fifteen percent or less of patients with juvenile rheumatoid arthritis (jra) have positive latex fixation tests (lft); whereas, approximately % have hidden rheumatoid factor (rf) ( s igm r f in the peripheral blood probably bound with s igg and, therefore, not detectable by the standard lft). in this study, a complement-dependent hemolytic assay was used to determine the presence of hidden rf. sera of children with seronegative jra, with seropositive jra, children with connective tissue diseases, with leukemia, and normal children were separated by gel filtration at ph . to obtain the igm-containing fraction. these igm fractions were subjected to the complement-dependent hemolytic assay in which sheep erythrocytes (srbc) are coated with reduced, alkylated, and acid-treated rabbit igg anti-srbc antibody and are hemolyzed by guinea pig complement in the presence of s igm rf. thirty-seven of patients with jra, of which of polyarticular jra, of pauciarticular jra, and of systemic jra, had titers > : . none of normal controls and only of disease controls had titers > : . the median titer of all jra patients was : and healthy and disease controls, : . estimates of the significance of the differences between the median titers of jra patients and of the controls were obtained by mann-whitney analysis. they were significant at p < . . when data from patients with active disease were analyzed separately, the median titers for polyarticular jra were : , pauciarticular : , and systemic : . patients with inactive disease did not have significantly different titers from controls. the active polyarticular and pauciarticular values were significant at p < . and p < . . these results demonstrate: ( ) % of seronegative jra patients have hidden rf by this procedure; ( ) the hemolytic assay is more sensitive than the lft or sheep cell agglutination tests (scat) in determining the presence of hidden rf in jra; and ( ) activity of disease correlated with higher titers in the hemolytic assay, and the assay was superior to the lft or scat as an indicator of disease activity. this study was devised to answer in relation to articular cartilage proteoglycans (pgs) whether degradation products or products of incomplete synthesis are detectable in the earliest pathological lesions of this rabbit model of osteoarthritis. an ultramicro transport method for obtaining s value polydisperse profiles for pg aggregates (pgagg) and subunits (pgs), and miniaturized extraction, dialysis and cscl density gradient ultracentrifugation techniques have allowed study of these pgs and various pg products within the s value range of - from histologically defined sites. thirty new zealand white rabbits (weighing - . kg) were subjected to partial medial meniscectomy and killed - weeks postoperatively under nembutal anesthesia. micro-dissected medial femoral condyle samples included ) ulceration, ) mm rim surrounding the ulcer often with fibrillation, and ) cartilage from the medial and lateral femoral condyle which occasionally revealed fibrillation but usually appeared to be normal. amounts of cartilage equivalent to ) above were obtained from the contralateral (left) knee for use as a primary control and from sham operated (right) knees for use as a second control. the cartilage from rabbits was required to obtain a pool of - mg wet cartilage for each zone - above. these cartilages were diced and pgs extracted, dialysed, and partitioned. for pg extracted with . a gucl from left femoral articular cartilage of control knees, the profile shows a subunit peak with weight average sediment coefficient s; o of about and an aggregate peak extending from up to s with an average of . similar profile data on the tibia cartilage provided an average sediment coefficient of . about / of pg was in aggregated form and the rest pgs. interestingly, the undissociated pgagg revealed an s value range of - . pgs extracted without dissociation in . m gucl gave similar data. in regard to the profile of s values of pgs from small ulcers, the weight average sediment coefficent s: o of the peak was . peaks indicative of pg products with lesser values were found in pooled samples with large ulcerations. a similar study run after isolation of pgs in a dissociative csci gradient confirmed this result. in conclusion, the major abnormality detected early was a disturbance in pg aggregation, and later pgs degradation. we recently observed the development of glomerulonephritis in patients with sicca syndrome (ss) who did not fulfill the diagnostic criteria of systemic lupus erythematosus. these patients developed ss to years prior to the onset of glomerulonephritis. two were diagnosed as having membranoproliferative glomerulonephritis by light and electron microscopy, and was found to have membranous glomerulonephritis. although immunofluorescent studies were not available, all clearly had microscopic evidence of immune complex (ic) deposition. moreover c' levels were decreased in all three a t the time of onset of renal disease, and all had high levels of circulating ic as determined by the lz i clq precipitation test. these findings raised the possibility that ic plays a role in the development of certain aspects of tissue injury in ss. for these reasons patients with ss were studied for the presence of circulating immune complexes with the lasi clq precipitation test. increased ' ' ' clq binding activity (clqba) ( %) was found in ( %) patients (mean clqba = %, range - %). thirty-five patients ( %) had rheumatoid factor (rf) present in their serum. analysis by spearman rank correlation revealed a positive association between the clqba and the titer of r f as determined by the bentonite flocculation test (bft), (rho = . , p < . ). it was found that the highest positive correlation between clqba and bft titer existed for those patients with ss alone (rho = . , p < o.oos), and then decreased for those patients with ss plus extraglandular disease (rho = . , p < . ) and even more for patients with ss plus rheumatoid arthritis (rho = . , . < p < . ). there was no apparent correlation between clqba and bft titer in patients with ss plus another connective tissue disease. pretreatment of sera with . m mercaptoethanol to dissociate igm into monomers resulted in the eradication of rheumatoid factor as measured by bft but caused only slight to moderate reductions in clqba. thus circulating immune complexes exist in many patients with ss, are distinct from rheumatoid factor, and may contribute to certain aspects of tissue damage in this disease. we have recently described a high ( %), but not absolute, association of sicca syndrome (ss) with the hla-dw allelle, which is in linkage disequilibrium with hla-b (nejm : , ) . in this study we determined the blymphocyte antigens of patients ( females and males, ages to ) and normal controls. in addition to ss, of the patients have rheumatoid arthritis and have systemic lupus erythematosus. immunoglobulin-bearing lymphocytes were separated from heparinized peripheral blood on goat anti-human igg (fab) coated plastic plates and tested with antisera in complement dependent cytotoxicity tests. the antisera used were obtained from multiparous women and absorbed with pooled platelets to remove hla-a, -b, and -c antibodies. two antisera, and ags, reacted with the b-lymphocytes from all the ss patients compared to and % of the normal controls, respectively. four additional antisera ( , , , and ) reacted more frequently ( , , , and %) with the patients' b-cells than with those of controls ( , , , and %) . the remainder of the antisera tested had the same frequency of reactivity in this disease as in normals. t o determine if these antisera recognized the same or different antigens, their reactions with the lymphocytes from the normal controls were compared by the x ' test. antisera correlated with both and but not with ags or . antisera ags correlated only with . similar statistical analysis was done for the patients. the antisera and ags were excluded because of their % prevalance in ss. in the ss patient group antisera , , and were associated with each other but not with . this association can be due t o either cross-reactivity or linkage disequilibrium. family studies are in progress to determine whether ss patients can be heterozygous for and ags and, if so, whether they can be present in trans position. assuming that the b-lymphocyte antigens in humans are coded by loci of an ir region, our results suggest that two immune response genes may be involved in the pathogenesis of ss. in addition, the absolute association of and ags antigens with these patients should provide a n additional aid in diagnosis of ss. glucocorticoid administration is associated with induction of osteopenia in man and in some laboratory animals. there is debate about dose and duration required, and whether bone loss is primarily trabecular o r cortical. accordingly, adult female rabbits were divided into groups: received saline, received . mg cortisol/kg body weight (wt), and received . mg cortisol/kg wt. all received equal volumes subcutaneously once daily early in the morning. xylenol orange ( mg/kg days before kill) and tetracycline ( mg/ kg days before kill) were injected as bone markers. rabbits were given food and water ad libitum, and weights were measured every - days. bone mineral content (bmc) and width (w) were measured by photon absorptiometry before treatment and every weeks. measurement site was the humerus cm proximal to the bent elbow (all cortical bone). results are shown in the table. histopathology. the amount of bone was reduced in cortisol-treated rabbits. in the humerus this was localized to the endosteal surface. in the lumbar vertebrae the endosteal side of the ventral cortex was primarily affected with some thinning of trabeculae. calculation of rates of formation (f) and resorption (r) showed f was essentially nil in the cortisol groups. r was identical in the saline and low-dose cortisol rabbits, while r was accelerated in the . mg/kg treated rabbits. analysis of bmc and cross sections from the same site of the humerus for bone area showed a good correlation (r = . ). summary. rabbits treated with cortisol in the doses used developed generalized bone loss. this loss appears to involve more cortical than trabecular bone. bone marker studies show that the main effect is interference with f, and, with the larger dose, acceleration of r. in vivo measurement of bmc accurately reflects the progression of the bone loss. intermediate complexes are polymers of igg-rheumatoid factor which sediment between the . s and s components of normal serum. they are best identified by analytical ultracentrifugation. their presence in serum can be inferred by a double "gullwing" precipitin line of the igg on immunoelectrophoresis due to accumulation of the molecular aggregates of igg around the well. in this study we have shown they can also be detected by evaluation of the difference between the electrophoretically determined y-globulin concentration and the igg concentration determined by radial immunodiffusion (rid). molecular aggregates of igg are quantified by serum protein electrophoresis, but they are underestimated by r i d because their effective or molar concentration is low relative to the monomeric igg standard. therefore, when molecular aggregates of igg are present, the difference between the electrophoretically determined y-globulin and the igg measured by r i d is abnormally high. sixty-one consecutive blood donor sera and hypergammaglobulinemic sera from patients with diseases known not to be associated with the presence of intermediate complexes o r in which the presence of intermediate complexes was excluded by analytical ultracentrifugation made up the reference population. eleven sera with known intermediate complexes were examined. the mean and % tolerance intervals (covering % of the population with % confidence) of the reference population for the difference between the electrophoretically determined y-globulin concentration and the igg as measured by r i d was . f . g/dl. eight of i patients' sera with known intermediate complexes fell outside the upper limit. all sera which fell within the % intervals had concentrations of intermediate complexes less than . g/ dl. in addition, the degree of deviation from the reference mean showed a direct linear correlation with the level of intermediate complexes present. if the electrophoretically determined y-globulin concentration minus the igg concentra-tion by r i d is greater than .o g/dl, intermediate complexes should be presumed to be present in concentrations greater than . g/dl. synthesis of antibodies to dna (anti-dna) in systemic lupus erythematosus (sle) is postulated t o result from a lack of control mechanisms which normally suppress autoantibody production. we investigated these mechanisms by measuring anti-dna synthesis by normal and sle peripheral blood lymphocyte (pbl) populations stimulated with pokeweed mitogen (pwm). pbl isolated from patients with sle and controls were further separated into tand b-cell enriched fractions by density sedimentation of spontaneous rosettes formed by aet treated sheep red blood cells and t lymphocytes. a portion of the t-cell fraction was irradiated with rads to inactivate were cultured with normal or sle-b cells ( . x iv) at ratios these studies show that sle-b cells from some patients are capable of synthesizing anti-dna which is best demonstrated in co-culture with suppressor inactivated, irradiated t cells. normal-t cells suppress the response, whereas suppression exerted by sle-t cells is variable from patient to patient. anti-dna the antibody produced by rheumatoid arthritis (ra) synovial lymphocytes may be directed at a uniquejoint antigen inciting the inflammation, and thus may be useful to identify the antigen. we have made permanent r a synovial lymphoblastoid cell lines capable of producing unlimited quantities of antibody. r a synovial membranes were obtained surgically, finely minced in medium (rpmi , % fetal calf serum, glutamine, antibiotics), placed in mm linbro plates, infected with epstein-barr virus, incubated ( oc, air + % co,) and fed weekly. typical lymphoblastoid cell lines developed in of ( %) cultures after a mean days, range - . once established, the cell lines were put in large flasks and medium changed completely times a week. eight of the permanent lines were lost after a mean duration of days (range - ) due to bacterial contamination or poor viability. the latter was due to suboptimal growth support by specific lots of rpmi or serum; subsequently these were pretested. the remaining lymphoblastoid lines now have a mean duration of days (range - ), during which their mean volume was ml (range - ). maximum volume was . liters, but could be expanded indefinitely in liters or larger flasks on shaker platforms at low speed. mean cell doubling time was hours, range - . igg concentration in harvested supernatant medium was measured sequentially by a radioimmunoassay described previously. mean igg production by each line was , , , , and pg igg/day, decreasing slowly with time. adjusted for cell concentration, mean production was . , . , . , . , and . pg igg/iv cells/day. total production to date was . , . , . , . , and . milligrams. the culture producing the lowest amount may be producing a predominance of another immunoglobulin type. in of lines igg production tended to be significantly higher when cell concentration was lower (r = . - . ; p < . ). these ra lines produced more igg than previously reported for lymphoblastoid lines derived from other human tissues ( - bg/lp cells/day). they also produced - times more igg than our previous batch organ cultures of ra synovia. their increased igg production may be due to improved culture conditions and/or intrinsic immunologic hyperactivity of ra lymphocytes. these cultures may provide an excellent source of antibody specific for the antigen(s) inciting ra. recently we reported that partial disuse of a joint produced a defect in proteoglycan (pg) aggregation in human articular cartilage indistinguishable from that seen in osteoarthritis. the present study examines the rapidity with which defective pg organization develops after total immobilization of a limb, and the reversibility of the defect. the right hind limb of dogs was immobilized in a cast for days to weeks, at which time the animals were killed. during this period the dogs ambulated freely on legs but bore no weight on the immobilized limb. in some cases the cast was removed after weeks and the dogs then ambulated fully for up to weeks prior to sacrifice. cartilage from the distal femur of the immobilized and the contralateral control knees was cultured for hours in ham's f- nutrient mixture containing iwo fetal calf serum and s ,. pgs were extracted with m guanidinium chloride (guhci) and purified by successive cesium chloride density gradient centrifugations in . m and . m guhci, ie, under conditions favoring forma-tion of pg aggregates and disaggregation of pgs, respectively. after only days of immobilization a s , incorporation into pgs was suppressed by , and this diminution in pg synthesis persisted through weeks of immobilization. after weeks of immobilization no evidence of pg aggregation could be found. at that time pgs from the second gradient were the same size as those from the first gradient (sepharose b k., = . ) and showed no shift in their sepharose b elution profile after incubation with hyaluronic acid (ha) in vitro, indicating that pg-ha interaction had not occurred. however, week after removal of the cast, aggregates had again formed in the cartilage and were as large in hydrodynamic size as those in control cartilage. these results emphasize the importance of joint motion in maintenance of the normal organization of cartilage pgs. the pg aggregation defect which occurs with immobilization alters hydraulics of the cartilage, especially with impact loading, and may thus predispose to chondrocyte injury. in the course of examining host defenses against infection in patients with systemic lupus erythematosus (sle), we have found a previously undescribed serum inhibitor of complement (c )-derived -chemotactic activity (ctxa). serum from of patients, when activated with zymosan, failed to attract polymorphonuclear leukocytes (pmn) comparably to normal zymosan-treated serum (zts) (measured by the "leading front" method of zigmond and hirsch). incubation of normal pmn with these sera did not affect their random motility or subsequent chemotactic response to normal zts. whereas levels of c and c (measured immunochemically) were modestly low in these sera, no gross abnormalities involving alternative complement pathway activation could be detected. when preincubated with normal zts ( : ) at " for minutes, these sera caused significant inhibition ( - ooo/o) of ctxa. they also inhibited the ctxa of columnpurified c -derived peptides (from normal zts), but had no effect on the ctxa of either the synthetic peptide, n-formylmet-leu-phe or the bacterial chemotactic factor from e coli. the inhibitor in these patients' serum was heat-stable ( °c for minutes) and acted specifically on c -derived ctxa (not on pmn). mixing (without preincubation) of patient serum with normal zts failed to cause inhibition of ctxa. the inhibitor also acted reversibly; molecular sieve chromatography dissociated heat-stable inhibitory activity (a single peak with an apparent molecular weight of - , daltons) from normal amounts of c -derived ctxa in patients zts and in mixtures of normal zts incubated with patient serum. further characterization of the inhibitor has revealed it to be a basic protein (pi between and ) which can be inactivated completely by treatment with pronase. despite its effect on c derived ctxa, the inhibitor did not influence two other c derived biologic activities in zts: pmn lysosomal enzyme releasing activity and pmn aggregating activity. this heatstable inhibitor, uniquely specific for c -derived ctxa in serum from some patients with active sle, may account, in part, for increased susceptibility to infections caused by pyogenic microorganisms. the evidence that type c viruses are involved in systemic lupus erythematosus (sle) is conflicting. we tried to detect type c expression in a total of sle patients during various collaborative studies over the past four years. forty tissues ( placenta or gestational products, spleen, kidney, other) and/or cell cultures derived from them were tested a total of times using different methods. seventyfive percent of the tissues were tested by or more methods and % by or more. type c virus isolation was attempted using four distinct protocols: culture with sedimentation of sh-uridine-labeled virions, cocultivation with viral rna-dependent d n a polymerase assay, cocultivation with focus formation assay for helper virus rescue of the defective murine sarcoma virus genome (a albino), and triple cell fusion with viral polymerase assay. thirty-four tissues from patients were tested a total of times with negative results except for one type c isolate in a recent experiment. detection of both type c interspecies and primate species antigens was attempted using different radioimmunoassays in separate laboratories (g. j. todaro, h. p. charman), and indirect immunofluorescence (r. c. mellors). twenty-two tissues from patients were tested a total of times with negative results. electronmicroscopy of gestational products from patients revealed type c-like particles in placentas, but also in of normal controls (m. imamura). type c-related sequences were not found in cellular d n a from patients using hybridization to a murine type c cdna probe (g. s. aulakh). various false-positive results were also encountered in most of the studies. the virus isolate-positive patient has not yet been tested by other methods, but the patients with type c-like particles were each tested by to other methods with negative results. thus of the total tests were positive on of the tissues from patients. these combined collaborative studies are the most comprehensive yet done in sle. if type c expression is enhanced in sle, it is not regularly demonstrable using current methods. a prospective clinical, serologic and histopathologic study was performed on consecutive patients suspect for sjiigren's syndrome (ss) referred to an interdepartmental university clinic during the years - . one hundred female and male patients met at least two of the following three criteria: ) lymphocyte focus sco.re greater than one (fs > ) on labial salivary gland (lsg) biopsy, ) keratoconjunctivitis sicca (kcs), ) associated extraglandular connective tissue or lymphoproliferative disorder. of the ss patients, lsg biopsy fs > occurred in %, definite kcs in %, and associated extraglandular disease in %. rheumatoid arthritis was present in %, scleroderma in %, systemic lupus erythematosus in %, and polymyositis in %. lymphoproliferative disease or connective tissue abnormalities not fulfilling criteria for a coexisting connective tissue disease (ctd) were present in % of patients. patients with extraglandular disease and kcs almost always had a fs > ( : patients), whereas patients with extraglandular disease and a fs > did not necessarily also have kcs (only : patients). we conclude that lsg biopsy is more sensitive than kcs for detection of ss in patients with an underlying connective tissue or lymphoproliferative disorder, and may help establish a diagnosis in patients with clinically undiagnosed autoimmune or lymphoproliferative disease. moreover, lsg biopsy is far superior t o clinical symptoms or signs of salivary gland dysfunction which are not specific for ss. in fact, only % of patients with any symptoms suggestive of ss actually had the disease confirmed. local glandular disorders, anxietydepressive syndromes, and parasympatholytic drugs were common causes of oral and/or ocular complaints. our study suggests a new definition of ss as an autoimmune exocrinopathy based on the utility and diagnostic value of lsg biopsy. in view of the hla-b association and unique precipitating antibodies to nuclear antigens (ha, ss-a, ss-b) found in ss, autoimmune exocrinopathy might be considered a genetically and serologically distinct connective tissue disease related t o but separable from other connective tissue diseases. rheumatoid arthritis or a coexisting connective tissue disease occurs in only a minority of patients with autoimmune exocrinopathy and should not be a requirement for diagnosis. degradation of collagen at sites of tissue injury and inflammation is effected by the dual action of collagenase and nonspecific proteases present in neutrophils, macrophages, and other cells. monocytes eventually accumulate at such sites and as macrophages perform important phagocytic functions. mechanisms whereby monocytes are attracted to areas of in-flammatory reactions are incompletely understood, although several different chemotactic factors have been described. we have measured the chemotactic response of normal human peripheral blood monocytes to different types of human collagens and collagen degradation peptides by a modified boyden technique. chemotactic activity (ctx) expressed as monocytes per oil immersion field for various preparations tested were as follows: type i collagen ( . p m collagenase retained chemotactic activity. additional studies were undertaken with synthetic tri-and dipeptides containing amino acids common to the three different collagens. peptides containing proline or hydroxyproline (for example, gly-pro, gly-hyp, pro-hyp, gly-pro-hyp, and gly-pro-ala) were chemotactic for monocytes at concentrations ranging between lo-' and -bm. these data suggest that peptides generated as a result of degradation of collagen by collagenase and other proteases might function to chemotactically attract monocytes to sites of tissue damage and inflammation in vivo. although it is known that a small percentage of patients with sjbgren's syndrome (ss) may develop malignant lymphoma, the frequency of ss in lymphoma has not been established. therefore, consecutive untreated patients with biopsy-proven non-hodgkin's lymphoma were screened to establish the prevalence of ss. patients were defined as having ss if they exhibited objective evidence of keratoconjunctivitis sicca and xerostomia. all were assessed by history, physical examination, schirmer test, rose bengal staining of the cornea and bulbar conjunctiva, and if possible a serial salivary scintiscan, serologic studies, and a lip biopsy. of the patients seen, were identified as having ss. of these, all had a history of xerostomia and keratoconjunctivitis sicca, while had a positive schirmer test, had positive staining with rose bengal, and had a markedly abnormal salivary gland scan. three had a positive anti-nuclear antibody (ana) and anti-salivary duct antibody. of the with ss, had adequate lip biopsies. of these, were normal, and showed mild, non-specific lymphocytic infiltration, while was highly iuggestive of ss with lymphocytic infiltration and salivary gland atrophy. five of the had musculoskeletal complaints, had classic or atypical raynaud's phenomenon, and had a concurrent diffuse connective tissue disease ( had scleroderma and had probable rheumatoid arthritis). in summary, of patients screened, had ss as defined above. of these, had positive schirmer tests, had positive rose bengal staining, and had abnormal salivary scans. three had a positive ana, had anti-salivary duct antibody, and had abnormal lip biopsies. five had musculoskeletal complaints, had raynaud's phenomenon, and had a concurrent diffuse connective tissue disease. thus, ss in patients with non-hodgkin's lymphoma appears to be more common than is generally appreciated. hydroxyapatite crystals have been recently suggested as a cause of crystal induced synovitis in humans. we have performed the following studies in an attempt to develop an experimental model to further study the relation of hydroxyapatite to inflammation. articular calcification was induced in six-week-old nzw rabbits with techniques similar to those described by selye in other tissues. eight rabbits were given a single dose of , u oral vitamin d, and the next day left knees were injected with i mg feci,. right knees were injected with saline. four rabbits received intraarticular feci, without vita-min d. cartilage and synovial membrane were studied by light and electron microscopy at , , and days. synovianalysis, roentgenograms, and microradiography were also done. six other six-week-old rabbits were given vitamin d orally , u for days without any intraarticular injection. six controls were followed without vitamin d and all were killed at days for studies as above. mild synovial inflammation was seen in feci, injected joints without vitamin d. in rabbits given vitamin d there was tissue necrosis in the feci, injected joint with synovial calcification visible by light microscopy by the fifth day. calcifica-tions were all characteristic of hydroxyapatite by electron microscopy and could be seen in interstitium, occasionally on collagen fibers, and in vacuoles of synovial cells. small amounts of iron were seen in phagocytes without relation to the calcification. calcification increased over days. cartilage was not calcified except for a small surface deposit at days in one rabbit. synovial fluids had only very low leukocyte counts with predominance of mononuclear cells. joints not injected with iron were normal. the rabbits given vitamin d for days all developed round, mid-zone articular cartilage calcifications similar to those spontaneously occurring in older rabbits. there was no synovial calcification, inflammation, or joint effusion. by electronmicroscopy all calcium deposits were in the interstitium and were hydroxyapatite-like needles. many chondrocytes showed degenerative changes. thus, different patterns of articular calcification can be produced in rabbits' knees with techniques described. acute crystal associated inflammation was not demonstrated. crystals formed and sequestered in synovial or cartilage tissue appear to be tolerated without inflammation. studies of systemic lupus erythematosus (sle) families and populations suggest that a gene(s) linked to the major histocompatibility complex (mhc) influences sle. we have examined the mhc in sle families, patients, and controls by determining the cytotoxicity of antisera which detect mhcdetermined antigens expressed selectively on b lymphocytes. b lymphocytes from sle patients and controls were tested against a panel of pregnancy sera, and reaction frequencies of individual sera were compared. twenty-eight of the sle patients were also typed with a panel of hla-drelated sera from the th international histocompatibility workshop, hla-drw types assigned, and compared with workshop controls. the hla-drw types and individual serum reactivities which were increased in the sle population are shown. sle families shows that one hla haplotype is usually shared among those individuals with sle and other autoimmune abnormalities in a given family. exceptions exist, however, indicating that the haplotype itself is neither necessary nor sufficient for the expression of autoimmunity. this study demonstiates that certain mhc-related bcell alloantigens, possibly products of immune response genes, are increased in sle. family study indicates that the requirements for sle development are not limited t o the mhc, however, and probably involve additional genetic and/or environmental factors. clinical observations of increased osteoarthritis (oa) with menopause and studies of oa in experimental animals suggest that androgens worsen and estrogens ameliorate oa. further studies have demonstrated that estrogens suppress and androgens increase s incorporation into nonarticular cartilage. using a rabbit partial meniscectomy model of experimentally induced oa, we compared nontreatment ( rabbits), estradiol valerate . mg/kg intramuscularly every weeks ( rabbits), and testosterone cypionate mg/kg intramuscularly every weeks ( rabbits) on the development of oa and on proteoglycan (pg) synthesis by cartilage. animals were killed weeks post partial meniscectomy, and osteoarthritic lesions were noted. knee sections processed for histologic examination were stained with h and e and safranin- with fast green counterstain. cartilage metabolism in the groups was examined by in vitro measurement of ""so, incorporation into articular cartilage after hour incubation in dulbecco's modified eagle medium. medial and lateral components of femoral and tibia knee joint surfaces were studied separately. normal unoperated knees served as additional controls. frequency and severity of osteoarthritic lesions were the same in all groups. osteoarthritic and normal articular cartilage from estradiol-treated animals revealed statistically significant reduction in s s incorporation as compared to untreated animals. androgens had no significant effect on s incorporation. femoral s s , incorporation was uniformly greater than tibial , incorporation in all groups (p < . ). estradiol did not ameliorate nor testosterone worsen oa. both normal and osteoarthritic articular cartilage were susceptible to estradiol suppression of proteoglycan synthesis. the poor correlation between severity of oa and rate of pg synthesis may require reevaluation of the role of the latter in the oa disease process. variations in cartilage metabolism from different surfaces (femoral versus tibial) may relate to known differences in susceptibility of joints to development of oa. we have been analyzing antibody activities in sera from individuals working in various laboratories and in sera from a normal population. compared to the normal sample, the prevalence of elevated anti-dna activity was significantly greater in samples of sera from over personnel in systemic lupus erythematosus (sle) laboratories (p < . ), from laboratories involved with nucleic acids (p < . ), and from routine hospital laboratory personnel (p < . ). evidence that this anti-dna activity was due to gammaglobulin was obtained by the presence of y -d n a bound to igg in a radioautograph. in addition, differences were found in the prevalence of dna reactivity between the normal sample and the samples from the three laboratory groups when anti-human gammaglobulin was used as the precipitating agent. also, we have isolated the material from a serum containing high dna reactivity by a dna-cellulose affinity technique, and the fraction with dna binding activity contained only igg. lymphocytotoxic activity was also increased in the sera from sle laboratory personnel compared to the other laboratories and to the normal sample (p < . ), as previously reported (xiv international congress of rheumatology, abstract p. , ). immunoglobulin levels were analyzed by immunofluorescence. there was no significant difference in the igg and igm levels, but the mean iga level of the sle laboratory personnel was . f . mg/ml compared to . * . mg/ml in the normal sample ( p < . ). when sera from of the laboratory personnel with the highest anti-dna activities were compared to normal sera, additional abnormalities were found. not only was there a significant difference in the anti-dna activity (p < . ) and the iga levels ( p < . ), but also the mean igm level in the laboratory personnel ( . f . mg/ml) was significantly greater than in the normal sera ( . f . mg/ml)(p < . ). no difference in the mean igg levels was found. of additional specific antibody activities quantitated by radioimmunoassay, one of these (anti-bovine gammaglobulin) was elevated more frequently in the laboratory personnel than in the normals (p < . ). the data suggest that laboratory personnel tend to have an increased immune reactivity, particularly those working with sle sera. this condition might be due to laboratory exposure to a stimulus, causing an immune response that includes autoantibody production. in most cases, amyloid arthropathy (aa) has been associated with multiple myeloma or demonstrable paraproteinemia. various modes of presentation and laboratory features have been described. we have analyzed clinical, laboratory, and radiographic features of patients with aa seen over a -year period. none of these patients had identifiable diseases known to cause secondary amyloidosis. presenting symptoms were those of carpal tunnel syndrome ( : ) and/or swollen hands with stiffness ( : ) . periarticular tenderness of the hands and thickened palmar tendons were noted. pitting edema of the hands, at times massive, was noted in of . knee and elbow effusions were present in patients. sedimen-tation rates, rheumatoid factor, and antinuclear antibodies were normal or negative. serum and urine protein electrophoresis and immunoelectrophoresis failed to detect any paraprotein. joint fluids ( ) were noninflammatory except for one aspirated during an acute attack of pseudogout. radiographs showed degenerative changes ( : ) and chondrocalcinosis ( : ) . synovium obtained by open biopsy ( : ) revealed deposits of material which displayed metachromasia after crystal violet staining. in cases, electron microscopy was performed and revealed typical fibrils in synovium. deposits were localized to perivascular and subsynovial locations. attention is called to the presentation of aa as edema of the hands with or without median nerve compression. edematous hands and seronegative arthritis should raise the possibility of aa. these patients are also unusual in that aa was not associated with dysproteinemia and absence of a paraprotein should not discourage invasive measures to document aa. the associated chondrocalcinosis has been previously recognized in only one case report. chondrocalcinosis in of patients may be a chance occurrence in an elderly population (age range - ) or may cause aa by chronic local inflammation. antimalarial therapy for connective tissue disease has been limited by its potential retinal toxicity. the present study was undertaken to assess visual problems in patients treated with long-term hydroxychloroquine given in standard dosage. all patients who received both treatment and ophthalmologic evaluation a t albany medical college were included and consisted of patients treated for a t least year. each patient was examined a t baseline and then every months for visual acuity, central fields using a red test object, funduscopic abnormalities, accommodation, corneal deposits using a slit lamp, and keratoconjunctivitis by shirmer test. electro-oculograms (eog) were performed in patients who had received high total doses or had abnormal central fields suggesting toxicity. almost all patients received hydroxychloroquine in the maximal daily dose of mg. total dose ranged from to g (median ) and duration of treatment from to months (median ). diseases treated were rheumatoid arthritis in patients, systemic lupus erythematosus in , juvenile rheumatoid arthritis in , mixed connective tissue disease in , and other in . ophthalmologic toxicity was minimal. no patients were precluded from taking hydroxychloroquine at baseline evaluation. no corneal deposits or accommodation defects were found. three patients had abnormalities in central fields: paracentral scotomata and/or minor field restrictions. all had rheumatoid arthritis. toxicity occurred after total doses of , , and g. visual changes were completely reversible in the first patient who was the only one in the series with funduscopic changes. she has now received a total dose of g and has normal fundi, central fields, and eog. the second patient continues to have central field restriction but has had resolution of paracentral scotomata, a normal eog, and no visual complaints. the third had reversal of central field defects but had an abnormal eog when subsequently tested. we conclude that hydroxychloroquine in a dosage of mg/day is safe from significant ophthalmologic toxicity if followed by appropriate testing, and we find n o evidence from an association of increased toxicity with higher total dose or with the diagnosis of systemic lupus erythematosus. the metal-dependent neutral proteoglycanase, extracted from , grams of human articular cartilage, occurred in four electrophoretic forms. these were purified , -fold and produced single proteolytically active bands on disk electrophoresis. these forms were separated by preparative flat-bed isoelectric focusing and had approximate isoelectric points of . , . , . , and . . gel filtration and sds gel electrophoresis showed that they have an apparent molecular weight of - ,ooo and are composed of subunits of - , . gel filtration and dialysis indicated their tendency to disaggregate and reaggregate into monomers and dimers. their proteoglycanase activity passed through visking tubing and the passage continued on repeated dialysis with fresh buffer. this was prevented to a large extent by dialysis against zinc or cobalt ions. all the forms degraded the protein core of proteoglycan subunit optimally at ph . and were inhibited almost completely by addition of o-phenanthroline or passage through the chelating resin, chelex. however, they differed in their inhibition by edta, the most cationic forms being the least inhibited. also, the most cationic forms had n o activity on casein, histone, and the link proteins, indicating a relative specificity for degrading proteoglycan. these enzyme forms which are active at the ph in cartilage matrix may have an important role in proteoglycan degradation in osteoarthritis. bxsb mice (developed by dr. e. d. murphy, jackson laboratory) spontaneously evolve a lupus-like disorder similar to the disease of nzb/nzw (b/w) mice. however, in contrast to b/w mice, the bxsb disease is more severe in males who die of immune complex nephritis at a mean age of months. we have compared the formation of antibodies to dna in bxsb and b/w mice. the early appearance of autoimmunity in male bxsb mice is associated with a premature switch from igm to igg serum antibodies to dna. this is analogous t o the early switch from igm to igg antibodies in female b/w mice which is associated with accelerated disease and impending death. we have also measured the spontaneous synthesis of antibodies to dna by spleen cells cultured for hours. culture supernatants contain immunoglobulin which binds dna specifically as determined by radioimmunoprecipitation. two month old male bxsb mice produce more antibody to d n a in culture than d o age-matched female mice of bxsb, b/w, balb/c, or c b / strains. anti-dna production in culture increases with age in b/w mice. older female b/w mice subjected to prepubertal castration and treated with androgen produce significantly less anti-dna in culture as compared to sham or estrogen-treated controls (p < . ). the effect of hormone treatment on bxsb mice is currently under investigation. these results suggest that the male-dominant disease of bxsb mice is reflected in early synthesis of antibodies t o d n a which is apparent both in vivo and in vitro. female the rheumatoid nodule : clinical significance although the subcutaneous nodule (scn) is the extraarticular hallmark of rheumatoid arthritis (ra), its clinical significance has not been established. it was our thesis that the presence of scn would serve to identify the subset(s) of rheumatoid patients with systemic and highly immunoreactive disease. all patients with definite ( ) and classic ( ) rheumatoid arthritis who had an initial unit admission during the interval january through june were selected for study. primary reasons for admission included active synovitis ( %), orthopedic surgery ( %), and a miscellany ( %) of therapeutic complications and intercurrent illness. of the patients, ( %) had scn on admission or by documented history. medical record analysis per protocol revealed patients with and without scn were comparable in terms of demography, duration, and activity of ra. similarly, systemic features did not differ significantly between two groups, as shown in the table. with regard to sero-reactivity, patients with scn and the anodular group were also alike in terms of the presence of rheumatoid factors ( % versus %), antinuclear factors ( % versus %), and hypocomplementemia ( % versus %). there was a tendency only for those with scn to have higher latex test titers. thus, contrary to expectations, subcutaneous nodules cannot be relied upon to screen out those patients with rheumatoid variants and systemic disease. twenty-four patients ( women, men, mean age mg daily, versus placebo for months, while continuing years) with definite rheumatoid arthritis were entered into a baseline non-steroidal antiinflammatory therapy and/or preddouble-blind study using the immunomodulator levamisole, nisone up to mg/day. assessment criteria included articular index, grip strength, feet walking time, duration of morning stiffness, subjective pain relief, sedimentation rate, and latex fixation titers. to date, patients have completed the initial phase. eight o f on levamisole improved while the remaining patient, who did not appear to respond, flared after drug discontinuation. two patients on levamisole were excluded because of skin rash. four of on placebo appeared to improve but not to the degree seen with levamisole. the mean changes in articular index, number of swollen joints, and duration of morning stiffness were statistically significant a t months for levamisole treated patients but not for placebo (p < . ). subjective pain scale responses were significantly better among levamisole versus placebo, but n o differences were observed between the groups in grip strength or feet walking time. five levamisole treated patients converted to seronegativity after months. sedimentation rates, however, remained stable in both groups: skin rashes developed in patients on levamisole, and were discontinued; in , rash was unrelated, and continued on a lower dose. of interest, of on placebo had rashes. preliminary investigations of delayed skin hypersensitivity, lymphocyte responsiveness, phagocytic function, and cutaneous inflammatory responses failed to show any correlation with clinical responsiveness. levamisole is a potentially therapeutic agent in rheumatoid arthritis, but its mode of action is yet to be determined. since synovial fluid cells in patients with systemic lupus erythematosus (sle) have not previously been examined by electron microscope, we have studied sle joint effusions with emphasis on the occurrence of le cells and tubuloreticular structures (trs) and on correlations with clinical and light microscopic findings. all patients fulfilled at least ara preliminary criteria for sle and had antinuclear antibodies in their serum. three patients had drug-induced sle. synovial fluid volumes varied from . - cc; leukocyte counts ranged from - , but only were over , . polymorphonuclear leukocytes predominated in effusions including the one with , cells. cultures were all negative. monocytes and large sudan positive macrophages were prominent. joint fluid le cells were found in patients while had extracellular hematoxylin bodies, and had a variety of other smaller eosinophilic, hematoxyphilic, or cellular inclusions. le cells were identified by electron microscope in patients. the major and often sole visible constituent of the inclusion was a clump of short filaments about nm in diameter. these lay in vacuoles which also contained acid phosphatase. these filaments which appear to be products of nuclear chromatin also were seen extracellularly surrounding some cells and in smaller vacuoles. small acid phosphatase positive vacuoles also contained cell cytoplasmic debris, intact nucleii, erythrocyte fragments, and large amounts of finely granular protein-like material. the finely granular material was shown t o contain igg by immunoperoxidase electron microscope staining. t r s were found in synovial fluids and were predominantly in mononuclear cells with dense bodies and rough endoplasmic reticulum. they were infrequent in small lymphocytes or transformed lymphocytes with polyribosomes. two l e cells had trs. buffy coat blood cells contained trs in the cases studied. synovial fluid cell trs were seen in of inflammatory and noninflammatory synovial fluid controls but no control joint fluids had the filamentous le inclusions seen by electron microscope. n o correlation of any synovial fluid finding with therapy, effusion duration, or disease severity has been found. synovial fluids in sle contain trs, many phagocytic cells, and can have higher leukocyte counts and pmn percentages than often appreciated. le inclusions in joint fluid are composed predominantly of filaments derived from nuclear chromatin. the sera of patients fulfilling the ara criteria for systemic lupus erythematosus (sle) were studied by immunodiffusion and the modified farr technique for the presence of antibodies to the following antigens: ss-a, ss-b, rana (rap antigen), rnp, sm, scl- , and ds-dna. the incidence of antibodies to ss-b, rnp, sm, and ds-dna was similar to those previously reported. the incidence of anti-scl-l antibody was %, a finding which has not been reported pre-viously. the incidence of r a p ( %) was higher than the % previously reported. the incidence of anti-ss-a antibody was %. this antibody was found in high frequency in primary sjbgren's syndrome ( %) in the past but was not found in the small number of sle patients studied at that time. in this study where a large number of sle patients were studied and anti-ss-a antibody was present, only % had any evidence of keratoconjunctivitis sicca based on schirmer's tests. this indicates that not only is anti-ss-a an antibody "marker" for primary sjogren's syndrome, but it is relatively common in sle as well and furthermore, can exist in sle in the absence of keratoconjunctivitis sicca. serial studies were also performed on some patients. titers of anti-ss-a antibody were determined. these varied from neat to and correlated with disease activity. anti-ss-a often paralleled anti-ds-dna levels; however, anti-ss-a in some cases was an earlier predictor of clinical flares. thus, anti-ss-a antibody may be helpful not only in the management of sle patients, but with further investigation, aid in determining the pathogenesis of this disease. the phenanthridine dye ethidium bromide (eb) intercalates with native double-stranded d n a (dsdna) resulting in an enhancement of fluorescence when assayed fluorometrically. contamination of dsdna preparations by singlestranded dna (ssdna) does not affect the assay because there is no fluorescence enhancement when ssdna is added to eb. igg purified by deae column chromatography from the sera of normal volunteers: systemic lupus erythematosus (sle) sera with d n a binding activity as measured by the millipore filter (mpf) radioimmunoassay, and sle sera without d n a binding activity were tested for their ability to compete with eb for binding to dsdna. in of of the sle with mpf-dna binding activity, there was greater than inhibition of the fluorescence normally shown when eb binds dsdna. in contrast, of control igg preparations and of of the sle igg from non-dna binders had any such effect. furthermore, the decrease in fluorescence due to the inhibition of eb binding to dsdna was linear with in-creasing amounts of igg, thus allowing direct quantitation of anti-dna activity. since the binding constant of eb is known to be > x -'m, this assay measures high avidity antibodies and is not affected by non-specific, low-avidity d n a binding molecules. the specificity of anti-dna antibodies in the eb assay was shown when absorption with excess nucleoside monophosphates did not alter the inhibition of eb binding in unabsorbed samples. however, the synthetic polynucleotides poly da, poly dt, poly dc, poly da:dt, and poly dg:dc were able t o absorb dna binding activity. therefore, the eb assay has demonstrated several features which make it an important adjunct in studying the specificity and binding characteristics of anti-dna antibodies. in addition, the assay is inexpensive, is easily and quickly performed, and it does not require the use of radiolabeled substrates making it practical for further development in investigative and clinical use. degradation of collagen in tendon, bone, and cartilage by collagenase is a significant factor contributing to the morbidity of the inflammatory arthritides. although considerable information is available as to the effect of drugs and mediators of inflammation on collagenase production and activity, the mechanisms during aging in the collagen fibril causing increased resistance to collagenase degradation are poorly understood. in this study, the effect of collagen cross-linking on collagenolysis was measured with an in vitro model system consisting of purified chick calvarium collagen fibrils and lysyl oxidase, the cross-linking enzyme. chick calvatia collagen fibrils were cross-linked by incubation with lysyl oxidase for varying time. controls consisted of collagen incubated without enzyme or with lysyl oxidase plus / -aminopropionitrile, an irreversible inhibitor. the fibrils were subsequently measured for nascent cross-link content or incubated with purified rheumatoid synovial collagenase, and the rate of collagenolysis was measured. after synthesis of approximately . schiff base cross-links per collagen molecule, a ten-fold resistance to collagenase digestion was observed. inhibition of collagenase by edta prevented digestion. synthesis of collagen fibrils with higher cross-link content resulted in further resistance. these results demonstrate that the increased resistance of collagen to collagenolysis observed during fibril maturation in vivo is due to synthesis of native, unreduced schiff base cross-links. the cross-link content at which resistance to degradation develops is significantly less than that which affects fibril tensile strength in vivo. this suggests that resistance to collagenase is the earliest physiological effect of cross-linking in vivo. the rate of cross-link synthesis may be a significant factor regulating the rate of net collagen deposition in normal and pathologic states. vascular abnormalities have been shown to play an important pathogenic role in progressive systemic sclerosis (pss), especially in the group of patients who develop rapidly progressive kidney disease. as part of a study of the kidney in pss, we have also performed comprehensive investigations of coagulation parameters in order to evaluate the possibilities that: ) coagulopathy occurs in these patients; and ) coagulation abnormalities may be related to the development of renal disease in pss. seven patients with pss and normal renal function, as judged by normal blood pressure, creatinine clearance, and absence of proteinuria, were enrolled in the study. coagulation parameters studied included prothrombin and partial thromboplastin times, fibrin split products, fibrin monomer, sonoclot@ (which reflects fibrin monomer formation in recalcified whole blood), thromboelastograph (which reflects fibrin polymer formation), and platelet aggregometry. in addition, each patient underwent percutaneous renal biopsy. patients with pss showed platelet hyperreactivity t o collagen but not to other agents ( % aggregation versus control of . % p < . ). the following abnormalities, indicative of a hypercoagulable state, were found: increased rate of clotting by thromboelastograph in patients; increased rate of clotting by sonoclot in patients; and premature onset of clotting by thromboelastograph in patients. the patient with the most hypercoagulable profile has subsequently developed mild proteinuria. five patients had renal biopsy findings on light microscopy suggestive of pss kidney disease, including vascular intimal fibrosis, sclerosis, and fibrinoid necrosis. four of these were hypercoagulable by one or more tests; both patients with normal biopsies had normal coagulation profiles. these data suggest that: ) platelet-collagen interactions may be abnormal in pss; ) certain patients with pss manifest laboratory evidence of hypercoagulability; and ) hypercoagulability appears to correlate with renal biopsy abnormalities in pss patients with normal renal function. normal human mononuclear cells spontaneously lyse in vitro targets derived from cell lines of tumor origin. the natural killing of k- , a tumor cell line derived from a patient with chronic myelogenous leukemia, varies in patients with systemic lupus erythematosus (sle) ( % f %) and controls ( % f %). t o evaluate the possible role of serum factors, normal peripheral blood mononuclear cells were incubated with sle sera, normal control sera, or media alone in the natural killing assay. . x l(p ficoll-hypaque purified mononuclear cells after pretreatment with sera or media were incubated for hours with x ' s chromium ("cr) labeled k- cells. cytotoxicity was measured by the release of into the supernatant. the mean cr release in the presence of normal human sera was % f % of that with media alone. in the presence of sle sera, mean cr release was % & % of that with media alone. the magnitude of natural killing suppression by sle sera did not correlate with complement levels, ana titer, or d n a binding. the natural killing suppression by an individual sle serum did not correlate with its inhibition of the antibody dependent cell mediated cytotoxicity of chicken red cells. the factors in sle sera responsible for suppression of natural killings are non-dialyzable, precipitable with % (nh,),so,, and removable by absorption with anti-ig antibody. the factors are excluded by a g- column. the major portion of the suppressive activity was found in fractions of igm or greater density on sucrose gradients. these findings are compatible with factors in sle sera, most likely immune complexes or antibodies, capable of inhibiting the natural killing of tumor cell lines. these factors may prevent cell mediated cytotoxic destruction of malignant cells in vivo and may explain the increased association of sle and malignancy noted by canoso et af. (arthritis rheum : , ). full evaluation of any hyperuricemic patient requires these problems can be largely avoided by assessing uric acid assessment of uric acid excretion, but the standard -hour excretion per ml of glomerular filtrate. this simple, physiurine method presents many problems. ill-timed and inologically sound parameter is obtainable from single, untimed complete collections may combine with bacterial con-urine specimens by multiplying urinary uric acid by plasma tamination and crystal precipitation to cause significant errors. creatinine and dividing by urinary creatinine (all in mg/ ml). to minimize possible diurnal effects on uric acid excretion, all samples were taken in the morning. to evaluate the precision of the method, normal men collected two -hour urines. spot urine specimens with concurrent serum samples were also obtained on two separate mornings. uric acid was measured spectrophotometrically, and creatinine was measured by standard autoanalyzer technique. the coefficient of variation (sd/x) was i% for paired, quantitative -hour urine uric acid determinations, while spot, mid-morning assessments of uric acid excretion per ml of glomerular filtrate had a more satisfactory coefficient of %. since most laboratories do not employ the enzymatic spectrophotometric method, urine uric acid determinations were also performed by a colorimetric, autoanalyzer technique. these values correlated well (r = . ) with the more specific spectrophotometric findings, but were an average of % higher. samples from normal, adult men, the mean urinary excretion of uric acid in mid-morning was . f . mg/ ml (sd), while gouty men (studied between attacks and off hypouricemic drugs) had a mean of . f . mg/ ml. included in the latter group were significant overexcretors with values of . , . , . , . , and . mg of uric acid per ml of glornerular filtrate. we believe that this assessment of mid-morning serum and urine samples effectively identifies overexcretors of uric acid. in addition, it is more convenient, more physiological, and more precise than the conventional -hour method. (supported by nih grant am .) in mixed connective tissue disease (mctd), a newly described rheumatic syndrome, no comprehensive histopathologic descriptions exist. we have followed mctd children over a range of - years, (mean years); have died. three autopsies and renal and muscle biopsies were reviewed. among adults with mctd, tissues from autopsies, and kidney, muscle, and lung biopsies were reviewed. in children, proliferative vascular lesions, with intimal and medial thickening and luminal narrowing but without fibrinoid or inflammatory change, affected large vessels (aorta, coronary, renal), and small arterioles of many organs. inflammatory infiltration, predominantly plasmacytic, was marked in skeletal muscle : , liver : , salivary glands : , intestine : , and heart : . distinctive lesions of esophagus (atrophy of inner muscle layer), thymus (hyperplasia), kidney (membranous change), liver, lung, and salivary glands differed significantly from those expected in childhood systemic lupus erythematosus, polyarteritis, or scleroderma. many of these organs were not clinically involved during life. histologic estimates of numbers of t and b lymphocytes in spleen and lymph nodes, and degree of plasmacytosis (with hyperglobulinemia), differ from systemic lupus erythematosus and juvenile rheumatoid arthritis. in adults with mctd, muscle changes included: diffuse inflammatory infiltrate : (perior endomesial, and perior intravascular). by atp-ase, : i i had type i fiber predominance. on muscle immunofluorescence, : had vascular, sarcolemmal basement membrane, or granular fiber staining, with igg or igm. in kidneys there were included: mesangial proliferation , focal-local change , membranous , membrano-proliferative i , proliferative vessels , normal . lung tissue ( ) revealed: vascular proliferation , vascular medial hypertrophy , and interstitial fibrosis . in mctd, children and adults have similar lesions: inflammatory lesions may predominate early, but can occur late. immunofluorescent data suggest an immune basis for injury; the late predominance of proliferative vascular lesions suggests that vascular sclerosis is a serious complication. the findings of many significant histologic lesions, without clinical signs or symptoms, suggest that features of this multi-system disease evolve slowly, and that the full spectrum of mctd is not yet known. with ra and normal subjects. after isolation of the lymphocytes by isopycnic sedimentation, the cells were placed in tissue culture and half were infected with ebv. every days for month the supernatant fluid was removed from each culture and fresh medium was added. total igm and igm-rf secreted into the supernatant were measured by solid phase radioimmunoassay. independently the cells were examined for transformation and numerically graded. all infected cultures produced igm-rf, which correlated with a high grade of transformation. the amount of rheumatoid factor (rf) produced by ebv infected normal, but not rheumatoid, lympho- the incubation of pmns with a chemotactic factor (cf), in the absence of a gradient, prevents the cells from responding with directional migration when, after washing, they are challenged with a gradient of the same or a different chemotactic factor (deactivation and cross-deactivation). structurally unrelated cfs, some shown to bind to distinct cell receptors, can cross-deactivate, which suggests that deactivation is not due to receptor blockade. we propose that deactivation is the result of generalized microtubule assembly induced by c f incubated with the cells in the absence of a gradient, thus rendering the cells incapable of responding to a c f gradient with distinctive localized assembly, a proposed requirement of normal chemotaxis. if this scheme is correct, the simultaneous preincubation of pmns with suitable concentrations of colchicine, a microtubule disrupting agent, and a c f should protect the cell against deactivation and colchicine-induced suppression of chemotaxis. human neutrophils were preincubated, ' at oc, with colchicine to -'m); the chemotactic factors gly-his-gly (iopg) or crystalinduced chemotactic factor (ccf) pg alone; colchicine and either chemotactis factor; or hanks. cells were washed and tested for chemotactic response against the c f using a radioassay that utilizes y r labeled neutrophils. preincubation of cells with either chemotactic factor or colchicine alone resulted in a dose-dependent inhibition of chemotaxis. when cells were preincubated with both chemotactic factor (gly-his-gly iopg or ccf pg) and suitable concentrations of colchicine ( or io-o), a reversal of the inhibition of chemotaxis was noted. deactivation reappeared when the balanced ratio between colchicine and c f was altered. preincubation of c c f with colchicine had no direct effect on its chemotactic activity, and colchicine ( - m) did not alter the specific binding of radiolabeled c c f to neutrophils. additionally, both c c f and gly-his-gly induced microtubule assembly by electron microscopy. functional evidence is presented with two distinct chemotactic factors, which suggests that the basis for deactivation is overpolymerization of microtubules that prevents the pmns from responding to a chemotactic gradient with directional migration. fever is a common occurrence and frequent reason for hospitalization of patients with systemic lupus erythematosus (sle). to assess the frequency, causes, and clinical and laboratory characteristics of febrile episodes in hospitalized patients with sle, the medical records of admissions of sle patients during the year interval to were reviewed. eighty-three febrile episodes, defined as a n oral temperature oc and one blood culture drawn for evaluation of fever, occurred in patients. the febrile episodes were classified: group a-clinically active sle only ( ). group b-documented infection (l ), and group c-miscellaneous ( ). clinically active sle accompanied episodes in b and in c. treatment a t the time fever developed included steroids in , , and % and cytotoxic agents in , , and % of episodes in a, b, and c, respectively. infectious causes of fever were bacterial septicemia ( ), localized bacterial infections ( ), herpes zoster ( ), and miliary tuberculosis ( ). miscellaneous causes were procedure-related fever ( ). drug fever ( ), addisonian crisis (i), myocardial infarction (i), and unidentified ( ). the initial clinical impression was correct in of infectious episodes. two episodes of bacterial septicemia were unexpected; one occurred in a patient with concommitantly active sle. the clinical impression was correct in of episodes in a; infection ( ), acute appendicitis (i), and drug fever ( ) were suspected in the others. comparing a and b, patients' age, disease duration, and fever patterns were similar. shaking chills were more frequent with infection (p < . ) but were present in % of a. laboratory studies helpful in identifying patients with infection were leukocytosis > x ip/mm (p < . ), neutrophilia > x ip/mm (p < . ) and normal dna binding (p < , ). four deaths occurred despite appropriate therapy: in a with necrotizing lupus pneumonitis, and in b with gram negative sepsis, of whom had active sle. in summary, infection accounted for % of febrile episodes in hospitalized patients with sle. bacterial septicemia was found in % and was associated with a high mortality. the initial clinical impression was usually correct and the wbc count, absolute neutrophil count, and dna binding were helpful in distinguishing between infection and active sle. renal involvement in progressive systemic sclerosis (pss) is the most devastating form of the disease with one year mortality close to % unless hemodialysis or transplantation is successful. at present, there are no reliable predictive factors which allow us to separate those who will eventually develop renal disease from those who will not. because of this, we have studied patients with pss who were normotensive and had normal renal function (mean serum creatinine . mg%, mean creatinine clearance cc/min, mean protein excretion mg/ hours) and normal urinalysis. studies included percutaneous renal biopsies, evaluation of the renin-angiotensin system, and a cold pressor test to try to determine renal vascular reactivity. five of renal biopsies demonstrated distinct vascular lesions on light microscopy with intimal fibrosis in , hyaline intimal sclerosis in , and arteriolar fibrinoid necrosis in . electron microscopy of the vessels showed changes similar to those on light microscopy. in addition, wrinkling of the glomerular basement membrane (gbm) with expansion of the mesangium by gbm-like material was seen in of biopsies. although nonspecific, these changes are suggestive of ischemia in the kidney and are compatible with the vascular changes of pss. immunofluorescence studies revealed c in vessels in all biopsies. plasma renin activity (ppa) was performed in of patients. of the patients with abnormal biopsies, had elevated plasma renins ( . ng/ml/hr) at the time of biopsy and the fifth patient has subsequently developed significant elevation. the patients with normal biopsies had normal plasma renins ( . ng/ml/hr). cold pressor testing with pra determination resulted in a mean maximal increase of . ng/ ml/hr in those with abnormal biopsies, . ng/ml/hr in those with normal biopsies, and . ng/ml/hr in control subjects. these findings indicate that renal histologic vascular involvement may precede the onset of clinical renal disease in pss and correlates well with pra elevations and increased responsiveness of the renal vasculature. followup of these patients will determine the clinical significance of these findings. ninety-six patients with classic or definite rheumatoid arthritis were consecutively screened for the presence of early ocular manifestation of disease. each patient was followed for an average of . months and received an average of ophthalmologic exams at an average of month intervals. none of the patients demonstrated ocular lesions definitely attributable to rheumatoid arthritis. eighteen patients developed new ocular pathology consisting of chronic blepharitis, chronic iritis, corneal subepithelial defects, keratitic precipitates, keratitis, corneal abrasion, and abnormal tear film. twenty-one patients of this group and normal adult controls were screened for keratoconjunctivitis sicca. slit lamp examination, rose bengal staining, shirmer's testing, and tear lysozyme concentration were measured in each patient. tear lysozyme concentration was indirectly determined spectrophotometrically by measur-ing lysis of the dried cell walls of micrococcus lysodeikticus. patients with rheumatoid arthritis demonstrated abnormal rose bengal staining (rheumatoid arthritis, patients; control, none), significant decreased tear production (rheumatoid arthritis, . f . mm wetting shirmer strip; control, . f . mm wetting schirmer strip) p < . , and markedly increased tear lysozyme concentration (rheumatoid arthritis, . f . pg/ml; controls . f . pg/ml) p < . . thus, unlike patients with severe sjagrens syndrome who have low tear lysozyme concentrations, patients with rheumatoid arthritis demonstrate significant increases in tear lysozyme levels. this new finding, as yet unexplained, may represent the earliest lesion in the development of keratoconjunctivitis sicca in patients with rheumatoid arthritis. nzb/nzw mice develop autoimmune disease similar t o that of humans with lupus systemic erythematosus. treatment with cyclophosphamide (cp) protects against the disease but often is associated with a high incidence of tumors. tumors resulting from direct chemical carcinogenic activity increase with rising cumulative dose and time after exposure and are of diverse types. immunosuppression itself is reported to facilitate tumor growth or, perhaps, oncogene expression and has been primarily associated with sustained immunosuppression, predominantly with tumors of reticuloendothelial (re) origin. we compared tumor development in nzb/nzw mice given different cp regimens (see table) . reticuloendothelial tumors increased with daily dose but did not correlate with cumulative dose or duration of treatment. smaller numbers of other (non-re) types of neoplasms occurred in all groups. our results ) suggest that re tumor development in autoimmune disease is more dependent on continuous, high doses of cytotoxic drug than on cumulative dose or duration of treatment and ) are compatible with the possibility that re tumors result from immunosuppression or oncogene expression. no. the success of colchicine in the treatment of acute gouty arthritis is well established. however, the usefulness of this drug in the therapy of the pseudogout syndrome has been less enthusiastically regarded. in general, assessments of such therapy refer to lower success rates and somewhat inconsistent results. nevertheless the use of other antiinflammatory drugs to treat acute pseudogout is sometimes militated against by the presence of associated conditions commonly seen in the age group of these patients, including congestive heart failure, gastrointestinal disease, or neurological deficits. we have treated consecutive patients with the acute arthritis of pseudogout with a standard regimen of colchicine by the intravenous (iv) route. there were males and females, ages to , and all presented with acute mono-or oligoarticular arthritis. in all cases typical calcium pyrophosphate dihydrate (cppd) crystals were identified by red-compensated polarized light microscopy of the synovial fluid. none of the patients had elevated serum uric acid, except which was on diuretics, and synovial urate crystals were absent. synovial fluid white counts varied from to / mma with - % polymorphonuclear leukocytes; of the had radiologic evidence of chondrocalcinosis. all patients were treated within hours of the onset of the acute arthritis. colchicine was usually given at a dose of . mgm iv over a period of minutes followed by . mg iv every hours for the next - hours. total iv colchicine dosage varied from to . mgm. an excellent response occurred in - hours in of patients and in - hours in the other , with complete resolution of inflammation in all . we conclude that ) iv colchicine may provide effective and consistent therapy for the acute arthritis of pseudogout; and ) because of this, prompt response of an acute arthritis to iv colchicine remains an insufficient clinical criterion for the diagnosis of gout. the high pyrophosphate (ppi) concentrations observed in osteoarthritis (oa) and chondrocalcinosis (cc) synovial fluid (arthritis rheum : , ) suggest that ppi may be a precursory ion involved in human articular mineral formation, whether in midzone sites in cc-cappi deposition-or in "tidemark" cartilage in oa. why predominantly hydroxyapatite and often some cappi (bjelle, personal communication) are seen in oa, and cappi is deposited in cc, might hypothetically depend in part on moderate and severe ppi hydrolase(s) deficiencies. such deficiencies are postulated to be associated with the calcifying subcellular apparatus in oa and cc articular cartilage, respectively. this postulated ppi deficiency would be relative to normal calcifying growth cartilage in which ppi in mineral is present in only trace amounts (wuthier et al., calc tiss res , ). to examine this hypothesis, articular cartilage from patients with oa ( male, female, age - ), with cc ( male, female, age - ), and normal controls was assessed for various phosphohydrolase activities. cartilage was sliced in a cryostat, homogenized, and extracted in triton x- . the extract was centrifuged and the supernatant, once dialyzed, was passed through a de- column and enzyme fractions were eluted according to prevjously described techniques. although total protein and dna content were about equal from all cartilage samples, alkaline phosphatase activity was found in the following ratios: normal: cc: oa = i : : . in all samples, peaks of alkaline phosphatase activity were eluted similar to the findings of arsenis et al. in calf growth cartilage (calc tiss res , ). uniquely, with cc some of the total activity ( %) was found in the void volume of both peaks. characterization of alkaline phosphatase using various metals, inhibitors, and substrates was similar for all samples tested. however, analysis of the ratios of ppiase: alkaline phosphatase for growth cartilage (arsenis, above) versus oa and cc samples ranged from : to : respectively. in conclusion, the high ppiase to alkaline phosphatase ratio in growth cartilage compared to this ratio in cc and oa supports the view that reduced ppiase activity might play a role in ) the probable elaboration of ppi into synovial fluid in oa and cc; ) the in vitro elaboration from articular cartilage of ppi into eagle's medium in patients with oa and cc (j clin invest : , ); and ) provision of ppi ion for variable cappi crystal deposition. despite the prominence of neuropsychiatric features in systemic lupus erythematosus (sle), no satisfactory method exists for the diagnosis and monitoring of central nervous system (cns) involvement. this study describes the application of a new technique for studying both cerebral metabolism and blood flow in sle patients by using inhaled molecular -oxygen and -oxygen labeled carbon dioxide. tracer amounts of -oxygen are inhaled, and after a period of equilibration, the brain is scanned with a gammacamera. the image produced represents cerebral metabolism. the procedure is repeated using carbon -dioxide, and the resultant image represents cerebral blood flow. twenty-eight scans were performed on sle patients who had been classified as having clinically definite cns disease ( ), clinically probable cns disease ( ), or no clinical evidence of cns disease ( ). the scans, which were reported blind, were classified as normal (showing a full pattern of both cerebral blood flow and metabolism), as showing a major abnormality, or as showing a minor abnormality. the results are shown in the table. scan abnormalities were seen in of the studies, usually afecting several cortical areas. in patients in whom multiple recordings were made, improved -oxygen scan appearances correlated with clinical improvement. -oxygen brain scanning appears t o offer a highly sensitive non-invasive technique for the identification and study of cerebral involvement in sle. neutrophils stimulated at sites of inflammation release lysosomal enzymes. experimentally, a variety of agents are capable of stimulating neutrophils to release their lysosomal enzymes. neutrophil membranes contain fc receptors for igg, and thereby possess surface bound igg. in these studies we examined the hypothesis that specific antibodies could combine with surface bound igg, perturb the cell membrane, and provoke inflammatory responses of neutrophils. antisera were raised to immunoglobulins g , m, a, d, e, and isolated y chains, fc and f(ab'), of igg. these were rendered monospecific by solid phase immunoabsorbents. isolated normal human peripheral blood neutrophils were incubated with specific antisera and the release of lysosomal / -glucuronidase, a-mannosidase, and lysozyme were measured. non-lethal release of lysosomal enzymes was observed with antisera to whole igg, iga and t o y and fc of igg. the igg fraction of anti-igg antiserum also stimulated non-lethal release, thereby suggesting that the response was not complement-mediated. specific igg and iga receptors were detected on neutrophils using immunofluorescent labeled antibodies. n o such receptors were detected for igm and igd. these results indicate that inflammatory responses of neutrophils can be triggered directly by anti-immunoglobulins, and suggest one mechanism for initiation of the inflammatory response in patients with diseases characterized by the presence of antibodies to igg and other immunoglobulins. incubation of polymorphonuclear leukocytes (pmn) with immune complexes trapped in micropore or collagen membranes results in the phenomenon of "frustrated phagocytosis," that is, enhanced release of lysosomal hydrolases. w e have previously shown immune complexes irreversibly trapped in joint collagenous tissues in antigen-induced rabbit experimental arthritis and in rheumatoid arthritis. the present experiments were designed t o investigate in vitro interactions between pmn and joint collagenous tissues obtained from rabbit joints with experimental arthritis or control tissues from saline or monosodium urate crystal-injected joints. experimental and control articular cartilage samples and menisci obtained from such animals were incubated for hour with normal pmn isolated from rabbit peritoneal exudates or blood. after fixation, the tissues were examined by electron microscopy. fresh cartilage and menisci from arthritic joints showed only few damaged pmn near the articular surface. after incubation with pmn, large numbers of pmn attached to the articular surface were seen. in areas of superficial erosion, the pmn invaded the tissue several cell diameters below the lamina splendens. degranulated pmn were observed with immunoelectron microscopy in scattered areas to phagocytose amorphous material containing rabbit ig. following addition of pmn t o control tissues, only a few pmn became attached to the articular surface. in monosodium urate injected joints, after incubation with pmn, these cells were found attached to the surface in moderate numbers, but no invasion into the tissues was seen. these studies indicate that immune complexes trapped in joint collagenous tissues may induce the phenomenon of "frustrated phagocytosis" leading to enhanced release of lysosomal hydrolases. similar studies using rheumatoid joint tissues are in progress. (suppported by nih grant am .) half of the girls. intervals between onset of symptoms and diagnosis ranged between month and years (median months). twenty-eight children were white, black, native american, asian, and of other races. most frequent presenting findings were fever and malaise, musculoskeletal complaints, skin rash, and renal disease. unusual presentations included cholecystitis, isolated nephrotic syndrome, and chorea. three patients presented with isolated thrombocytopenia. serious pulmonary manifestations occurred in patients; died within months of disease onset. clinical evidence of nephritis occurred in of boys and of girls. thirty-six patients had or more renal biopsies. three patients with normal urinalyses had histologic nephritis by biopsy. therapy consisted of prednisone ( of patients) and either azathioprine or chlorambucil ( of patients). since therapy has been geared to normalizing serum hemolytic complement values as well as controlling clinical manifestations of disease. chronic renal failure developed in patients; only had had adequate therapy early in disease. one renal failure patient received a renal transplant, and are on hemodialysis. six girls and boys have died ( ' ). seven patients had active lupus at time of death with severe uncontrolled multisystem lupus ( ), pulmonary lupus ( ), central nervous system lupus (l), and renal failure ( ). one patient with active sle also had a myocardial infarction (age ) as a contributing cause of death. two patients died while in clinical remission, of a myocardial infarction (age ) and of clostridial sepsis with hemolysis. infection played a major role in deaths (fungal brain abscess, acute staphylococcal endocarditis, disseminated herpes infection, clostridial sepsis). thirty-three patients continue to have active sle requiring treatment; are in remission and are not on medications; i patient was unavailable for followup in . in patients with systemic lupus erythematosus (sle), abnormal unfractionated lymphocyte responses to phytohemagglutinin (pha) can be corrected by removal of adherent mononuclear (m) cells (arthritis rheum , ) . the present study demonstrates that this abnormal response occurs when ) autologous adherent mononuclear cells or ) cell-free supernatant fluids from autologous and allogeneic adherent cells are added to t-cell cultures, and that ) suppression does not occur when adherent cells are pre-treated with indomethacin. ficoll-hypaque (fh) separated peripheral blood mononuclear cells from normals and from patients with sle were further fractionated into t cells by passage over an antihuman f(ab), column ( % e-rosettes) and m cells by plating on glass ( % peroxidase-staining). cell number was constant in all experiments. supernatants from normal controls and from sle patients were obtained after hour culture of fh, t, and m cells in rpmi containing ab serum. normal and sle t cells were tested for pha response in the presence and absence of autologous and allogeneic m cells or cell-free su-pernatants from fh, t, or m-cells. autologous but not allogeneic m cells suppress t cell responses. cell-free supernatants derived from m cells of consecutive experiments with sle patients caused an average % suppression of normal t-cell response to pha (mean peak cpm relative to control). suppression was not due to media exhaustion. supernatants from normal m cells caused an average % suppression. supernatants from both normal and sle t cells did not suppress either autologous or allogeneic t-cell responses. when m cells of patients with sle were cultured in the presence of pg indomethacin/ml, the obtained supernatant was not inhibitory. the data indicate that poor in vitro unfractionated lymphocyte response to pha in patients with sle is mediated by a soluble indomethacin-sensitive product derived from the adherent mononuclear cells, possibly prostaglandin. since supernatants from normal adherent cells were also inhibitory when m-cell concentration was held constant, it is likely that the difference between sle and normal lymphocyte responses is quantitative rather than qualitative. renal crisis in scleroderma, with malignant hypertension and rapidly progressive renal failure, is perhaps the most ominous of clinical syndromes in the rheumatic diseases. survival with medical management has not been reported, although a few patients have survived with renal dialysis or transplantation. we report patients surviving scleroderma kidney with medical management alone for periods of , , and years. patient with malignant hypertension in the fourth year of his classic systemic sclerosis was hospitalized with blood pressure of / , generalized seizures, visual acuity decrease to virtual blindness, and striking grade hypertensive retinopathy. creatinine rose to a high of . . extraordinarily vigorous antihypertensive treatment with massive doses of drugs reduced blood pressure to low normal ranges. renal function years later is stable with a creatinine of . mg . patient developed her malignant hypertension after years of classic scleroderma, with blood pressure / , grade hypertensive retinopathy, generalized seizures, a creatinine of . , and urine protein of gms per hours. plasma renin was ng/dl. aggressive antihypertensive treatment with drugs reduced blood pressure to the low normal range, and over the following years renal function has improved to a creatinine of . . patient developed malignant hypertension after several years of classic scleroderma. blood pressure was / , creatinie rose to . , hypertensive grade eye changes were noted, and renal biopsy confirmed afferent arteriolar necrosis. hemorrhage following renal biopsy resulted in transient hypotension, with subsequent control of blood pressure in the low normal range with combination antihypertensive drug therapy. her renal function remains stable with a creatinine of . mg% after years. skin lesions improved following hypertensive control in of these patients. the common denominator in these successfully managed patients appeared to be aggressive and determined reduction of blood pressure to low normal ranges, with later reliance upon propranolol in of the patients. the percentage of patients who will respond to such management is unknown, but aggressive early treatment of hypertension appears indicated. the multi-system involvement and varied clinical presentation of systemic lupus erythematous (sle) make its treatment difficult and often controversial. to determine present clinical practices, rheumatologists and nephrologists, selected randomly from the directory of medical specialists, were surveyed for their management practices for hypothetical sle patients. responding physicians follow over patients with sle. patients were managed as follows: ( ) joint and skin involvement only: ninety-four percent of respondents chose asa ( - tabs/day) with % also using hydroxycholoroquin. ( ) early clinical nephritis: seventy-eight percent would initially treat with prednisone, usually at about mg/ kg/day but with % choosing . mg/kg. ( ) active nephritis with rising creatinine: ninety percent would initially employ prednisone, at doses approximating mg/kg, and % would also use immunosuppressives. ( ) serologicalfiare in an asymptomatic patient: forty-six percent would treat with prednisone and % would withhold treatment. (three percent demanded a renal biopsy). ( ) central nervous system sle: uniformly treated with prednisone, at least mg/kg, with % also employing immunosuppressants. ( ) late stage arotemic in-active sle: fifty-nine percent elected to treat with prednisone. ( ) prednisone taperingpractices: from a starting dose of mg/day, physicians tapered to a mean of mg/day after months and mg/day after months. thirty-three percent used a divided dose initially, and % used alternate day schedules later. ( ) flare while tapering: all physicians increased medication, with % increasing prednisone dose, % adding an immunosuppressant, and many doing both. rheumatologists and nephrologists were similar in treatment of most problems. however, nephrologists used immunosuppressive agents twice as frequently in early disease but treated late stage nephritis less vigorously than did rheumatologists. nephrologists also used fewer divided daily doses of prednisone and used alternate day schedule sooner and more frequently. treatment was highly case specific. substantive disagreement occurred when there was serological disease without clinical disease, and in treatment of end-stage nephritis. central nervous system episodes and early progressive nephritis were uniformly treated aggressively. immunosuppressive agents were employed only by a minority of respondents. antibody-dependent cell-mediated cytotoxicity (adcc) was studied in adult patients with rheumatoid arthritis (ra) and in healthy controls. three effector cell populations from the peripheral blood were studied. these included a mixed mononuclear population ( - % latex positive), a monocyte-depleted fraction (less than % latex positive), and a monocyte enriched fraction ( - % latex positive). the target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody (igg fraction); multiple effector: target ratios were studied. there was no significant difference in lymphocytotoxic antibody is found in the serum of patients with systemic lupus erythematosus (sle). the antibody is defined by its reaction with lymphocytes, but it also reacts with antigens on human brain tissue. the existence of an antibody in sle serum recognizing both brain and lymphocyte antigens prompted a search for a similar substance in the cerebrospinal fluid (csf) of sle patients, with and without central nervous system (cns) disease. csf was obtained from patients with sle in the course of evaluations for fever, headache, or manifestations of cns dysfunction. an independent clinical analysis of the sle patients and their hospital records revealed that of the had cns dysfunction attributable to sle. criteria for cns disease were one or more of the following: seizures, transverse myelitis, chorea, ataxia, hemiparesis, psychosis, or hallucinations. multiple abnormalities were present in of the patients, with l patient having , and patients having of the above abnormalities. a population of patients without rheu-matic disease who were being evaluated for a variety of neurological abnormalities served a s controls. the csf was tested in a dye-exclusion microcytotoxicity assay using peripheral blood lymphocytes from normal donors as targets. cytotoxicity was defined as greater than % killing of lymphocytes from or more of the normal donors. nine of the sle patients'had lymphocytotoxic activity in their csf compared to of controls. when lymphocytotoxicity of csf was compared with presence or absence of lupus cns disease, a positive correlation was found (x' = . , p < . ). the mean % lymphocytotoxicity in the cnfs did not correlate with the serum titer of lymphocytotoxic antibody (r = . ). cytotoxic activity to human lymphocytes has been identified in the csf of patients with cns manifestations of sle. if the cytotoxicity is cross-reactive with neuronal antigens, as is the serum antibody, it may have a pathogenetic role in cns lupus. the role of bone marrow derived cells in determining genetic variation in susceptibility to experimentally induced amyloidosis in mice was investigated. mice of the amyloid susceptible cba strain were lethally irradiated and repopulated with marrow cells from the highly resistant a strain. lethally irradiated cba mice reconstituted with syngeneic marrow served as controls. to determine the effects of irradiation per se on amyloid production, normal cba mice, as well as a strain mice that were irradiated and reconstituted with a marrow, were also studied. following weeks of daily subcutaneous casein injections, spleen sections stained with congo red were scored for the degree of amyloidosis by independent blind observers using the following point scale: grade i-a rim of amyloid around or more splenic follicles; grade -a rim of amyloid in more than % of splenic follicles; grade -a rim of amyloid in all splenic follicles; grade -diffuse splenic amyloid with bridging be-tween almost all follicles and some distortion of the splenic architecture. thirty percent of cba mice given a marrow failed to develop amyloid, with an average score of . for this entire group. in contrast, control mice given syngeneic marrow all developed significantly more amyloid, with an average score of . . non-irradiated cba mice developed a n intermediate severity of lesions. irradiated a mice remained amyloid free. the progression of amyloidosis in cba mice was significantly retarded by grafting of marrow from the resistant a strain, even though irradiation seemed to accelerate amyloid production in cba mice. the resistance of the a strain to amyloidosis was not affected by lethal irradiation. these data demonstrate that bone marrow derived cells are an important factor in the pathogenesis of amyloidosis and that the genetic difference between susceptible and resistant mouse strains lies, at least partially, in their hematopoietic tissues. the biochemical features of families with purine nucleoside phosphorylase (pnp) deficiency and t-cell immunodeficiency disease were compared. the erythrocyte enzyme from brothers in family had . % normal pnp activity. plasma urate values in these patients are . mg/dl and plasma inosine levels are . and . p m . urine samples contain uric acid and mg/gm creatinine, inosine . and . millimoles/gm creatinine, and guanosine . and . milli-moles/gm creatinine. when compared t o normal, their enzymes showed a) a -fold increase in the apparent km for inosine, b ) a diminution of the isoelectric p h from normal values of . to . to . to . , c) an inability of inosine to protect the mutant enzyme against thermal inactivation as compared to the protection of the normal enzyme, d ) a loss of the normal near optimum activity a t ph . , and ) a normal value for the stokes radius. the enzyme from a patient in family has . % of normal activity. this patient has a plasma urate of . mg/dl and a plasma inosine of w m . urine samples contain uric acid mg/gm creatinine, inosine . millimoles/gm creati-nine, and guanosine . millimoles/gm creatinine. the enzyme protein had a ) a to -fold increase in the apparent km for inosine, and b ) only minor changes in enzyme activity over a ph range compared to normal. these observations suggest that a ) the degree of abnormality in uric acid and nucleoside concentrations in the plasma and urine reflect the severity of the enzymatic deficiency, b) structural alterations of the mutant pnp proteins result from structural gene mutations and genetic heterogeneity in the disease pnp deficiency. since pnp activity was found in every human tissue assayed, the systemic involvement of pnp deficiency can be accounted for. recently we reported the finding of an antibody, pm-i , directed toward one of the nuclear acidic protein antigens (napa), in sera of patients with polymyositis. this study was undertaken to investigate the relationship between pm-i antibody, polymyositis syndromes, and other rheumatic diseases. sera were tested against nuclear extracts by immunodiffusion and counterimmunoelectrophoresis for the presence of antibodies to pm- and other napa. clinical data were then obtained and analyzed. fifty-two patients were identified who had polymyositis syndromes as defined by significant muscle weakness, elevated serum muscle enzymes, myopathic electromyogram, and muscle biopsy typical of polymyositis. of these, had polymyositis, had polymyositis-scleroderma overlap, and had derrnatomyositis. pm-i antibody was found in % of polymyositis, % of polymyositis-scleroderma, and % of dermatomyositis. polymyositis syndromes, a difference in the prevalence of the clinical characteristics was noted as shown in the table. blinded serum exchanges of sera between medical centers revealed the prevalence of the pm-i antibody to be % in childhood dermatomyositis ( of ), % in adult dermatomyositis ( of ). and % in polymyositis ( of ). patients with other rheumatic diseases were negative for pm-i antibody ( of ). we conclude that the pm-i antibody has a high specificity for polymyositis and that it may identify a subset of polymyositis patients who have other connective tissue disease manifestations. pm-i positive pm-i negative clinical characteristic n = n = the pm- antibody was found rarely ( < i % ) in lo patients other muscular disorders such as myasthenia gravis, muscular pulmonary disease dystrophy, and polymyalgia rheumatica. among patients with an association between viral infection and joint inflammation has repeatedly been reported. our recent studies showed that interferon inducer, double stranded polyinosinate-polycytidylate (poly l:c), as well as human interferon, stimulated both prostaglandin e (pge) and hyaluronic acid production by human cultured synovial fibroblasts. the present study was performed in order to evaluate further the role of interferon and poly i:c in joint inflammation. polynucleotides, mouse interferon, or phosphate buffered saline were injected intraarticularly in -week-old wistar derived male rats by methods previously described. animals were killed hours later and the synovia were removed for histological examination and determination of pge. injection of poly i:c or mouse interferon induced an inflammatory response. poly a:u was only slightly active in this respect. by contrast, pbs or single stranded polyinosinate (poly (i)) or polycytidylate (poly (c)) did not induce any significant inflammatory response. rat knee joints injected with either poly i:c ( , , or pg/joint) or interferon ( or lo p/joint), appeared macroscopically swollen and edematous and contained an increased amount of viscous fluid. microscopically inflamed synovium contained variable amounts of inflammatory cells, most of them polymorphonu-clear. synovial pge levels were increased by poly i:c, poly a:u, and interferon but not by poly (i) or poly (c). mouse interferon also induced an inflammatory response when injected in mouse knee joint. we suggest that interferon may be a mediator in the initiation of inflammation by viruses. nineteen patients with musculoskeletal complaints after jejuno-ileal bypass for morbid obesity were reviewed for evidence of connective tissue disease and immunologic abnormalities. presence of arthritis or arthralgias and extraarticular connective tissue symptoms, elevated westergren sedimentation rate (esr), and absence of other significant forms of arthritis were criteria for diagnosis of postintestinal bypass arthritis. eleven of the patients met these criteria. seven were females. six of the i had joint swelling and had only arthralgias. all had moderately elevated esr. extraarticular manifestations include erythema nodosum ( patients), pleural effusion ( patients), and carpal tunnel syndrome ( patient). synovial fluid analysis in showed mild to moderate inflammation. serum rheumatoid factor was present in patient in low titer while patients had positive ana in titers ranging from : to : . in patients the ana reverted to negative with therapy, and in the third patient the ana became negative after the bypass was reversed. four had hypercomplementemia, had elevated serum igg, and had ele-vated serum iga. none of the patients demonstrated cryoglobulins, but had soluble immune complexes by complement inhibition technique. synovial biopsy in case showed chronic inflammation and iga and c' deposition by immunofluorescence. hla typing of of the patients with post bypass arthritis showed no trend. in i patient the length of bypassed bowel was reduced, and in another the bypass was reversed. in both cases the arthritis remitted. two patients required low dose prednisone therapy for the arthritis; the others were treated effectively with nonsteroidal antiinflammatory agents. these patients had an inflammatory arthropathy often accompanied by features suggestive of connective tissue disease, such as erythema nodosum, pleural effusions, and circulating ana. although none had cryoglobulins, which were previously reported by others, had circulating immune complex. this suggests that immune mechanisms are involved in the pathogenesis of arthritis in some, if not all, of these patients. honig et a (arthritis and rheum , recently reported that patients with active systemic lupus erythematosus (sle) usually do not have markedly increased serum concentrations (> pg/ml) of c-reactive protein (crp) and that such a finding suggests the presence of superimposed infection rather than active lupus. since a semi-quantitative capillary precipitin technique was employed in that study, in which only very high crp concentrations were regarded as positive, we investigated the significance of lesser increases in serum crp concentration in patients with sle, using a quantitative radial immunodiffusion method sensitive to . pg/ml. we retrospectively determined crp concentrations in serum samples collected from a group of patients with sle over a mean period of months. thirtytwo episodes of significant increase in serum crp concentration were detected. careful review of clinical findings associated with each crp peak revealed that these episodes were associated with active lupus without infection in instances (median crp concentration pg/ml, mean . , range . - . ) in instances, elevations of serum crp concentration were attributed to proven or suspected superimposed infection or bone fracture (median pg/ml, mean . , range . - . ). three crp elevations of . , . and . pg/ml occurred at a time when there was clinical evidence of neither lupus activation nor infection. there were instances of onset or exacerbation of lupus activity at times when serum samples did not show elevated crp levels. in of these, very active disease was present; crp elevations were noted in the next samples obtained, and days later. in a third patient with both infection and lupus activity, crp levels were found elevated days later. these data indicate that moderate to marked increases in serum crp concentration may occur in the course of sle in association with activation of disease, as well as in association with infection or other cause of tissue necrosis. rarely no obvious clinical cause can be found. occurrence of serum crp elevation in patients with sle does not differentiate between lupus activity and infection. and immunologic study of the post-intestinal bypass arthritis-dermatitis syndrome medical versus surgical management of ischemic necrosis of bone in systemic lupus intestinal bypass surgery has become a successful means of effecting weight reduction in morbid obesity. as a complication of surgery, a fraction of patients develop an arthritis and tenosynovitis which may be associated with circulating cryoproteins. we have had the opportunity to study patients with post-jejunoileostomy arthritis, all of whom had, as a striking feature of the illness, a widespread dermatitis.the patients were all female. the arthritis developed from to months after surgery and predilected both large and small joints of the upper and lower-extremities. associated with the arthritis were erythematous macules, ranging in size from to mm in diameter. these lesions developed into papules over days, subsequently became pustular-vesicular, and predilected arms, legs, trunk, and face. lesions were found in different stages of evolution.cryoglobulins consisting of igg, igm, and c , c were found in of patients. immune complexes (raji cell) were found in of patients, including patient with absent cryoglobulins. synovial fluid analysis in patients revealed california wbc ranging from to with from to % pmn. immune complexes (raji cell) were found in of synovial fluids.excisional biopsies of the skin lesions were performed in patients. there were no areas of fat necrosis. small capillaries and venules were infiltrated with pmn in all cases.immunofluorescent stains revealed deposits of igg and c -c in vessel walls in of patients. fluorescein conjugated antisera against bacterial pathogens revealed no staining in patient. treatment with oral antibiotics was initiated in all patients but was not invariably successful in decreasing the dermatitis or arthritis. in patients, intravenous antibiotics were also unsuccessful, but oral prednisone was promptly followed by decreased dermatitis-arthritis. in responsive patients, the serum cryoprotein titer decreased; the kinetics of this decrease and the reappearence of the cryoprotein after stopping antibiotic-prednisone treatment was studied.post-intestinal bypass surgery may cause a systemic immune complex disease primarily involving joints and skin. each met at least sle classification criteria, had clinical criteria of disease activity, and had elevated serum dna binding ( - % by farr assay). three had low serum hemolytic complement levels ( - u). their drug regimen, including prednisone ( - mg/day), remained stable for i days prior to, during, and for days after frentizole therapy. rash ( of ), synovitis ( of ), mucosal ulcers ( of ), and pleurisy ( of ) improved during frentizole therapy. synovitis worsened in case. the only toxicity was transient sgot and sgpt elevation in cases. bone marrow and skin tests were not suppressed by the drug. frentizole had no effect on hemoglobin concentration, creatinine clearance, or urinary protein ex-response. other laboratory findings (mean f sem) are summarized in the table. these results suggest that frentizole is an active, relatively non-toxic immunoregulatory drug in sle patients. current long-term studies should define its role in the therapy of sle and other autoimmune diseases. clinical and laboratory findings of children with systemic lupus erythematosus (sle) prospectively followed since have been summarized. mean disease duration was . years (range months to years, median years). there were girls and boys, less of a female preponderance than that of most adult series. youngest age for onset of systemic complaints was years. ten of boys had disease onset prior to sexual maturation, whereas disease began after menarche in adcc activity between patient cells and control cells when either the mixed mononuclear population or monocyte-depleted population were studied as effectors. the monocyteenriched fraction from patients with ra, however, mediated a significantly increased degree of cytotoxicity (table) . enhanced cytotoxicity was more evident at low effector: target ratios and was independent of phagocytosis. adcc may be important in ra since it reflects both humoral and cell-mediated immune mechanisms. the enhanced effector function of the peripheral blood monocyte in this system may be an indication that mononuclear phagocytes are "activated" in patients with ra. the ara preliminary criteria for the classification of systemic lupus erythematosus (sle) were developed before the widespread use of tests for anti-dna antibodies and serum c levels. analysis of our data suggests that these tests should be included in the ara criteria and could replace of the present manifestations which are less specific and less sensitive than the serum c level and circulating anti-native dna antibody level (a-dna).three thousand three hundred thirty-four sera from sle patients were studied. only patients observed by us and whose sera were tested both during periods of remission and disease activity were included in this study. an average of serum samples per patient was studied. the average period of observation was . months. patients had at least of the manifestations of the ara criteria. c levels and a-dna were studied from normal individuals, patients with rheumatoid arthritis followed by us, and from patients with other diseases.two of the non-sle patients ( %) had a-dna level (expressed as % dna bound) of more than % ( %, %). thus, a a-dna of > % binding had a specificity for sle of %. eighty-two percent of the sle patients had a-dna of > % binding as their highest value-a sensitivity of %. none of the non-sle patients had a c of < mg%-a specificity of %. fifty-five percent of the sle patients had a c level of < mg% as their lowest c -a sensitivity of %.these data were analyzed using the method employed to select the manifestations of the ara criteria. the average rate of correct classification (the arithmetic mean of the sensitivity and specificity) (arcc) for a-dna of > % binding is . and for c of < mg% is . the arcc for the items which comprise the manifestations of the ara criteria varied from . to . . of these, only the presence of le cells had a arcc greater than that for a-dna. if c and a-dna data had been available at the time the ara criteria were developed, they would have been included. the arcc for the mean of the items combined for the cns manifestations and the arcc for the mean of the hematologic items were lower than those for either c or a-dna. thus, the c and the a-dna could replace these manifestations. the data suggest that the ara criteria should be revised.the significance of ischemic necrosis of bone (inb) in sle and the cause of the bony lesion are not established. in this study, patients with sle and inb have been compared to patients with sle alone; and, in those with inb, the cause of the osseous lesion has been analyzed in relation to medical versus surgical management.those clinical features of sle significantly correlated with inb include raynaud's phenomenon ( % versus %, p < o.ool), myositis ( % versus %, p < o.ol), and vasculitis ( % versus %, p < . ). the course of inb sites in the patients initially grouped into medical ( patients; sites) and surgical (i patients; sites) is shown in the table.the initial orthopedic procedure was uniformly core decompression. all patients were receiving corticosteroids. the failure of medical management alone is shown by the persistence of symptoms ( or %), x-ray progression ( or %), and need for reconstructive surgery ( or %). in stages i and of the surgical group, only sites ( %) were symptomatic, sites ( %) progressed on x-ray, and only ( %) required further surgery. none in stage i advanced.differences in followup intervals would not appear to explain these statistically significant differences.thus, in the steroid-treated sle patient, especially with raynaud's, myositis, and/or vasculitis, the risk of inb is major and the need for early diagnosis essential if progression of the bony lesion is to be retarded by orthopedic intervention. acute abdominal syndromes have been increasingly recognized as major life-threatening events in patients with rheumatic disease. early diagnosis is critical to the institution of appropriate medical/surgical management and may be impeded by the masking effects of antiinflammatory, especially steroidal, therapy. it is our purpose here to report those features, clinical and laboratory, which appear to relate specifically to intraabdominal arteritis, our major cause of an acute abdomen in those with systemic lupus erythematosus (sle) and polyarteritis (pa).during the past years, of patients admitted to the unit with sle and of patients with pa developed an acute abdomen. in i ( %) of those with sle and all with pa, the abdominal event was secondary to mesenteric arteritis. the remaining patients with sle had polyserositis ( ), pancreatitis ( i ) and, in the other patient, an undiagnosed recurrent syndrome which responded each time to increased corticosteroids alone. when patients with sle and an acute abdomen were compared to the patients without abdominal syn-dromes, the significant discriminating features in the abdominal group were increased peripheral vasculitis ( % versus %, p < . ), thrombocytopenia ( % versus %, p < o.ooos), and presence of rheumatoid factor by the latex test system ( % versus %, p < . ). of the patients with pa, peripheral neuropathy was present in all, thrombocytopenia developed concomitant with the abdominal syndrome in all, and rheumatoid factor was uniformly present in the patients tested ( : , : , i : , ). eight of those with sle and the with pa died from the abdominal catastrophe. six of the surviving sle patients represent those most recently seen.thus, in the patients with sle and pa, the presence of thrombocytopenia and circulating rheumatoid factor are poor prognostic signs and may pathogenetically predispose the patient to intraabdominal arteritis. outcome can only be favorably altered by early recognition and prompt institution of appropriate medical and/or surgical management. key: cord- -n sih authors: villard, viviane; agak, george w.; frank, géraldine; jafarshad, ali; servis, catherine; nébié, issa; sirima, sodiomon b.; felger, ingrid; arevalo-herrera, myriam; herrera, socrates; heitz, frederic; bäcker, volker; druilhe, pierre; kajava, andrey v.; corradin, giampietro title: rapid identification of malaria vaccine candidates based on α-helical coiled coil protein motif date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: n sih to identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. the corresponding synthetic peptides are expected to mimic structurally “native” epitopes. indeed the chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. these antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. this strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. human plasmodium falciparum (pf) infection is a dramatic public health problem. today approximately forty percent of the world's population is at risk of malaria. malaria causes more than million acute clinical cases, and at least one million deaths annually. ninety percent of malaria deaths occur in sub-saharan african countries mostly among young children and pregnant women (http://rbm.who.int). thus, there is an urgent need of a malaria vaccine. however, vaccine discovery, in general and particularly in malaria, is still a very empirical process. in fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. for protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. in addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [ , ] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. proper protein folding and solubility remain a limitation in numerous cases. thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge. we addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. we focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. lsa- [ ] , lsa- [ ] , msp- [ ] , and msp- [ ] and other pathogens [ ] . msp- , another leading malaria vaccine [ ] , also contains predicted a-helical coiled coil regions (unpublished results). our choice was based on the following considerations. first, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. this motif can be easily identified by bioinformatic analysis. secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [ ] . thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. in addition, these domains are short (about residues) and can be rapidly produced by chemical synthesis. furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. here, we focus on the pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis. the screening of the pf genome [ ] using generalized sequence profiles [ ] identified several hundred proteins containing putative a-helical coiled coil motifs. through proteome and transcriptome data [ ] [ ] [ ] we assessed which of these molecules are expressed in the pf parasite erythrocytic stage. the combined analysis/assessment identified over segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (table s ) . out of these a-helical coiled coil fragments, in general - amino acids long, present either in the same protein or in different ones, were chemically synthesized and hplc purified. among them, longer peptides (up to amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigens , and ; table s ). the selected antigens were then tested in elisa assays for reactivity with three panels of sera obtained from adult donors from burkina faso, tanzania and colombia, respectively. to our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (tables and s ). in this manner, proteins were identified whose lengths varied from to , amino acids. twenty-one peptides with the highest prevalence of responders and elisa mean od value were selected for further studies. variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. the high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution. indeed, circular dichroism (cd) studies of selected peptides associated with biological activities (tables and ) indicate that they predominantly assume an a-helical conformation in water. peptides , and ( figure s a ) exhibit a cd pattern characteristic of a high a-helical content, whereas the remaining peptides show cd profiles similar to that shown for peptide ( figure s b ) or intermediate between those shown in figures s a and s b characteristic of a partial a-helical organization. when analyzed by size exclusion chromatography on fplc columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (mw and kda, respectively). the cd and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by nmr and ultra-centrifugation studies. to test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from papua new guinean adults. the serum pools were first tested in elisa assays against peptides that were the most antigenic (table ) ; from these, peptide-specific antibodies were purified from the most positive serum pool and tested again in elisa. these antibodies all reacted with parasite native proteins in infected red blood cells as shown by ifat ( figure a ; table ). reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by ifat competition assays with the corresponding peptide ( figure a ). the specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (glu)-rich sequences which are known to generate cross reactivity among several malarial glu-rich proteins [ ] . cross-reactions were systematically investigated using each of the affinitypurified antibodies on each of the peptides. results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (table s ) . to determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, elisa competition assays were performed. to this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. only homologous peptides competed best whereas peptides having sequence similarity did not (figures a, b , and s b), or at a much higher concentration ( figure s a ) including the shorter glu-rich peptides derived from the cterminus of peptide and (figures a and b) . finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide. antibodies corresponding to the selected peptides were tested for direct and cell-mediated anti-parasite activity. clinical experiments have shown that the antibody-dependent cellmediated inhibition (adci) of p. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [ ] . twelve peptide-specific antibodies proved able to induce a strong (more than %) and intermediate (lower than %) monocyte-dependent parasite killing (table ) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. the effects were in the range observed with antibodies from african adults who have the highest natural protection known against malaria. therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria. sixteen peptides -twelve targeted by adci positive antibodies and four controls-were used to immunize cb f mice ( table ) . eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by ifat ( figure b ; table ). as seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in ifat assays (data not shown) and by ifat competition assays with the corresponding peptide ( figure b ). anti-peptide mouse antibodies, which are positive in ifat, are also specific for the homologous peptide but not for the sequence related peptides , and (table s ) . thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the n-and c-terminal ends, b) stabilizing the a-helix as suggested by cooper et al. and lu and hodges [ , ] and/or c) use of other adjuvants. genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. however available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (plasmodb . , and unpublished sequencing data). a few peptide dna sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation. with regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for adci assays ( table ) , of the proteins contain a pentapeptide conforming to the pexel consensus [ , ; , ] , but that none of these have a position within the amino acid sequence that conforms to the location of known active pexel motifs (see materials and methods and membrane segments, and none of them has a gpi anchor. only one protein contains a signal sequence. fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (table s ). the prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [ ] . further investigations will be required to determine the actual localization of the corresponding antigens. the predicted localization is a priori surprising for molecules able to trigger an adci activity. however, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering adci provided they defined at least two epitopes [ ] , which is the case for a-helical coiled coil heptad repeats. therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link fc-c receptors on monocytes to achieve pf parasite killing. in conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. in fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. in principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens. the pf d genome [ ] was used for the bioinformatics analysis. the generalized sequence profile method and the pftools package [ ] were used to search for the short a-helical coiled coil domains. the coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. two profiles containing four and five heptad repeats were used for the analysis. the cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known d structures. subsequently, the coiled coil figure . immunofluorescence microscopy analysis of pf d parasites with peptide specific antibodies. acetone/methanol-fixed schizonts and merozoites were reacted with a: human peptide specific, affinity purified antibodies obtained with peptides and (table ) and b: sera from mice immunized with peptide (table ) . grey: bright field images; blue staining: indicates dapi nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by cy -conjugated anti-human or anti-mouse igg specific antibody. merge picture is an overlay of the blue and red fluorescence channel. doi: . /journal.pone. .g regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. these proteins were also analyzed by the coils program [ ] . the selected a-helical coiled coil containing proteins were further tested on their possible surface location and gpi anchoring by using the following programs: identification of potential signal peptides, secretomep and signalp (http://www. cbs.dtu.dk/services/) [ ] ; transmembrane spanning regions (tmpred http://www.ch.embnet.org/software/tmpred_ form.html and tmhmm http://www.cbs.dtu.dk/services/ tmhmm; [ , ] ), and gpi-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [ ] ) and prediction of sub-cellular localization (ptarget http://bioinformatics. albany.edu/,ptarget; [ ] ). to identify the pexel-like motifs in sequences of the selected proteins we used the following pattern [ [deq] that represents a combination of the pexel patterns indicated in recent papers [ , ] . the presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of p. falciparum (www.plasmodb.org; [ ] ). peptides were synthesized on the advanced chemtech (hatley st george, uk) ac t omega multi channel synthesizer and the applied biosystem synthesizer a and a (foster city, ca) using solid-phase fmoc chemistry. crude peptides were purified by rp-hplc (c preparative column) and analyzed by mass spectrometry (maldi-tof; applied biosystem, foster city, ca). chemicals and solvents used for peptide synthesis were purchased from fluka (buchs, switzerland) and novabiochem (laufelfinger, switzerland). circular dichroism (cd) spectra of peptides were recorded on a jasco j- spectrometer (jasco corporation, tokyo, japan) equipped with a temperature controller and a . cm path length cuvette. the measurements were made in water at ph . and uc and at a peptide concentration of . mg/ml. the sera from burkina faso were collected in the village of goundry located in the central mossi plateau, between and km north of the capital ouagadougou, in the province of oubritenga. the climate is characteristic of areas of sudanese savannah, with a dry season from november to may and a rainy season from june to october. malaria transmission is very high during the rainy season and markedly seasonal. ethical clearance was obtained from the ministry of health, burkina faso. after obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season . the tanzanian sera came from a large-scale community based study undertaken in kikwalila village, kilombero district, morogoro region from to . blood samples from adults (. years) were taken by finger prick and the serum was kept at - uc until use. research and ethical clearance for the study was obtained by the tanzanian commission for science & technology. the colombian sera were collected in buenaventura the main port on the colombian pacific coast after human informed consent, during a cross sectional survey carried out from february to may within the framework of a project supported by the colombian research council, colciencias. the area has unstable transmission of both p. falciparum and p. vivax malaria. ethical clearance to draw blood from human volunteers was human purified antibodies were used at mg/ml; ifat was not performed on ring stages. valle. blood was taken by venipuncture into tubes containing edta and sera fractionated and stored frozen until use. the sera from adults from papua new guinea (png) pooled for affinity purification were collected in the maprik district of the east sepik province, during a cross sectional survey in july within the framework of the malaria vaccine epidemiology and evaluation project (mveep) supported by the united states agency for international development [ ] . the area is highly endemic for malaria. ethical clearance for mveep was obtained from the png medical research advisory committee. blood was taken by venipuncture into tubes containing edta. the pool of immune african globulins (piag) used for adci was prepared from immune individuals living in endemic areas and negative control igg (n-igg) was obtained from a pool of more than french adult donors with no history of malaria. briefly, the igg fractions from both positive and negative controls were purified using a size exclusion trisacrylh gf m (pall bioseprah; pall life sciences, ny) column followed by an ionic exchange deae ceramic hyperdh f column (pall bioseprah). purified igg were then extensively dialyzed against rpmi and kept at uc until use. cb f mice were injected times with mg of the indicated peptide in montanide isa at the base of the tail on day , and . bleeding was performed days after the second and third immunization. elisa was performed according to lopez et al. [ ] and anti human igg-or anti mouse igg conjugated to alkaline phosphatase was used (sigma, st louis, mo) as second antibody. individual human sera from , and adults donors from burkina faso, tanzania and colombia respectively were used at : dilution. serum was considered positive if the optical density (od) reading was higher than the mean od value+ standard deviation (sd) of the negative controls (individual serum samples from to naïve swiss donors) or if the od ratio of the mean of duplicate experimental values to the mean od of the negative control was higher than . for mouse sera, the end point value was determined as the last dilution of the mean od value+ standard deviation (sd) of the negative control (non immune sera). elisa competition assays were performed by incubating either each of the selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the antigens over the indicated range of concentrations for minutes at room temperature prior to addition to the elisa peptide-coated plate wells. antigen-sepharose conjugate preparation: mg of antigen was dissolved in ml of coupling buffer ( . m nahco containing . m nacl, ph . ). the cnbr-sepharose b (amersham bioscience ab, uppsala, sweden) was activated by swelling in mm hcl and then washed with coupling buffer. the antigen solution was added to the gel and the mixture was stirred for h at rt. after the coupling reaction, excess antigen was washed away with coupling buffer. the unreacted activated groups were blocked by treatment with ethanolamine ( . m; ph . ) for min at rt. the gel was then washed with sodium acetate buffer ( . m; ph . ), followed by coupling buffer. the antigensepharose beads were either used or stored at uc in pbs ( x) containing mm azide. isolation of specific antibody: pooled human serum was diluted five times with pbs ( x) containing . m sodium chloride and mixed with antigen-sepharose conjugate. this mixture was then stirred gently on a wheel o/n at uc. after centrifugation the supernatant was collected and stored at uc for further use. the antigen-sepharose beads were then washed with ml of trizma base tris ( mm containing . m nacl, ph . ) then with ml of tris ( mm, ph . ). the elution of bound antibody was achieved with glycine ( . m, ph . ). the fractions obtained were instantly neutralized with tris ( m, ph . ), dialyzed against phosphate buffer ( . m, ph . ) and the antibody concentration was determined by the absorbance of the solution at nm. slides coated with pf sporozoites were dried at rt for minutes, fixed with % acetone at uc for minutes, washed times in pbs- . % tween , dried carefully and blocked with ml/ well of pbs- % bovine serum albumin (bsa) for minutes at rt. slides coated with pf merozoites were fixed with % acetone at uc for minutes and dried o/n at rt. the appropriate antibody or serum dilutions prepared in pbs- % bsa were distributed ( ml/well) and incubated for h at rt in a humid chamber. after washing with pbs- . % tween- , goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to cy (molecular probes) diluted / in pbs- % bsa or anti-human igg (fc specific) fitc conjugate (sigma) diluted / in evans blue solution ( / ) was added ( ml/slide) and incubated for h at rt in a humid chamber in the dark. slides were washed as above, covered with % glycerol, sealed and read using a fluorescence microscope (leica dmirb dc ). the uganda palo alto strain (fup/c) was cultured in rpmi- supplemented with . % albumax i (gibcobrl-invitrogen, san diego, ca). for adci assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over h, by floatation on % porcine skin gelatin type a (sigma). blood monocytes (mn) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (lecourbe blood bank, paris, france). peripheral blood mononuclear cells (pbmc) were separated on ficoll density gradients j prep (techgen, les ulis, france) and washed in ca + and mg + free hbss buffered with mm hepes (both from gibcobrl-invitrogen). cells were then distributed on polystyrene -well flat-bottomed culture plates (tpp, trasadingen, switzerland) and adherent mn were selected by incubation for h at uc, in a humidified % co atmosphere. more than % of the adherent cells obtained in this manner were mn as estimated by the non-specific esterase test (a-naphtyl acetate esterase; sigma). mn from each donor were tested prior to adci assays and only those without direct inhibitory effect were used in assays. to wells containing mn purified as described above, ml of an asynchronous parasite culture at . % parasitemia and % hematocrit were added. wells were then supplemented with test or control antibodies (ab) and the total volume adjusted to ml with culture medium. after h and h, ml of culture medium were added to each well and after h the adci assay was stopped and the final parasitemia was determined by light microscopy on giemsa-stained smears by counting $ , red blood cells. for each ab tested, duplicate wells included the following controls ) non-specific monocytic inhibition, both mn+parasite, and mn+n-igg+parasites and ) direct inhibition by control or test igg, both n-igg+parasites, and test abs+parasites. piag and n-igg were used at a final concentration of mg/ml as positive and negative controls respectively. immunopurified tests abs were used at mg/ml. the specific growth inhibitory index (sgi) which considers the parasite growth inhibition due to the effect of test abs cooperating with mn was calculated as follows: sgi = [ (% parasitemia with mn and test abs/% parasitemia test abs)/(% parasitemia with mn and n-igg/% parasitemia n-igg)]. figure s cd spectra of the peptides (s a) and (s b) found at: doi: . /journal.pone. .s ( . mb tif) figure s elisa inhibition assay using anti-human peptide specific antibodies. binding of peptide specific antibodies to peptides (s a) and (s b) absorbed on elisa plates was inhibited by incubating specific antibodies ( - mg/ml) with peptides , and (s a) and peptides and (s b), respectively (see material and methods). peptides , and share nnm or mnn as sequence similarity while peptides and do not exhibit any apparent sequence similarity. table s structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in adci (table ) . found at: doi: . /journal.pone. .s ( . mb doc) identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology and genomics plasmodium falciparum liver stage antigen- is well conserved and contains potent b and t cell determinants protection against plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein antigen plasmodium falciparum merozoite surface protein displays multiple targets for naturally occurring antibodies that mediate monocyte-dependent parasite killing template-based coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus phase randomized double-blind safety and immunogenicity trial of plasmodium falciparum malaria merozoite surface protein fmp vaccine de novo design of alpha-helical proteins: basic research to medical applications genome sequence of the human malaria parasite plasmodium falciparum a flexible motif search technique based on generalized profiles transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis a proteomic view of the plasmodium falciparum life cycle the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum crossreactive antigens between life cycle stages of plasmodium falciparum antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes mapping of conformational b cell epitopes within alpha-helical coiled coil proteins a de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins targeting malaria virulence and remodeling proteins to the host erythrocyte a host-targeting signal in virulence proteins reveals a secretome in malarial infection proteomic analysis identifies novel proteins of the maurer's clefts, a secretory compartment delivering plasmodium falciparum proteins to the surface of its host cell multi-character population study of the vir subtelomeric multigene superfamily of plasmodium vivax, a major human malaria parasite a maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells a novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes fcgammarii and fcgammariii predicting coiled coils from protein sequences improved prediction of signal peptides: signalp . tmbase-a database of membrane spanning proteins segments predicting transmembrane protein topology with a hidden markov model: application to complete genomes prediction of potential gpimodification sites in proprotein sequences ptarget [corrected] a new method for predicting protein subcellular localization in eukaryotes plasmodb: the plasmodium genome resource. a database integrating experimental and computational data the malaria vaccine epidemiology and evaluation project of papua new guinea: rationale and baseline studies a synthetic malaria vaccine elicits a potent cd (+) and cd (+) t lymphocyte immune response in humans. implications for vaccination strategies the authors wish to thank luis rodrigues and florela penea for the synthesis and purification of peptides and thomas smith for discussion and all of the blood donors. key: cord- -jqgervt authors: fenner, frank; bachmann, peter a.; gibbs, e. paul j.; murphy, frederick a.; studdert, michael j.; white, david o. title: laboratory diagnosis of viral diseases date: - - journal: veterinary virology doi: . /b - - - - . - sha: doc_id: cord_uid: jqgervt tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species in some different viral families that infect the eight major domestic animal species. if antigenic types within an individual viral species are considered and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is, therefore, not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. tests for the specific diagnosis of a viral infection in an animal are of two general types: ( ) those that demonstrate the presence of the virus, and ( ) those that demonstrate the presence of specific viral antibody. the provision, by a single laboratory, of a comprehensive service for the diagnosis of viral infections of domestic animals is a formidable undertaking. there are about individual viral species, in some different viral families, that infect the eight major domestic animal species (cattle, sheep, goat, swine, horse, dog, cat, and chicken) . if antigenic types within an individual viral species are considered, and the number of animal species is broadened to include turkey, duck, and zoo and laboratory animals, then the number of individual viruses exceeds . it is therefore not surprising that few single laboratories could have available the necessary specific reagents, skills, and experience for the diagnosis of such a large number of infections. one consequence of this great variety of viruses is that veterinary diagnostic laboratories tend to specialize, e.g., in diseases of food ani- mais, or companion animals, or poultry, or in "exotic" viruses. within these specialized laboratories there is considerable scope for the development of rapid diagnostic methods that short-circuit the need for the isolation of viruses, which is expensive, time-consuming, and rarely necessary. many viral diseases can be diagnosed clinically, others with the assistance of the pathologist; but there are several circumstances under which laboratory confirmation of the specific virus involved is desirable or, indeed, essential. the industrialized countries of europe, north america, australasia, and japan are free of many devastating diseases of livestock that are still enzootic in other parts of the world, such as foot-and-mouth disease, african swine fever, rinderpest, and fowl plague. all industrialized countries maintain or share the use of specialized biocontainment laboratories (such as those at plum island in the united states and pirbright in the united kingdom) devoted to diagnosis and research on such "exotic" viruses. clearly it is of the utmost importance that the clinical diagnosis of a suspected exotic virus should be confirmed quickly and accurately (see chapter ). several animal viral diseases such as rabies, rift valley fever, and eastern, western, and venezuelan encephalomyelitis are zoonotic and are of sufficient human public health significance to require the maintenance of specialized diagnostic laboratories. for example, confirmation of the diagnosis of rabies in a skunk that has bitten a child provides the basis for postexposure treatment of the human patient (see chapter ). confirmation and early warning of an equine encephalomyelitis virus epizootic allows implementation of mosquito control and other measures such as restriction of the movement of horses. for diseases in which there is lifelong infection, such as bovine and feline leukemia, equine infectious anemia, and herpesvirus infections, a negative test certificate is often required as a condition of sale, particu-larly export sale, for exhibition at a state fair, or show, or for competition, as at race meetings. males used for semen collection and females used in embryo transfer programs, especially in cattle, and blood donors of all species, are usually screened for a range of viral infections to minimize the risk of transmission to recipient animals. for retrovirus infections, marek's disease, pseudorabies, and certain other diseases, it is possible to reduce substantially the incidence of disease or eradicate the causative virus from the herd or flock by test and removal programs. laboratory diagnosis is essential for the effective implementation of such operations. provision of a sound veterinary service in any state or country depends on a knowledge of prevailing viral diseases; hence, epidemiologica! studies to determine the prevalence and distribution of particular viral infections are frequently undertaken, usually based on the detection of specific antibody. many relatively nonspecific disease syndromes, such as respiratory disease (e.g., kennel cough and shipping fever), diarrhea, and some skin diseases, may be caused by a variety of agents, viral and bacterial. proper management of individual cases or infected herds or flocks may require specific viral diagnosis. specific diagnosis of the kind outlined above can be achieved by one of three methods: ( ) isolation and characterization of the causative virus, ( ) direct demonstration of virions, viral antigens, or viral nucleic acids in tissues, secretions, or excretions, and ( ) detection and measurement of antibodies (table - ). each group of methods has its place. viral isolation remains the benchmark against which other methods are measured, and is essential when decisions of major economic importance depend on the diagnosis, e.g., with suspected exotic diseases such as foot-and-mouth disease or fowl plague. on the other hand, the direct demonstration of virions or viral components may provide a much more rapid and cheaper method of specific diagnosis than viral isolation, particularly when large numbers of samples must be tested. epidemiologica! surveys, eradication programs, and the provision of certificates of freedom from specific infections are often based on serological methods or rapid tests for viral antigen. it requires at least as much effort, and often more, to process a negative specimen as it does one from which virus is isolated. the chance of isolating a virus depends critically on the knowledge, care, and attention of the veterinarian who collects the specimen (see plate - ). obviously such a specimen must be taken from the right place and at the right time. the right time is always as soon as possible after the onset of clinical signs; virus is usually present in maximum amount at about this time and diminishes, sometimes quite rapidly, in the ensuing few days. specimens taken as a last resort when days or weeks of empirically chosen antibiotic therapy have failed are almost invariably a waste of effort. the site from which the specimen is collected will be influenced by the clinical signs and a knowledge of the pathogenesis of the suspected disease (table - ). having collected the appropriate specimen(s), it should be properly labeled and sent to the laboratory, with a history, including the provisional diagnosis. where ambient temperatures are moderate and transit time to the laboratory is less than day, ice or cold packs (< °c) in a styrofoam box are frequently used. if the environmen tal temperature is high and transit times longer than a day, dry ice (- °c) may be used, although wet ice with provision to replenish it in transit is better. if exotic or zoonotic viruses are suspected, the styrofoam boxes must be replaced by or enclosed within sturdier, dou ble-walled containers with absorbent padding. appropriate permits must be obtained for interstate and international transportation, and in "blood: refers to clotted sample for serology and sample with anticoagulant added for other tests. large animals, - ml; small animals, - ml; others as appropriate. if possible remove clot before dispatch. such circumstances the collection and transport arrangements need to be discussed with the laboratory and/or the appropriate government regulatory agency. for particularly labile viruses such as respiratory syncytial virus, herpesviruses, or coronaviruses, it may be an advantage to take the cell culture to the animal. isolation and identification requires at least a week, usually longer, and it is expensive. however, it is probably the most sensitive available method, if properly collected material is used, and it provides material for further study. the sooner the specimen is processed and inoculated after arrival at the laboratory, the better. if delays of more than day are anticipated, the specimen may be frozen to - °c. swabs are processed by twirling them in the transport medium and expressing the fluid by pushing the swab firmly against the side of the container. feces are dispersed on a vortex mixer. tissue specimens are finely minced with scissors and ho mogenized in a glass or mechanical homogenizer. prior to inoculation, contaminating microorganisms are removed by filtering through membrane filters of average pore diameter . μιη, although such filters allow the passage of mycoplasmas. once virus is successfully isolated and grown to a high titer, the suspension can be refiltered through . μιη filters to exclude mycoplasmas. feces and tissue homogenates should be diluted at least : and centrifuged at g for minutes to obtain a supernate that can be filtered. if the concentration of virus is suspected to be very low, high concentrations of antibiotics may be preferable to filtration. whatever the origin of the specimen, some of the original sample and some of the filtrate should be retained at °c or frozen until the isolation attempt is finalized. virus can be grown from the suitably prepared specimen by inocula tion into cell cultures, laboratory animals, or the species of host animal from which the specimen was obtained. by far the most widely used substrate is cultured cells. choice of cultured cells. the choice of the optimal cell culture for the primary isolation of a virus of unknown nature from clinical specimens is largely empirical. primary or low-passage, homologous, monolayer cell cultures derived from fetal tissues probably provide the most sen sitive substrate for isolation of the greatest variety of different viruses. continuous cell lines derived from the homologous species are almost as good. often the nature of the disease from which the material was obtained will suggest what species of virus may be found, and in such cases the optimum cell culture for that virus can be chosen, in parallel, perhaps, with a second type of culture with a wide spectrum. cell lines offer some advantages and are available for most domestic animals ex cept avian species (table - ) . monolayer cell cultures for virus isolation should be grown in sealed containers, such as plastic flasks or glass tubes with screw caps. openculture systems such as petri dishes or microtiter trays should not be used because of the risks of cross-contamination. for some viruses, rolling the cultures on a drum improves isolation rates. special types of cultures are utilized for particular viruses. for exam ple, betaherpesviruses and gammaherpesviruses may be recovered from monolayer cultures of tissue taken directly from the diseased animal, whereas inoculation of established monolayer cell cultures with cell-free material may be negative. for some corona viruses and rhinoviruses that do not grow well in monlayer cultures, growth may occur in expiant cultures (i.e., small cubes of tissue from the trachea or gut), probably because these do not dedifferentiate in culture (see chapter ). recognition of viral growth. cultures are usually incubated at °c, despite the fact that the normal body temperatures of all domestic animal species are somewhat higher. cultures are observed daily for cytopathic effects. the speed and nature of the cytopathic effect caused by different viruses varies considerably. cytopathic effect must always be based on comparison with uninoculated cell cultures; this is particularly important for viruses requiring incubation periods of longer than a week. where none or a doubtful cytopathic effect is observed, it is usual to make a second or even a third ("blind") passage. when cytopathic effect is observed, there is a range of options: . the speed and appearance of the cytopathic effect, coupled with the case history, may immediately suggest the diagnosis. . after suitable manipulation, material from the cell culture may be examined by electron microscopy. . infected monolayers on glass coverslips or special slide/culture chambers may be fixed and appropriately stained, and the cells examined for inclusion bodies, syncytia or other characteristic cell changes. better, they may be stained with fluorescent antibody, which may provide an immediate definitive diagnosis. where prior experience and knowledge suggest it, such slide cultures may be included at the time of primary inoculation, with a consequent saving in time. table - ). their growth in monolayer culture may sometimes be recognized by means of hemadsorption. most viruses that hemagglutinate will also hemadsorb; the growth of paramyxoviruses, orthomyxoviruses, and, to a lesser extent, the flaviviruses and toga viruses, is routinely recognized in this way (see plate - ). nowadays laboratory animals play a minor role in the virus diagnostic laboratory. however, some virologists still regard intracerebral inoculation of baby mice as the method of choice for the isolation of rabies virus, flaviviruses, and toga viruses. the developing chick embryo occupies a special place. intraamniotic inoculation provides the most sensitive method for influenza viruses and for several avian viruses, and species diagnosis of orthopoxviruses can be made directly from the type of pock produced on the chorioallantoic membrane. in addition, chick embryos are extensively used as a source of primary monolayer cultures (fibroblasts, kidney cells) for the isolation of avian viruses. in veterinary medicine it is feasible to consider using the natural host species, especially susceptible young animals (e.g., calves, pigs, chicks), for the recovery of a virus from suspect material. such animals, if free of antibodies, must be considered a highly sensitive substrate. however, their use would now be contemplated only for viruses not yet cultivable, or where cell culture procedures were negative in circumstances that strongly indicated a viral etiology, and/or where there might be serious repercussions if the diagnosis were missed. a newly isolated virus can usually be provisionally allocated to a particular family, and sometimes to a genus or species, on the basis of the clinical findings, the host cell used for virus isolation, and the visible result of viral growth (cytopathic effect, hemadsorption, hemagglutination, electron microscopy of the cytopathogenic agent, etc.). definitive identification, however, usually depends on serological procedures. by using the new isolate as antigen against known antisera, e.g., in a complement fixation test, the virus can often be placed into its correct family or genus. having allocated it to a particular family (e.g., adenoviridae), one can then go on to determine the species or serotype (e.g., canine immunodiffusion antibody neutralizes infectivity of virion; inhibits cytopathology, reduces plaques, or protects animals antibody inhibits viral hemagglutination antigen-antibody complex binds complement, which is thereafter unavailable for the lysis of hemolysissensitized sheep red blood cells antibody-aggregated virions are visible by electron microscopy antibody labeled with fluorochrome binds to intracellular antigen; fluoresces by uv microscopy peroxidase-labeled antibody binds to intracellular antigen; colored precipitate forms on adding substrate enzyme-labeled antibody (or antigen) binds to antigen (or antibody); substrate changes color radiolabeled antibody (or antigen) binds to antigen (or antibody), e.g., attached to solid phase antibodies and soluble antigens produce visible lines of precipitate in a gel adenovirus ) by more discriminating serological procedures. this sequential approach is applicable only to families with a common family antigen. the range of available serological techniques is now extremely wide (table - ). some are best suited to particular families of viruses. each laboratory makes its own choice of favored procedures, based on considerations such as sensitivity, specificity, reproducibility, speed, convenience, and cost. currently most serological procedures are carried out with "hyperimmune" sera comprising a polyclonal mixture of antibodies, sometimes after they have been absorbed to eliminate antibodies of certain specificities. monoclonal antibodies with defined specificity are now becoming available. these make it possible to proceed quickly to very specific diagnosis even to the level of subtypes, strains,or variants, e.g., rabies viruses from different geographical areas. family-, genus-, and typespecific monoclonal antibodies are also being developed. as their properties are defined and they become commercially available, we can expect monoclonal antibodies to be widely used for all methods of serological identification. . immunofluorescence, used here for determining the site of assembly of components of influenza virus. antibody against the nudeoprotein antigen shows nuclear accumulation at hours after infection of chick cells. procedure: guinea pig antiserum to nudeoprotein antigen is added to a monolayer of infected chick cells, then fluoresceinconjugated rabbit anti-guinea pig igg. (courtesy dr. n. j. dimmock.) immunofluorescence. the simplest way of identifying a newly iso lated virus is by fluorescent-antibody staining of the infected cell monolayer itself (plate - ). this can provide definitive diagnosis within an hour or so of recognizing the earliest suggestion of cytopathic effect. immunofluorescence is best suited to the identification of monotypic genera, or genera of which only a single species affects that particular species of animal, or to epidemic situations when a particular virus is suspected; otherwise, replicate cultures must be screened with a range of antisera. the advantages and disadvantages of monoclonal anti bodies, in comparison with polyclonal or "absorbed" sera, discussed below in the context of radioimmunoassays, apply equally to other serological procedures, including immunofluorescence and neutrali zation. electron microscopy and immunoelectron microscopy. these pro cedures (see plate - ) are most useful in the rapid identification of cell culture virus isolates, as well as directly on specimens (see below). elec tron microscopy allows identification only to the level of family, whereas immunoelectron microscopy using suitable specific antibody may per mit finer distinctions to be made. complement fixation. for the complement fixation test, the acute and convalescent sera are heated ( °c for minutes) to inactivate complement, then serially diluted in a plastic tray. two to four units of antigen (e.g., a crude preparation of live or inactivated virus) are then added to each serum dilution together with units of complement, derived from a guinea pig. the reagents are allowed to interact at °c overnight, to allow the complement to become "fixed/' sheep erythrocytes, "sensitized" by the addition of rabbit antiserum to them ("hemolysin") are then added and the trays are incubated at °c for minutes. in those cups where the complement has been fixed by the virusantibody complex, the hemolysin fails to lyse the sheep erythrocytes; where complement is still present, they are lysed. crude cell culture supernatants often used for complement fixation tests contain not only mature virions but a range of soluble antigens, both structural and nonstructural. since many of these are shared by many or all viruses within a particular genus or family, e.g., mastadenovirus, they will cross-react with antibodies raised against any other member of the genus or family. this property makes complement fixation a useful method for preliminary screening of an isolate-to place it within the correct family or genus. immune-adherence hemagglutination is basically a somewhat simplified version of the complement fixation test; currently it is applied more often to the detection of antibody than that of antigen. hemagglutination and hemagglutination inhibition. virions of several viral families bind to red blood cells and cause hemagglutination. if antibody and virus are mixed prior to the addition of red blood cells, hemagglutination is inhibited (table - ; plate - ). the hemagglutination titer of certain viruses, e.g., canine distemper virus, may be increased by dissociation of the virions with detergents. antisera may have to be pretreated to remove nonspecific inhibitors of hemagglutination (see chapter ). the hemagglutination inhibition test is sensitive and, except in the case of the togaviruses, highly specific, since it measures antibodies binding to the surface protein most subject to antigenic change. moreover, it is simple, inexpensive, and rapid, and is therefore the serological procedure of choice for identifying isolates of hemagglutinating viruses. virus neutralization. the infectivity of a virus may be neutralized by specific antibody by a variety of mechanisms (see chapter ). serum must first be "inactivated" by heating at °c for minutes to remove nonspecific virus inhibitors. serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the "virus titer only" controls develop cytopathic effects (plate - ). antibody, by neutralizing the infectivity of the virus, protects the cells against viral destruction. in keeping with the general trend toward miniaturization, most neutralization tests are now conducted in disposable nontoxic sterile plastic trays with, say, flat-bottomed wells in each of which a cell monolayer can be established. virus-antiserum mixtures can be added to established monolayers, or, more usually, serum dilutions are made in the wells, a standard amount of virus is added, and the mixture incubated, after which cells are added. in the standard neutralization test the end point is read by cytopathic effect, the titer of the serum being defined as the highest dilution that inhibits the cytopathic effect. in a version of the neutralization test known as the plaque reduction assay, cell monolayers inoculated with virus-serum mixtures are overlaid with agar or methylcellulose and incubated until plaques develop (see plate - ); the end point is usually taken to be the highest dilution of serum reducing the number of plaques by at least %. if a newly isolated virus proves to be "untypeable," i.e., not neutralizable by antisera against any of the known serotypes, it may be a novel serotype, or it may indicate a mixed infection with two distinct viruses, or aggregation of virions in the specimen. aggregates can be removed by vigorous agitation, filtration, or, in the case of some nonenveloped viruses, dispersed with sodium deoxycholate, prior to repeating the neutralization test. for most routine diagnostic purposes it is usually not necessary to "type" the isolate antigenically, even to the degree just described. sometimes, however, important epidemiological information can be obtained by going even further, to identify differences between "variants" or subtypes within a given serotype (see table - ). short of determining the complete nucleotide sequence of viral nucleic acid, the most useful methods of doing this are by oligonucleotide fingerprinting of viral rna or the determination of restriction endonuclease fragment patterns of viral dna. with rna viruses, viral rna is labeled with p during replication of the virus in culture; the labeled rna is phenol-extracted from purified virions, digested with ribonuclease tl, and the resulting oligonucleotide fragments separated by two-dimensional polyacrylamide gel electrophoresis, or by cellulose acetate electrophoresis followed by deae-cellulose chromatography. autoradiography reveals a // fingerprint ,, unique to that particular viral strain. an example of the epidemiological use of this technique to trace the origin and spread of foot-and-mouth disease virus in europe in is described in chapter . similarly, viral dna prepared from virions or infected cells can be cut with appropriately chosen restriction endonucleases and the fragments separated by agarose gel electrophoresis. when stained with ethidium bromide or silver, restriction endonuclease fragment patterns (also called fingerprints) are obtained. the method has found application with all dsdna virus families, particularly in epidemiological studies, but also in understanding pathogenesis. depending on the viral family, the resolution of these methods is such that different isolates of the same viral species may be distinguishable, unless they come from the same epizootic. minor degrees of genetic drift, often not reflected in serological differences, can sometimes be detected in this way. the isolation and identification of a particular virus from an animal with a given disease is not necessarily meaningful in itself. fortuitous subclinical infection with a virus unrelated to the illness in question is not uncommon. koch-henle postulates (see chapter ) are as apposite here as in any other microbiological context, but are not always easy to fulfill. in attempting to interpret the significance of any virus isolation, one must be guided by the following considerations: . the site from which the virus was isolated is important; e.g., one would be quite confident about the etiological significance of equine herpesvirus isolated from the tissues of a -month-old aborted equine fetus with typical gross and microscopic lesions, or of distemper virus isolated from the cerebrospinal fluid of a dog with encephalitis, because these sites are usually sterile, i.e., they have no normal bacterial or viral flora. on the other hand, recovery of an enterovirus from the feces, or a herpesvirus from a nasal or throat swab may not necessarily be significant, because such viruses are often associated with inapparent infections at these sites. . interpretation of the significance of the isolation in such instances will be facilitated by recovery of the same virus from several cases of the same illness during an epizootic. . knowledge that the virus and the disease in question are often causally associated provides confidence that the isolate is significant. it is appropriate to conclude this section with some remarks about safety precautions in virus diagnostic laboratories in general and regulations about exotic viruses in particular. diagnostic virology is one of the less hazardous human occupations, but over the years a number of deaths have been caused by laboratory-associated infections. some of the commoner hazards are listed in table - . it is important to note that many of the procedures that may be dangerous for laboratory workers, particularly aerosolization, may also be sources of laboratory contamination-something that may give rise to mistaken diagnoses and sometimes a great deal of misdirected administrative action. precautions to avoid these hazards consist essentially of good laboratory technique, but special measures may be needed. mouth-pipetting is banned. gowns must be worn at all times, and gloves for anything besides personal hazard, exotic animal viruses pose special community risks such that major developed countries with large livestock indus- tries support special laboratories for their investigation. these are the so-called maximum containment laboratories, popularly designated by their location, e.g., plum island in the united states, pirbright in the united kingdom, and geelong in australia. "restricted" animal viruses in the united states and australia, the importation, possession, or use of which is prohibited or restricted by law or regulation, are listed in table - . we use the term direct identification, in contrast to virus isolation, to encompass a variety of methods that can be used to detect and often identify the etiological agent by the direct demonstration of virions or viral constituents in the tissues, secretions, or excretions of infected animals. although they do not provide the laboratory worker with a culture of the causative virus for further study, these direct methods have great advantages in terms of speed, cost, and the number of samples that can be examined. they can be subdivided into methods used to detect virions, viral antigens, or viral nucleic acids. the introduction of negative staining procedures, together with a realization that in many clinical situations the concentration of virions frequently exceeds the critical lower limit of per milliliter required for visualization in the electron microscope, has led to the use of this instrument for rapid viral diagnosis (plate - ). the procedure is particularly suited to enteric infections, in which a crude fecal suspension can be clarified by low-speed centrifugation, followed by high-speed centrifuga tion to yield a pellet for negative staining. in addition to its value in the recognition of known viruses, this technique has led to the discovery of new viruses of etiological importance in diarrheal diseases which were, and in some cases remain, uncultivable (e.g., some adenoviruses, astroviruses, caliciviruses, coronaviruses, parvoviruses, and rotaviruses). the procedure is also suited to viral infections of the skin and mucous membranes, the appropriate specimen being scabs, vesicular fluid, or scrapings made with a scalpel. also, as described earlier, electron microscopy can be used for the rapid identification of viruses isolated in cell culture, allowing immediate and definitive diagnosis to the family or sometimes the genus or species level. the sensitivity of electron microscopic methods can be enhanced by the use of immune serum, by a procedure known as immunoelectron microscopy. the sample, usually clarified by low-speed centrifugation, is mixed with antibody, and after overnight interaction, the immune complexes are pelleted by low-speed centrifugation and the pellet negatively stained. the antibody used may be serum from an old animal hyperimmune to a large number of viruses, or it may be type-specific polyclonal or monoclonal antibody, or such antibodies may be used sequentially. solid-phase immunoelectron microscopy procedures have also been developed, in which virus-specific antibody is first bound to the plastic supporting film on the copper grid. sensitivity is enhanced by a double-layering procedure, in which staphylococcal protein a (which binds the fc moiety of igg) is bound to the film, then virus-specific antibody, to which the sample is then added. . negative staining for electron microscopy. (a) direct staining: virions of bovine papular stomatitis virus. an electron-opaque stain ( % phosphotungstic acid, ph . ) was mixed with scrapings from the lesion and applied to plastic film supported by the copper electron microscope grid (xl , ). (b) immunoelectron microscopy: an isolate of foot-and-mouth disease virus type o was incubated with homotypic antiserum and stained with phosphotungstic acid (χΙΟΟ,ΟΟΟ). note aggregation of virions by antibody. [from e. p. ] . gibbs et al., vet. ree. , ( ) .] these methods are based on direct interaction between virions or viral antigens, in situ in tissues or in excretions or secretions, and specific antibodies which are prelabeled in some way so as to permit the ready recognition of the interaction. the methods are specified by the method of labeling used: immunofluorescence, immunoperoxidase staining, radioimmunoassay, or enzyme-linked immunosorbent assay (elisa). vir- al antigens can also be detected by such time-honored serological procedures as precipitation and complement fixation. immunofluorescence. its specificity, sensitivity, rapidity, and relative simplicity make immunofluorescence a procedure of singular importance in the rapid diagnosis of viral infections. the prototypic example of immunofluorescence is the diagnosis of rabies, for which it has been the standard test for more than years (see chapter ). it is now being used for a wide range of viruses. immunofluorescence can be applied to smears and frozen sections of tissues or organs. two alternative staining procedures are used: ( ) direct immunofluorescence, in which the antiviral antibody is conjugated to the fluorescent dye, fluorescein, and ( ) indirect ("sandwich") immunofluorescence, in which an antiimmunoglobin specific for the animal species providing the antiviral antibody is conjugated to fluorescein ( fig. - ) . for instance, an acetone-fixed smear or frozen tissue section is treated with virus-specific antibody (prepared, say, in rabbits), then rinsed before the second antibody, a fluorescein-conjugated anti-rabbit immunoglobulin made in goats, is added. indirect procedures have two significant advantages over direct-staining procedures: . if antibodies to different viruses are raised in a single animal species, e.g., rabbits, then only a single conjugated antibody is required. . the amount of bound labeled antibody is greatly augmented, hence the method is much more sensitive. although simple in principle, the effective use of immunofluorescence demands careful attention to many technical details if false positive results are to be avoided. in addition to immunofluorescent staining of specimens taken directly from clinical cases, the method is an important adjunct in the identification of viruses isolated in cell cultures. it may also be used in reverse, for the detection of antibody in serum. slides containing viral antigen, either smears, sections, or, more usually, cell cultures, are prepared in large numbers and stored at - °c. for use, they are flooded with the serum under test and a second fluorescein-conjugated antispecies antibody is used to detect the bound antibody. special care needs to be exercised in the application of immunofluorescence to herpesviruses, in that some herpesvirus-infected cells are known to express fc receptors on their plasma membrane; such receptors bind all igg molecules, not only those with herpesvirus specificity, hence additional controls are required. staining. an alternative method for locating and identifying viral antigen in infected cells employs an enzyme-labeled antibody. the procedure requires less expensive equipment than immunofluorescence-an ordinary light microscope is used-and produces a morphologically clearer, nonfading, permanent preparation. the procedures and principles are similar to those of immunofluorescence. the conjugated antibody, bound to antigen by a direct or indirect procedure, is detected by adding a substrate appropriate to the particular enzyme; in the case of peroxidase this is h mixed with a benzidine derivative which forms a colored, insoluble precipitate in the presence of enzyme. a disadvantage of the technique is that endogenous peroxidase present in the cells of many tissues, particularly leukocytes, produces false positive results. this problem can be circumvented by meticulous technique and adequate controls. in radioimmunoassay the label is a radioactive element, commonly i. the method is exquisitely sensitive, enabling viral antigens to be detected at concentrations as low as ~ m. many protocols for radioimmunoassay s have been devised. both direct and indirect methods can be used, the principles being the same as for immunofluorescent staining (fig. - which the "capture" antibody (or antigen) is bound to a solid substrate, typically a polystyrene tube or bead, or to the wells of a plastic microtiter plate. in the simplest format ( fig. - , left) the sample suspected to contain virus or viral antigen is allowed to bind to the bound antibody, then after washing, i-labeled antiviral antibody ("detector" antibody) is added. after a further washing, the bound labeled antibody is mea sured in a gamma counter. a more commonly used protocol is the indirect radioimmunoassay, in which the detector antibody is unlabeled but a further layer, i-labeled anti-igg, is added as "indicator" anti body. (the antiviral antibodies constituting the capture and detector antibodies must be raised in different animal species; see fig. - , right.) enzyme-linked immunosorbent assays (elisa). elisa (also known as enzyme immunoassay, eia) offers the same sensitivity as radioim munoassay without the inherent disadvantages of expensive isotopes of short half-life and the need for safe handling and disposal and a costly gamma counter. the basic principles are similar to those of radioim munoassay ( fig. - ) . antibody is bound to a solid phase, usually the wells of a microtiter tray. samples suspected to contain antigen are added to the wells. after an appropriate reaction time, the wells are rinsed and a second virus-specific antibody that has been conjugated to an enzyme is added. after allowing this to bind, the contents of the well are rinsed and a substrate for the enzyme is added. the assay is read by a color change in the substrate and can be made quantitative by serially diluting the antigen to obtain an end point or by photometrically reading the amount of color change, a reflection of the amount of enzymeconjugated antibody bound. as in radioimmunoassay, there are many variations in protocol, e.g., exploiting the very high affinity of avidin for biotin ( fig. - , right) . moreover, if antigen is bound to the plate first, the procedure is equally suitable for the detection and quantitation of viral antibody. elisa procedures have been developed for a wide variety of applications in veterinary medicine. at one level, kits have been marketed for the rapid diagnosis of a number of important viral diseases by veterinary practitioners themselves. at another level elisa procedures have been automated by the introduction of automatic dispensing, washing, and spectrophotometric reading and recording instruments, that permit hundreds of samples to be processed in a day, e.g., in the testing of swine for pseudorabies antibodies. immunodiffusion (precipitation-in-gel). if wells are cut in agar and antibody and antigen are placed in separate wells, the two diffuse toward each other (immunodiffusion) and form visible bands of precipitate (plate - ). several ingenious applications of the procedure have been developed and the test is widely used for the diagnosis of some diseases of domestic animals (e.g., bovine leukemia, equine infectious anemia; see chapter ). although now considered too cumbersome a procedure for general use in the rapid detection of viral antigen, the complement fixation procedure is still employed for the rapid and specific detection of foot-and-mouth disease viral antigen in vesicular fluid, providing both rapid diagnosis and specific typing of the virus involved. if dsdna is separated ("melted") into single strands by heat or alkali treatment, the single strands will, under appropriate conditions, reanneal to each other, or competitively to an identical or related complementary strand. if the original dna is labeled with either p or s, it may be used as a probe for the detection of related dna in infected cells. this procedure, known as in situ hybridization, can be made even more specific by using probes that are shorter than the full-length genome. it can also be made more sensitive by maximizing the amount of label incorporated into the probe. hybridization is detected by autoradiography. recently, nonradioactive hybridization procedures, based on the incorporation of biotin-conjugated nucleotides into the dna probe, have been developed; avidin, which binds strongly to biotin, is subsequently added. the avidin is detected by elisa, immunofluorescence, or immunoperoxidase staining. in situ hybridization procedures are particularly useful when viral dna is present in cells but is not expressed, as with integrated retroviral dna, or episomal dna in some papo va virus-infected cells. probes can be made highly specific by selection from a collection of cloned fragments of the whole viral genome. the probes are labeled by in vitro nick translation procedures, and are then applied to nitrocellulose blot transfers of the animal tissue (active hybridization) or to nitrocellulose blots taken from gels on which viral nucleic acid has been separated (southern blotting), or from nitrocellulose onto which viral nucleic acid-containing samples have been spotted (dot-blot hybridization). these procedures have proved of great value in virus research, but it remains to be seen to what extent nucleic acid probes and hybridization procedures displace other methods for rapid diagnosis of viral infections. it may have advantages over virus isolation in the case of viruses that are noncultivable, slow growing, dangerous, or nonviable as a result of suboptimal conditions of transport or storage. detection of viral antibody can be used for the diagnosis of viral infections, either in individual animals or in populations. the method is particularly useful in the latter context, since serum samples are readily obtained with simple equipment, in contrast to special requirements, time, and effort needed for collecting samples for virus isolation. furthermore, tests for antibody such as elisa lend themselves to automation, so that large numbers of samples can be tested. they form the basis of epidemiological surveys and of control and eradication programs, but have major limitations in diagnosis. for diagnosis in the individual animal, paired sera are tested for specific viral antibody, the first sample being taken when the animal is first examined (acute-phase serum), and the second sample - weeks later (convalescent-phase serum). a rise in antibody titer between the first and second samples is a basis, albeit in retrospect, for a specific viral diagnosis. sometimes the demonstration of antibody in a single serum sample is diagnostic of current infection, e.g., with retroviruses and herpesviruses, since these viruses establish lifelong infections. however, in such circumstances there is no assurance that the persistent virus was responsible for the disease under consideration. detection of antiviral antibody in presuckle newborn cord or venous blood provides a basis for specific diagnosis of in utero infections. it was used, for example, in showing that akabane virus was the cause of arthrogryposis-hydranencephaly in calves (see chapter ). since transplacental transfer of immunoglobulins is rare in domestic animals (see table - ), the presence of either igg or igm is indicative of exposure of the fetus to antigen. serological methods based on the detection of virus-specific igm may also be used for the specific diagnosis of recent viral infection, since antibodies of the igm class appear first after primary infection and de-clines to relatively low levels, compared to igg, by about months after infection. however, the method has not yet been much exploited in veterinary medicine. technical advances such as miniaturization (microtiter plates), automation for large numbers of samples, monoclonal antibodies, and the development of diagnostic kits such as latex agglutination assays for detecting specific igm, have resulted in a revolution in the approach to diagnostic serology in human medicine. the costs, coupled with the technical problems associated with the large number of animal hosts and their many viruses, have delayed the development of these procedures in veterinary medicine, but their use can be expected to expand considerably in the future. however, screening programs to establish regional or national prevalence rates for particular viruses, based on detection of specific antibody in single serum samples, are an essential feature in defining the epidemiology of viruses of domestic animals. biosafety in microbiological and biomedicai laboratories oligonucleotide fingerprinting of viral genomes diagnostic virology using electron microscopic techniques rapid virus diagnosis radioimmunoassay in diagnostic virology new developments in practical virology laboratory diagnosis of viral infections diagnostic procedures for viral and rickettsial infections manual of clinical microbiology recent advances in virus diagnosis guide to the collection and transport of virological specimens laboratory diagnosis of viral diseases laboratory safety: principles and practices manual of clinical laboratory immunology automated systems in viral diagnosis immunochemistry of viruses. the basis for serodiagnosis and vaccines enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects key: cord- - eq jth authors: chen, weizao; zhu, zhongyu; liao, huaxin; quinnan, gerald v.; broder, christopher c.; haynes, barton f.; dimitrov, dimiter s. title: cross-reactive human igm-derived monoclonal antibodies that bind to hiv- envelope glycoproteins date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: eq jth elicitation of antibodies with potent and broad neutralizing activity against hiv by immunization remains a challenge. several monoclonal antibodies (mabs) isolated from humans with hiv- infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. all known broadly neutralizing mabs (bnmabs) are immunoglobulin (ig) gs (iggs) and highly somatically hypermutated which could impede their elicitation. ig ms (igms) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to iggs. here we describe the identification and characterization of several human igm-derived mabs against hiv- which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of healthy donors. these antibodies bound with high affinity to recombinant envelope glycoproteins (gp s, envs) of hiv- isolates from different clades. they enhanced or did not neutralize infection by some of the hiv- primary isolates using ccr as a coreceptor but neutralized all cxcr isolates tested although weakly. one of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. it bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the cd binding site. these results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. further studies will show whether such a strategy plays a role in hiv infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. the newly identified mabs could be used as reagents to further characterize conserved non-neutralizing, weakly neutralizing or enhancing epitopes and modify or remove them from candidate vaccine immunogens. the ability of human immunodeficiency virus type (hiv- ) to rapidly generate mutants and evade immune response is the major obstacle for development of protective, prophylactic hiv- vaccines. therefore, candidate vaccine immunogens must be capable of eliciting broadly neutralizing antibodies (bnabs) that inhibit viruses from different genetic subtypes. several human monoclonal antibodies (hmabs) such as b [ ] , x [ ] , g [ ] , f and e [ , ] exhibit potent and broad hiv- neutralizing activity in vitro and can prevent hiv- infection in animal models [ ] . these bnabs target structures on hiv- envelope glycoprotein (env) that are crucial for virus-cell fusion. therefore, envs in various formats are potential candidate immunogens and have been evaluated in animal models and human clinical trials [ ] [ ] [ ] [ ] . however, neutralization efficacy of the resulting sera as broad as that by those bnabs has not been achieved by empirically using these envs as immunogens [ ] . it has been suggested that characterization of the epitopes of the bnabs could help design vaccine immunogens that would be able to elicit these bnabs or similar antibodies in vivo [ ] . although this approach is being vigorously pursued, none of the immunogens designed has yet efficiently elicited neutralizing antibodies with broad specificity. igm is the initial antibody that the host generates when an infectious agent or antigen is encountered. activation of igm-expressing b cells provides impetus for in vivo igm-to-igg isotype switch resulting in production of high-affinity neutralizing or non-neutralizing antibodies. it is therefore important to investigate the human igm repertoire for hiv- env-specific antibodies and understand how they interact with hiv- envs and impact on viral infection which could help design effective immunogens. previous attempts to select hiv-specific antibodies by use of non-immune libraries have resulted in antibodies with modest neutralizing activity and limited breadth of neutralization [ ] . studies by several groups show that human igm antibodies play important roles not only in shaping humoral immunity against hiv- but also in inducing cell-mediated response because of their pentameric binding nature as well as their very efficient activity to activate complement. torán et al. [ ] indicated that in vivo igm-to-igg isotype switch and affinity maturation may be important for protection and long-term survival in certain hiv- -infected individuals. sheppard et al. [ ] demonstrated that complement-deactivated serum from a healthy volunteer immunized with an hiv- clade c gp exhibited igm-associated neutralizing activity that was significantly enhanced in the presence of fresh normal human serum as a source of complement. bomsel et al. [ ] showed that a human anti-hiv- gp igm monoclonal antibody blocked the transcytotic route of hiv mucosal transmission in vitro. in this study, we describe identification and characterization of several human igm-derived mabs selected from phage-displayed naive human antibody libraries constructed from healthy donors. these antibodies have high affinity and cross-reactivity with hiv- gp s from different clades, and either neutralize or enhance weakly entry of virions pseudotyped with envs from hiv- primary isolates. these data provide additional insights into the possible immune response that hiv- infection or viral env-based immunization could elicit and help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. a large ( . × members) phage-displayed naive human antibody antigen-binding fragment (fab) library (designated m ) was constructed from peripheral blood b cells of healthy donors, spleens of healthy donors, and lymph nodes of healthy donors as described in materials and methods. the phagemid used for library construction, pzyd-n , was synthesized and briefly described previously [ ] . to approximately estimate the sequence diversity of m , clones were randomly selected from the library and sequenced. no identical sequences of heavy and light chains were found; more than % of the clones are productive. of the clones had kappa light chains and the other -lambda light chains. m was panned against several hiv- envs and human cancer-related antigens; significant enrichment was obtained with all the pannings (data not shown). these results indicate that the quality of the library is likely to be good. to identify human igm-derived antibodies specific for hiv- envs, we panned m against the gp of a clade b isolate, r (gp r ). one of the selected antibody clones, r h , contained a tga stop codon at the very beginning of the light chain due to a nucleotide deletion that resulted in a reading-frame shift. the heavy chain variable domain (vh) of r h , designated m , differs from the closest human germline sequence (vh - ) by mutations distributed in all frameworks (frs) and complementarity determining regions (cdrs) (figure a ). m contains mutations in nucleotide sequence of the variable (v) region resulting in mutations in amino acid sequence. we corrected the reading frame of the light chain by site-directed mutagenesis and the corrected antibody, designated r h m, was expressed at high levels in bacteria as an fab. fab r h m specifically bound with relatively high affinity to gp r (ec , ~ nm) and the gp of a clade c isolate, gxc (gp gxc ) (ec , ~ nm). because the antibody was initially selected as a functional heavy chain-only antibody, we hypothesized that the binding activity of r h m should be attributed mainly to the heavy chain of the antibody and the heavy chain could be paired with light chains derived from different germlines while retaining cross-reactivity with hiv- envs. we cloned m as an isolated antibody domain and found that it was highly soluble, stable, monomeric, and expressed at high levels in bacteria [ ] . in agreement with our hypothesis it specifically bound to gp r although with low affinity (ec , > nm). we further constructed a light chain-shuffling fab library ( × members) based on the heavy chain of r h . to increase the probability for selection of cross-reactive antibodies the library was panned sequentially against gp r and the gp of a clade f isolate, / / (gp / / ). five unique clones were identified that contained exclusively lambda light chains ( figure b) . they bound to gp r and gp / / with ec ranging from to nm (table ) while no significant binding to an unrelated antigen, bovine serum albumin (bsa), was observed (data not shown). one of the antibodies, m , also bound with high affinity to the envs of other three clade b isolates, gp bal, gp jrfl and gp con-s [ ] . these results suggest that m and possibly the other selected antibodies target conserved epitopes on gp . to test whether the five newly selected antibodies are capable of neutralizing hiv- primary isolates, we used viruses pseudotyped with envs from hiv- isolates representing clades a, b and c and using either ccr (r ) or cxcr (x ) or both (r x ) as a coreceptor. none of the antibodies exhibited potent broadly neutralizing activity. four antibodies (fabs m , m b, m c and m d) enhanced infection by bal even at very low antibody concentration while a positive control antibody, m [ ] , showed efficient neutralization and a negative control antibody, a , did not affect entry ( figure a ); m d enhanced infection by another clade b primary isolate, jrfl, at high concentrations ( figure b) . interestingly, the three antibodies that enhanced significantly infection by the bal weakly neutralized iiib which is a tcla clade b x isolate (figure c ). to find whether the activity of the antibodies is related to antibody size and how the viral infection could be affected by cross-linking of hiv- envs, we generated a single-chain fv fragment (scfv) (scfv m ) of m and a human igg fc-fusion protein (m fc) of scfv m ; m was selected for further characterization because its light chain was relatively less divergent from the germline ( figure b) and was the only one which did not contain any somatic mutations in the cdr of the light chain. when bal was tested, the enhancing activity of scfv m was comparable to that of fab m and slightly lower than that of m fc (data not shown). these data indicate that the antibody access to the epitope was not size-restricted and that the bivalency of the fc fusion protein strengthened the antibody activity probably due to avidity effects. the ability of m fc to modulate hiv- infection in vitro was further tested with six additional clade b isolates and an isolate each from clade a and clade c, respectively ( figure ) . enhancement was observed with jrcsf (clade b, r ) at very low m fc concentration and the enhancement was decreased with an increase in antibody concentration. m fc also conferred slight enhancement of infection by jrfl (clade b, r ) and ug . (clade a, r ) at about nm antibody concentration. it neutralized the clade b x isolates iiib and nl - . it also neutralized r , which is a clade b r isolate, displays some cd independence and is moderately neutralization sensitive, as well as gxc which is a clade c r isolate. no significant antibody activity was observed with the dual tropic clade b isolate . . these results indicate that the antibody activity in terms of neutralization or enhancement varies when different isolates are tested and is not significantly correlated with the coreceptor usage and the cd dependency of the viruses in entry. antibody, b (a) , and two cd i antibodies, m and m (b), in binding to gp bal and to gp bal -cd , respectively. an irrelevant human antibody, a , was used as a negative control. to approximately localize the antibody epitopes and begin to elucidate the underlying mechanisms of antibody neutralizing or enhancing activity, we measured the antibody binding to envs from different isolates alone and in complex with cd as well as the antibody competition with wellcharacterized antibodies. m fc bound to a single-chain polypeptide analogue of the hiv- gp -cd complex (gp bal -cd ) [ ] with threefold higher affinity (ec , ~ . nm) than to gp bal (ec , ~ nm) alone as measured by an elisa (figure a ). it did not react with scd suggesting that the antibody targeted a structure on gp that was outside the cd -binding site (cd bs) and could undergo cd -induced (cd i) conformational changes. the conformational changes on gp bal after cd binding were confirmed by a cd i bnab, m fc [ ] , which exhibited dramatically higher binding to gp bal -cd than to gp bal (figure a ). to find out whether the antibody bound to the conventional cd i epitopes, we used gp con-s , which was a synthetic env designed by aligning the consensus env sequences of group m [ ] . this env, by design, is composed by shorter v , v , v and v loops and therefore, might expose regions around the variable loops that might contain conserved neutralizing determinants. however, m epitope as a representative for cd i epitopes is still completely hidden on gp con-s because m fc did not bind in the absence of scd , it did bind with high affinity in the presence of scd (figure b) . however, m fc showed comparable binding to gp con-s with or without scd (figure b) suggesting that the antibody does not precisely target cd i epitopes but other structures on the gp . although m fc was not directed against the cd binding pocket, it strongly competed with the cd bs bnab, scfv b , which has binding surface larger than that of cd [ ] (figure a ). m fc also competed although weakly with two cd i antibodies, scfv m and m [ ] (figure b) . these results suggest that the epitope of m fc is in very close proximity to the cd bs and the coreceptor-binding site (corbs) which overlaps with cd i epitopes on gp ( figure ). figure . a schematic representation for the epitope (gray) of m on gp (blue). the red and white areas denote the cd bs and the corbs, respectively. the epitope (long dash-dotted circle on the left) of the cd bs antibody, b , is indicated in comparison with the cd -binding area. the dashed circle on the right denotes the corbs for some isolates that could partially overlap with the epitope of m . to estimate the possibility of rapid elicitation of these antibodies in vivo when an hiv- gp related immunogen is administered, we generated a germline-like scfv of m (scfv m gem) and measured its binding to envs from different isolates in the presence or absence of scd . no obvious interaction was observed with scfv m gem when several envs that bound the original m were tested (data not shown). previous studies showed that the binding activity of an antibody in the form of fab or scfv could be increased up to thousands of times when it is converted to dimeric formats such as an igg. we therefore fused scfv m gem to the fc portion of a human igg to gain possible avidity effects. still, the fc-fusion protein of scfv m gem (m gemfc) did not show measurable binding to the envs with or without scd (figure ) . these results suggest that significant somatic diversification is required for the m corresponding germline antibody to achieve recognition of the m epitope on gp . they also indicate that in some individuals certain igm antibodies could undergo somatic hypermutations to relatively high-affinity binders to the env in the absence of hiv- infection or immunization with env. figure . binding of the germline-like antibody of m to different gp s in the absence or presence of cd . the original antibody, m fc, was used as a positive control. vaccines based on recombinant envs have failed to prevent viral infection in human efficacy trials likely because of their inability to elicit neutralizing antibodies that are broad enough to combat the extremely high variability of the virus. recent success was reported but it is modest and needs to be repeated and further examined [ ] . to date only several bnabs have been isolated from hiv- patients suggesting difficulties to elicit such antibodies in vivo. however, the presence of epitopes recognized by these bnabs on envs argues that they are still a potential template for vaccine design. different strategies aiming at improving the presentation of neutralizing epitopes are rigorously pursued. analysis of the structures of envs shows that most of their surface is hidden from humoral immune responses by glycosylation and oligomeric occlusion [ , ] . the cd bs and the corbs on gp are also flanked by loop structures which may further limit the access by antibodies generated by the human immune system [ , ] . therefore, one strategy focuses on the use of modified envs, in which the variable loops have been deleted [ ] [ ] [ ] [ ] or glycosylation removed [ , ] or both [ ] in order to increase the exposure of the neutralizing epitopes. based on the finding that the neutralizing capacity of the antibodies is associated with their ability to bind to native trimeric envs on the virus but does not correlate with binding to isolated monomeric envs, another strategy focuses on preservation or re-construction of the functional trimeric envs [ , ] . the essential concept is that immunizing with a close mimic of the functional trimer will improve the chances of eliciting neutralizing antibodies. yet another strategy is to use fusion intermediates formed during virus entry [ , ] or their mimics [ ] . hiv- entry is initiated by binding of viral gp with the receptor cd and a coreceptor on the target cell surface. these interactions lead to intermediate env conformations that may include conserved structures useful for vaccine design. however, elicitation of desirable level and breath of neutralizing antibodies with the immunogens generated based on these strategies has not been achieved. while modification of envs based solely on the analysis of the interaction with neutralizing antibodies is not sufficient to create effective vaccines, we propose that the inherent capacity of the human immune system in response to hiv- infection should be explored. we have hypothesized that investigation of human igm repertoire for hiv- env-specific antibodies and understanding how they react with envs, evolve and impact on viral infection would help design effective hiv- immunogens. it has been previously found that igm-based response confers substantial effects on hiv- infection which are especially relevant in the context of acute infection [ ] [ ] [ ] . in this study, we describe the identification and characterization of several high-affinity human igm-derived mabs against hiv- from phage-displayed naive human antibody libraries constructed from healthy donors. although their potency in vitro appears minor the roles they could play in mediating hiv- infection in vivo should not be underestimated. igm antibodies are highly effective in activating complement system to destroy the virus or infected cells. the antibodies selected in this study target highly conserved regions on gp which are in very close proximity to the cd bs and the corbs. because these two regions on gp are vital for virus entry and harbor neutralizing epitopes, hiv- has evolved strategies such as steric occlusion to protect it from humoral immunity [ , ] . here we suggest that the possible high immunogenicity of the conserved non-neutralizing epitopes of these antibodies could further divert the immune system from responses to neutralizing epitopes. recent studies [ , ] showed that crossreactive non-neutralizing antibodies (igms and iggs) were elicited by immunizing mice with recombinant hiv- gp s suggesting the same possibility in humans. because the antibodies we selected are less divergent (totally about amino acid mutations in the v gene products for the heavy and light chains) (figure ) than the known bnabs which all contain extensive somatic mutations (on average > mutations) compared to germline antibodies (data not shown), they could be more easily elicited. in addition, these antibodies could directly block the access of some neutralizing antibodies generated by the human immune system, especially those targeting the cd bs or corbs. this was supported by our finding that m fc partially reversed the neutralization by a cd i antibody, m [ ] (data not shown). these results further support previous propositions to reduce the immunogenicity of unwanted epitopes when gp /gp is used as an immunogen. we found that the closest germline-like antibody to m does not bind an env (figure ) . we observed similar lack of binding of germline-like b , f and g to envs [ ] [ ] [ ] . moreover, we found that there was a lack of or no high-affinity binders to envs in the germline-like antibody repertoire of the human cord blood whereas high-affinity antibodies against other human infectious agents including sars coronavirus and henipaviruses could be easily selected (weizao chen, emily streaker and dimiter s. dimitrov, in preparation). the identified antibodies against sars coronavirus protein s exhibited nanomolar affinity and were very close to germline in sequence. by analyzing the previously identified antibody sequences we found that all of the cross-reactive neutralizing antibodies against hiv- were highly diversified from their germline sequences; in contrast, the antibodies against sars coronavirus and henipaviruses had only limited number of mutations [ ] [ ] [ ] . these results indicate that extensive somatic mutations could be required for high-affinity binding to conserved hiv- env structures. one could speculate that the maturation pathways for some hiv- antibodies are initiated by immunogens that are different from the envs; such immunogens could be used in combination with env-based immunogens to guide the immune system for elicitation of potent bnabs by additional somatic hypermutation and selection. this is in line with our proposition to elicit bnabs by using one or more primary immunogens that are different than envs but can lead to intermediates on the maturation pathways that can be subsequently further somatically hypermutated to matured bnabs [ ] [ ] [ ] . except for the conventional antigen-antibody interaction, gp s also bind to a small population of human antibodies containing products of mainly vh gene family and exhibit superantigen properties by activating the human b cells expressing such antibodies on cellular membrane [ ] . the core motif of the superantigen-binding site (sbs) on gp is a discontinuous structure spanning the v variable domain and the amino-terminal region flanking the c constant domain [ ] . the determinants on the antibody vh domains critical for gp binding are not clear yet while the putative important regions were identified in the fr and ; a modeling study showed that most of the potential contact residues were located on the face of the vhs opposite to the interface for interaction with light chain; other positions in the cdr and also influenced the binding [ ] . moreover, the gp -positive antibody vh genes showed > % similarity to the germlines suggesting that the somatic hypermutations may have adverse effects on gp binding [ ] . the antibodies described in this study contained vh - gene products ( figure a ) and therefore, their high binding could be due to the superantigen-like interactions. however, our results showed that the isolated vh of these antibodies, m , bound to gp s with affinity (ec , > nm) much lower than that (ec , - nm) of the fabs. in addition, the epitope of one (m ) of these antibodies was mapped to a structure in very close proximity to the cd bs and the corbs on gp but not to the defined sbs. these results suggest that the majority, if not all, of the binding activities of the selected antibodies should be contributed by the conventional antigen-antibody recognition. one should note that these antibodies may not represent the native ones because they were selected from phage libraries where the heavy and light chains of the antibodies are randomly recombined. however, previous studies [ ] [ ] [ ] have shown that selection of high-affinity antibodies from such libraries results in antibodies which are identical or very similar to those occurring in the host from which the libraries are made. it has been shown that at least in some systems the antigen selected antibodies such as the autoantibodies against thyroid peroxidase (tpo) from large random phage libraries have the same pairings as those from small libraries where the cognate pairing is preserved and that pairing between light and heavy chain is not promiscuous [ , ] . the study by chapal et al. [ ] directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairings, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to tpo. de wildt et al. [ ] analyzed the cognate pairings of heavy and light chain variable domains in human igg+ b cells from peripheral blood and established that the pairings are largely random. the antibodies we selected were originated from large families of genes -heavy chain from vh and light chain from vl ( figure ) . importantly, the pairings for two of them, m and m d, have been found in the very limited number ( ) of truly human antibodies previously analyzed [ ] . moreover, some (e.g., m and m b) of these antibodies are relatively close to their corresponding germlines (figure ). these data suggest the possibility of eliciting such antibodies in vivo during hiv- infection or gp -based immunization. we purchased the t cells from atcc. other cell lines and plasmids used for expression of various hiv- envs were obtained from the national institutes of health aids research and reference reagent program (arrrp). recombinant gp s were produced in our laboratories. gp bal and the single-chain fusion protein gp bal -cd were kindly provided by t. fouts (institute of human virology, baltimore; currently at profectus, baltimore, md). horseradish peroxidase (hrp)-conjugated anti-flag tag antibody and hrp-conjugated anti-human igg (fc-specific) antibody were purchased from sigma-aldrich (st. louis). m was constructed by using phagemid pzyd-n according to the reported protocols [ ] . the sources for amplification of antibody gene fragments are commercially purchased poly a+ rnas (bd biosciences, san jose, ca) from peripheral blood b cells of healthy donors, spleens of three donors and lymph nodes of healthy donors, respectively. m l was used for selection of antibodies against hiv- antigens conjugated to magnetic beads (dynabeads m- epoxy; dynal inc., new hyde park, ny) as described previously [ ] . , , . and µg of gp r were used in the first, second, third and fourth round of panning, respectively. clones that specifically bound to gp r were identified from the third and fourth round by using monoclonal phage elisa (mpelisa) as described [ ] . a light chain-shuffling fab library ( × members) was further constructed based on the heavy chain of r h . the light chain repertoire was also harvested from a naive human fab library ( × members) constructed from peripheral blood b cells of healthy donors [ ] , in addition to that from m . the new library was panned sequentially with two gp s from different clades. for the sequential panning, and . µg of gp r were used in the first and third round, respectively; antigens were alternated with and . µg of gp / / during the second and fourth round, respectively. clones that bound to both gp r and gp / / were identified from the fourth round by using mpelisa as described [ ] . the following primers were used: m f, '-tgg ttt cgc tac cgt ggc cca gcc ggc cca ggt gca gct ggtg- ' (sense); m fcr: '-gtg agt ttt gtc ggg ccc tag gac ggt cag ctt gg- ' (antisense). for construction of scfv, the full-length original or germline scfv gene fragment was synthesized (genscript, piscataway, nj), digested with sfii and cloned into pcomb x. to generate fc-fusion protein, the scfv gene was pcr (primers m f and m fcr) amplified by using the synthetic scfv gene fragment as a template. the new scfv products appended with sfii and apai restriction sites on both sides were digested and cloned into psectagb-fc. the scfv and fab were expressed in e. coli hb , as described previously [ ] . the bacterial pellet was collected after centrifugation at , × g for min and resuspended in pbs (ph . ) containing . million-unit polymixin b (sigma-aldrich). after min incubation with rotation at rpm at room temperature, it was centrifuged at , × g for min at °c. the supernatant was used for purification of scfv and fab by immobilized metal ion affinity chromatography (imac) by using ni-nta resin (qiagen, valencia, ca) according to manufacturer's protocols. fc-fusion proteins were expressed in free style cells. fectin (invitrogen, carlsbad, ca) was used to transfect free style cells according to the instructions of the manufacturer. three days post-transfection, the culture supernatant was harvested and used for purification by using nprotein a sepharose fast flow (ge healthcarezcomx, piscataway, nj). binding and competition elisas were performed as described previously [ ] . viruses pseudotyped with hiv- envs were prepared by cotransfection of - % confluent t cells with pnl - .luc.e-r-and psv d constructs encoding hiv- envs by using the polyfect transfection reagent (qiagen) according to manufacturer's instruction. pseudotyped viruses were obtained after h by centrifugation and filtration of cell culture through . µm filters. for neutralization, viruses were mixed with different concentrations of antibodies for h at °c, and then the mixture was added to about . × hos-cd -ccr (used for all r and dual tropic viruses) or hos-cd -cxcr cells grown in each well of -well plates. luminesence was measured after h by using the bright-glo luciferase assay system (promega, madison, wi) and a lumicount microplate luminometer (turner designs). mean relative light units (rlu) for duplicate wells were determined. relative infectivity (%) was calculated by the following formula: (average rlu of antibody-containing wells/average rlu of virus-only wells) × . a major finding of this study is the identification and characterization of several hiv- -specific human igm-derived mabs and their highly conserved epitopes. such antibodies were rarely investigated and reported. further characterization of these antibodies, their dynamics and epitopes could provide knowledge that in addition to its usefulness for basic understanding of immune responses to hiv- could also help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. recognition properties of a panel of human recombinant fab fragments to the cd binding site of gp that show differing abilities to neutralize human immunodeficiency virus type broadly crossreactive hiv- -neutralizing human monoclonal fab selected for binding to gp -cd -ccr complexes human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp of human immunodeficiency virus type broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type glycoprotein gp human monoclonal antibodies and engineered antibody domains as hiv- entry inhibitors extensively cross-reactive anti-hiv- neutralizing antibodies induced by gp immunization correlation between immunologic responses to a recombinant glycoprotein vaccine and incidence of hiv- infection in a phase hiv- preventive vaccine trial a clinically relevant hiv- subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection prevention of disease induced by a partially heterologous aids virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine aiming to induce broadly reactive neutralizing antibody responses with hiv- vaccine candidates antibodies, viruses and vaccines a human monoclonal antibody neutralizes diverse hiv- isolates by binding a critical gp epitope molecular analysis of hiv- gp antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv- -infected individual a functional human igm response to hiv- env after immunization with nyvac hiv c intracellular neutralization of hiv transcytosis across tight epithelial barriers by anti-hiv envelope protein diga or igm human domain antibodies to conserved sterically restricted regions on gp as exceptionally potent cross-reactive hiv- neutralizers construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain a group m consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype b and c hiv- primary viruses expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type gp -cd receptor complex improved breadth and potency of an hiv- -neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning crystal structure of a neutralizing human igg against hiv- : a template for vaccine design vaccination with alvac and aidsvax to prevent hiv- infection in thailand the antigenic structure of the hiv gp envelope glycoprotein structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing human antibody the ability of an oligomeric human immunodeficiency virus type (hiv- ) envelope antigen to elicit neutralizing antibodies against primary hiv- isolates is improved following partial deletion of the second hypervariable region immunogenicity and ability of variable loop-deleted human immunodeficiency virus type envelope glycoproteins to elicit neutralizing antibodies immunogenicity of dna vaccines expressing human immunodeficiency virus type envelope glycoprotein with and without deletions in the v / and v regions selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type envelope influence of n-linked glycans in v -v region of human immunodeficiency virus type glycoprotein gp on induction of a virus-neutralizing humoral response role of n-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type gp improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers crosslinked hiv- envelope-cd receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques purified complexes of hiv- envelope glycoproteins with cd and ccr (cxcr ): production, characterization and immunogenicity mutagenic stabilization and/or disruption of a cd -bound state reveals distinct conformations of the human immunodeficiency virus type gp envelope glycoprotein broadly reactive monoclonal antibodies to multiple hiv- subtype and sivcpz envelope glycoproteins. virology production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type envelope glycoproteins in a mouse model expressing human immunoglobulins all known cross reactive hiv- neutralizing antibodies are highly divergent from germline and their elicitation may require prolonged periods of time germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv- envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens maturation pathways of cross-reactive hiv- neutralizing antibodies immunoglobulin vh gene products: natural ligands for hiv gp identification of the b cell superantigen-binding site of hiv- gp structural basis of the gp superantigen-binding site on human immunoglobulins influenza virus hemagglutinin-specific antibodies isolated from a combinatorial expression library are closely related to the immune response of the donor original and artificial antibodies thyroid peroxidase autoantibodies obtained from random single chain fv libraries contain the same heavy/light chain combinations as occur in vivo recombinant thyroid peroxidase-specific autoantibodies. i. how diverse is the pool of heavy and light chains in immunoglobulin gene libraries constructed from thyroid tissue-infiltrating plasma cells recombinant thyroid peroxidase-specific autoantibodies. ii. role of individual heavy and light chains in determining epitope recognition analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire construction of a large naive human phage-displayed fab library through one-step cloning potent neutralization of hendra and nipah viruses by human monoclonal antibodies we thank xiaodong xiao in our group for helpful discussion. key: cord- - rfdkuw authors: chen, jiahui; gao, kaifu; wang, rui; wei, guowei title: prediction and mitigation of mutation threats to covid- vaccines and antibody therapies date: - - journal: nan doi: nan sha: doc_id: cord_uid: rfdkuw antibody therapeutics and vaccines are among our last resort to end the raging covid- pandemic.they, however, are prone to over , mutations uncovered by a mutation tracker. it is urgent to understand how vaccines and antibodies in the development would be impacted by mutations. in this work, we first study the mechanism, frequency, and ratio of mutations on the spike (s) protein, which is the common target of most covid- vaccines and antibody therapies. additionally, we build a library of antibody structures and analyze their d and d characteristics. moreover, we predict the mutation-induced binding free energy (bfe) changes for the complexes of s protein and antibodies or ace . by integrating genetics, biophysics, deep learning, and algebraic topology, we deduce that some of the mutations such as m i, s f, and s f may weaken the binding of s protein and antibodies, and potentially disrupt the efficacy and reliability of antibody therapies and vaccines in the development. we provide a strategy to prioritize the selection of mutations for designing vaccines or antibody cocktails. the expeditious spread of coronavirus disease pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has led to , , confirmed cases and , , fatalities as of september , . in the st century, three major outbreaks of deadly pneumonia are caused by βcoronaviruses: sars-cov ( ) , middle east respiratory syndrome coronavirus (mers-cov) ( ), and sars-cov- ( ) [ ] . similar to sars-cov and mers-cov, sars-cov- causes respiratory infections, and the transmission of viruses occurs among family members or in healthcare settings at the early stages of the outbreak. however, sars-cov- has an unprecedentedly high infection rate compared to sars-cov and mers-cov [ ] . considering the high infection rate, high prevalence rate, long incubation period [ ] , asymptomatic transmission [ , ] , and potential seasonal pattern [ ] of covid- , the development of specific antiviral drugs, antibody therapies, and effective vaccines is of paramount importance. traditional drug discovery takes more than ten years, on average, to bring a new drug on the market [ ]. however, developing potent sars-cov- specified antibodies and vaccines is a relatively more efficient and less timeconsuming strategy to combat covid- for the ongoing pandemic [ ] . antibody therapies and vaccines depend on the host immune system. recently studies have been working on the host-pathogen interaction, host immune responses, and the pathogen immune evasion strategies [ ] [ ] [ ] [ ] [ ] [ ] , which provide insight into understanding the mechanism of antibody therapies and vaccine development. the immune system is a host defense system that protects the host from pathogenic microbes, eliminates toxic or allergenic substances, and responds to an invading pathogen [ ] . it has innate immune system and adaptive immune system as two major subsystems. the innate system provides an immediate but non-specific response, whereas the adaptive immune system provides a highly specific and effective immune response. once the pathogen breaches the first physical barriers, such as epithelial cell layers, secreted mucus layer, mucous membranes, the innate system will be triggered to identify pathogens by pattern recognition receptors (prrs), which is expressed on dendritic cells, macrophages, or neutrophils [ ] . specifically, pprs identify pathogen-associated molecular patterns (pamps) located on pathogens and then activate complex signaling pathways that introduce inflammatory responses mediated by various cytokines and chemokines, which promote the eradication of the pathogen [ , ] . notably, the transmission of sars-cov- even occurs in asymptomatic infected individuals, which may delay the early response of the innate immune response [ ] . another important line of host defense is the adaptive immune system. b lymphocytes (b cells) and t lymphocytes (t cells) are special types of leukocytes that are the acknowledged cellular pillars of the adaptive immune system [ ] . two major subtypes of t cells are involved in the cell-mediated immune response: killer t cells (cd + t cells) and helper t cells (cd + cells). the killer t cells eradicate cells invaded by pathogens with the help of major histocompatibility complex (mhc) class i. mhc class i molecules are expressed on the surface of all nucleated cells [ ] . the nucleated cells will firstly degrade foreign proteins via antigen processing when viruses infect them. then, the peptide fragments will be presented by mhc class i, which will activate killer t cells to eliminate these infected cells by releasing cytotoxins [ ] . similarly, helper t cells cooperate with mhc class ii, a type of mhc molecules that are constitutively expressed on antigen-presenting cells, such as macrophages, dendritic cells, monocytes, and b cells [ ] . helper t cells express t cell receptors (tcr) to recognize antigen bound to mhc class ii molecules. however, helper t cells do not have cytotoxic activity. therefore, they can not kill infected cells directly. instead, the activated helper t cells will release cytokines to enhance the microbicidal function of macrophages and the activity of killer t cells [ ] . notably, an unbalanced response can result in a "cytokine storm," which is the main cause of the fatality of covid- patients [ ] . correspondingly, a b cell involves in humoral immune response and identifies pathogens by binding to foreign antigens with its b cell receptors (bcrs) located on its surface. the antigens that are recognized by antibodies will be degraded to petites in b cells and displayed by mhc class ii molecules. as mentioned above, helper t cells can recognize the signal provided by mhc class ii and upregulate the expression of cd ligand, which provides extra stimulation signals to activate antibody-producing b cells [ ] , rendering millions of copies of antibodies (ab) that recognize the specific antigen. additionally, when the antigen first enters the body, the t cells and b cells will be activated, and some of them will be differentiated to long-lived memory cells, such as memory t cells and memory b cells. these long-lived memory cells will play a role in quickly and specifically recognizing and eliminating a specific antigen that encountered the host and initiated a corresponding immune response in the future [ ] . the vaccination mechanism is to stimulate the primary immune response of the human body, which will activate t cells and b cells to generate the antibodies and long-lived memory cells that prevent infectious diseases, which is one of the most effective and economical means for combating with covid- at this stage. as mentioned above, secreted by b cells of the adaptive immune system, antibodies can recognize and bind to specific antigens. conventional antibodies (immunoglobulins) are y-shaped molecules that have two light chains and two heavy chains [ ] . each light chain is connected to the heavy chain via a disulfide bond, and heavy chains are connected through two disulfide bonds in the mid-region known as the hinge region. each light and heavy chain contain two distinct regions: constant regions (stem of the y) and variable regions ("arms" of the y) [ ] . an antibody binds the antigenic determinant (also called epitope) through the variable regions in the tips of heavy and light chains. there is an enormous amount of diversity in the variable regions. therefore, different antibodies can recognize many different types of antigenic epitopes. to be specific, there are three complementarity determining regions (cdrs) that are arranged non-consecutively in the tips of each variable region. cdrs generate most of the diversities between antibodies, which determine the specificity of individuals of antibodies. in addition to conventional antibodies, camelids also produce heavy-chain-only antibodies (hcabs). hcabs, also referred to as nanobodies, or vhhs, contain a single variable domain (vhh) that makes up the equivalent antigen-binding fragment (fab) of conventional immunoglobulin g (igg) antibodies [ ] . this single variable domain typically can acquire affinity and specificity for antigens comparable to conventional antibodies. nanobodies can easily be constructed into multivalent formats and have higher thermal stability and chemostability than most antibodies do [ ] . another advantage of nanobodies is that they are less susceptible to steric hindrances than large conventional antibodies [ ] . considering the broad specificity of antibodies, seeking potential antibody therapies has become one of the most feasible strategies to fight against sars-cov- . in general, antibody therapy is a form of immunotherapy that uses monoclonal antibodies (mab) to target pathogenic proteins. the binding of antibody and pathogenic antigen can facilitate either immune response, direct neutralization, radioactive treatment, the release of toxic agents, or cytokine steam inhibition (aka immune checkpoint therapy). the sars-cov- entry of a human cell facilitated by the process of a series of interactions between its spike (s) protein and the host receptor angiotensin-converting enzyme (ace ), primed by host transmembrane protease, serine (tmprss ) [ ] . as such, most covid- antibody therapeutic developments focus on the sars-cov- spike protein antibodies that were initially generated from patient immune response and t-cell pathway inhibitors that block t-cell responses. a large number of antibody therapeutic drugs are in clinical trials. currently, most antibody therapy developments focus on the use of antibodies isolated from patient convalescent plasma to directly neutralize sars-cov- [ ] [ ] [ ] , although there are efforts to alleviate cytokine storm. a more effective and economical means to fight against sars-cov- is vaccine [ ] , which is the most anticipated approach for preventing the covid- pandemic. a vaccine is designed to stimulate effective host immune responses and provide active acquired immunity by exploiting the body's immune system, including the production of antibodies, which is made of an antigenic agent that resembles a disease-causing microorganism, or surface protein, or genetic material that is needed to generate the surface protein. for sars-cov- , the first choice of surface proteins is the spike protein. there are four types of covid- vaccines, as shown in figure . ) virus vaccines use the virus itself, in a weakened or inactivated form. ) viral-vector vaccines are designed to genetically engineer a weakened virus, such as measles or adenovirus, to produce coronavirus s proteins in the body. both replicating and non-replicating viral-vector vaccines are being studied now. ) nucleic-acid vaccines use dna or mrna to produce sasr-cov- s proteins inside host cells to stimulate the immune response. ) protein-based vaccines are designed to directly inject coronavirus proteins, such as s protein or membrane (m) protein, or their fragments, into the body. both protein subunits and viral-like particles (vlps) are under development for covid- [ ] . among these technologies, nucleic-acid vaccines are safe and relatively easy to develop [ ] . however, they have not been approved for any human usage before. however, the general population's safety concerns are the major factors that hinder the rapid approval of vaccines and antibody therapies. a major potential challenge is an antibody-dependent enhancement, in which the binding of a virus to suboptimal antibodies enhances its entry into host cells. all vaccine and antibody therapeutic developments are currently based on the reference viral genome reported on january , [ ] . sars-cov- belongs to the coronaviridae family and the nidovirales order, which has been shown to have a genetic proofreading mechanism regulated by non-structure protein (nsp ) in synergy with nsp , i.e., rna-dependent rna polymerase (rdrp) [ , ] . therefore, sars-cov- has a higher fidelity in its transcription and replication process than other single-stranded rna viruses, such as the flu virus and hiv. even though the s protein of sars-cov- has been undergoing many mutations, as reported in [ , ] . as of september , a total of mutations on the s protein has been detected on complete sars-cov- genome sequences. therefore, it is of paramount importance to establish a reliable computational paradigm to predict and mitigate the impact of sars-cov- mutations on vaccines and antibody therapies. moreover, the efficacy of a given covid- vaccine depends on many factors, including sars-cov- biological properties associated with the vaccine, mutation impacts, vaccination schedule (dose and frequency), idiosyncratic response, assorted factors such as ethnicity, age, gender, or genetic predisposition. the effect of covid- vaccination also depends on the fraction of the population who accept vaccines. it is essentially unknown at this moment how these factors will unfold for covid- vaccines. it is no doubt that any preparation that leads to an improvement in the covid- vaccination effect will be of tremendous significance to human health and the world economy. therefore, in this work, we integrate genetic analysis and computational biophysics, including artificial intelligence (ai), as well as additional enhancement from advanced mathematics to predict and mitigate mutation threats to covid- vaccines and antibody therapies. we perform single nucleotide polymorphism (snp) calling [ , ] to identify sars-cov- mutations. for mutations on the s protein, we analyze their mechanism [ ] , frequency, ratio, and secondary structural traits. we construct a library of all existing antibody structures from the protein data bank (pdb) and analyze their two-dimensional ( d) and three-dimensional ( d) characteristics. we further predict the mutation-induced binding affinity changes of antibody and s protein complexes using a topology-based network tree (topnettree) [ ] , which is a state-of-the-art model that integrates deep learning and algebraic topology [ ] [ ] [ ] . after identifying mutations that are potentially disruptive to antibody and s protein interactions, we further infer their threats to vaccines based on antibody binding site analysis, mutation-induced disruptive free energy, and mutation occurrence frequency. we combine frequency and free energy change to prioritize mutation threats and guild the development of future vaccines and antibody therapies. as a fundamental biological process, mutagenesis changes the organism's genetic information and servers as a primary source for many kinds of cancer and heritable diseases, which is a driving force for evolution [ , ] . generally speaking, virus mutations are introduced by natural selection, replication mechanism, cellular environment, polymerase fidelity, gene editing, random genetic drift, gene editing, recent epidemiology features, host immune responses, etc [ , ] . notably, understanding how mutations have changed the sars-cov- structure, function, infectivity, activity, and virulence is of great importance for coming up with life-saving strategies in virus control, containment, prevention, and medication, especially in the antibodies and vaccines development. genome sequencing, snp calling, and phenotyping provide an efficient means to parse mutations from a large number of viral samples [ , ] (see the supporting material (s )). in this work, we retrieved over , complete sars-cov- genome sequences from the gisaid database [ ] and created a real-time interactive sars-cov- mutation tracker( https://users.math.msu.edu/users/weig/sars-cov- mutation tracker.html) to report over , single mutations along with its mutation frequency on sars-cov- as of september . figure is a screenshot of our online mutation tracker. it describes the distribution of mutations on the complete coding region of sars-cov- . the y-axis shows the natural log frequency for each mutation at a specific position. a reader can download the detailed mutation snp information from our mutation tracker website. as mentioned before, the s protein has become the first choice for antibody and vaccine development. among , complete genome sequences, unique single mutations are detected on the s protein, and the h-index of s protein is [ , ] the number of unique mutations (n u ) is determined by counting the same type of mutations in different genome isolates only once, whereas the number of non-unique mutations (n nu , i.e., frequency) is calculated by counting the same type of mutations in different genome isolates repeatedly. table lists the distribution of snp types among unique and non-unique mutations on the s protein of sars-cov- worldwide. it can be seen that c>t and a>g are the two dominated snp types, which may be due to the innate host immune response via apobec and adar gene editing [ ] . moreover, non-degenerated mutations occurred on the s protein receptor-binding domain (rbd), which are relevant to the binding of sars-cov- s protein and most antibodies as well as ace . additionally, mutations occurred on the s protein domain (residue id: to ) are relevant to the binding of another antibody ( a ) and sars-cov- s protein. furthermore, since antibody cdrs are random coils, the complementary antigen-binding domains must involve random coils as well. table lists the statistics of non-degenerate mutations on the secondary structures of sars-cov- s protein. here, the secondary structures are mostly extracted from the crystal structure of c l [ ] , and the missing residues are predicted by raptorx-property [ ] . we can see that for both unique and non-unique cases, the average mutation rates on the random coils of the s protein have the highest values. particularly, the a>g-(d g) mutation on the random coils has the highest frequency of . if we do not consider the a>g-(d g) mutations, then the unique and non-unique average rates on the random coils of s protein still have the highest values ( . and . ), indicating that mutations are more likely to occur on the random coils. consequently, the natural selection of mutations may tend to disrupt antibodies. table : the statistics of non-degenerate mutations on the secondary structure of sars-cov- s protein. the unique and non-unique mutations are considered in the calculation. n u , n nu , ar u , ar nu represent the number of unique mutations, the number of nonunique mutations, the average rate of unique mutations, and the average rate of non-unique mutations on the secondary structure of s protein, respectively. here, the secondary structure is mostly extracted from the crystal structure of c l, the missing residues are predicted by raptorx-property. we construct a sars-cov- antibody library of d antibody structures deposited in the pdb. among them, the binding sites of antibodies are on the rbd of the s protein. while another antibody, a [ ] , has a distinguished binding domain. additionally, mr -k y is a mutant of antibody mr [ ] . we align antibody structures, excluding mr -k y, with sars-cov- s protein in figure . ace is included as a reference. clearly, except for antibody a , all other structures bind to the s protein rbd. it is interesting to note that a locates on a different domain. the pdb ids of these complexes can be found in figure . [ ] , cr [ ] , ey a [ ] , and a , all the other antibodies have their binding sites spatially clashing with that of ace . notably, the paratope of h [ ] does not overlap with that of ace directly, but in terms of d structures, their binding sites still overlap. this suggests that the bindings of antibodies are in direct competition with that of ace . theoretically, this direct competition reduces the viral infection rate. for such an antibody with strong binding ability, it will directly neutralize sars-cov- without the need of antibody-dependent cell cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), or other immune mechanisms. the paratopes of s , cr , and ey a on the rbd are away from that of ace , leading to the absence of binding competition [ , , ] . one study shows that the adcc and adcp mechanisms contribute to the viral control conducted by s in infected individuals [ ] . for cr , one research indicates that it neutralizes the virus in a synergistic fashion [ ] . for ey a, the hypothesis is that the binding of ey a could inhibit the glycosylation of ace [ ] . a more radical example is a [ ] , it binds to the n-terminal domain (ntd) of the s protein (figure (h)), which is quite far from the rbd, it is speculated (i) the d structure of s protein rbd. the red, green, and blue represent for helix, sheet, and random coils of rbd, respectively. the darker color represents the higher mutation frequency on a specific residue. the antibodies are s ( m j) [ ] , cc . ( xc ) [ ] , cc . and cr ( xc ) [ ] , cc . ( xc ) [ ] , cc . and cr ( xc ), c ( xcm) [ ] , regn and regn ( xdg) [ ] , cv ( xe ) [ ] , fab - ( xey) [ ] , cr ( yla) [ ] , h -d ( yz ), cr and h -d ( z m) [ ] , h -h ( zbp), ey z and nanobody ( zcz) [ ] , ey z ( zer) [ ] , p b- f ( bwj) [ ] , bd ( byr) [ ] , b ( bz ) [ ] , cb ( c ) [ ] , a ( c l) [ ] , sr ( c v) [ ] , b ( c w), h ( cah) [ ] , mr -k y ( can) [ ] , bd- ( ch ), bd- ( ch ), bd- ( chb), bd- and bd- - ( che), bd- and bd- - ( chf), bd- - ( chh), cova - ( jmo) [ ] , and cova - ( jmp) [ ] . that a may neutralize sars-cov- by restraining the conformational changes of the s protein, which is very important for the sars-cov- cell entry [ ] . any antibody or drug that can inhibit serine protease tmprss priming of the s protein priming can effectively stop the viral cell entry [ ] . figure provides a visual illustration of antibody and ace competitions. it remains to know in the residue detail what has happened to these competitions. to better understand the antibody and s protein interactions, we study the residue contacts between antibodies and the s protein. we include the ace as a reference but excluding antibodies a and mr -k y. in figure , the paratopes of antibodies and ace were aligned on the s protein rbd d sequence, and their contact regions are highlighted. from the figure, one can see that, except for h , s , cr , and ey a, all the other antibodies have their antigenic epitopes overlapping with the ace rbd, especially on the residues from to of the sars-cov- rbd. therefore, these antibodies competitively bind against ace as revealed in figure . the next question is whether there is any connection or similarity between the antibody paratopes in our library, particularly for those antibodies that share the same binding sites. to better understand this perspective, we carry out multiple sequence alignment (msa) to further study the similarity and difference among existing antibodies. many antibodies are very similar to each other and can be described in a few groups. the first group includes bd- , cc . [ ] , cova - [ ] , cv [ ] , cc . [ ] , b [ ] , bd- , bd- , ey a, and regn [ ] , as well as cb [ ] . their identity scores to cb are . therefore, multiple sequence alignment suggests that the paratopes of the antibodies bd- , cb , cova - , cv , cc . , cc . , c , bd- , bd- , and b are almost identical. similarly, the paratopes of the antibodies h -h , h -d , nb are highly consistent. so are the antibodies regn , cova - , and p b- f . the above similarity indicates that the adaptive immune systems of individuals have a common way to generate antibodies. on the other hand, the existence of three distinct groups, as well as antibody a suggests the diversity in the immune response. note that we have also included ace in our msa as a reference but none of the existing antibodies is similar to ace , because they were created from entirely different mechanisms. to investigate the influences of existing s protein mutations on the binding free energy (bfe) of s protein and antibodies, we consider mutations occurred on the s protein rbd which are relevant to the binding of sars-cov- s protein and antibodies as well as ace . additionally, mutations occurred on the ntd of the s protein (residue id: to ) which are relevant to the binding of sars-cov- s protein and antibody a (pdb: c l). we predict the free energy changes following existing mutations using our topnettree model [ ] . the rbd mutations are computed which are in the distance of Å to antibodies. our predictions are built from the x-ray crystal structure of sars-cov- s protein and ace (pdb m j) [ ] , and various antibodies (pdbs wps [ ] , xc [ ] , xc [ ] , xc [ ] , xc , xcm [ ] , xdg [ ] , xe [ ] , xey [ ] , yla [ ] , yz , z m, zbp, zcz [ ] , zer [ ] , bwj [ ] , byr [ ] , bz [ ] , c [ ] , c l [ ] , c v [ ] , c w, cah [ ] , can [ ] , ch , ch , chb, che, chf, chh, jmo [ ] , and jmp [ ] ). the bfe change following mutation (∆∆g) is defined as the subtraction of the bfe of the mutant type from the bfe of the wild type, ∆∆g = ∆g w − ∆g m where ∆g w is the bfe of the wild type and ∆g m is the bfe of mutant type. therefore, a negative bfe change means that the mutation decreases affinities, making the protein-protein interaction less stable. we first present the bfe changes ∆∆g of sars-cov- s protein binding domain with antibody a in figure , which is the only complex that is not on the rbd in our collections of s protein and antibody complexes. most mutations have small changes in their binding free energies, while some of them have large changes. notably, out of mutations on the binding domain have positive bfe changes, which means that the mutations increase affinities and would make protein-protein interactions more stable. however, the majority ( %) of mutations have negative bfe changes, including high-frequency mutations, m i, s f, and s f. it is also noted that many mutations on the binding domain, such as g d and k n, have significant negative free energy changes. the mutations on the binding domain with negative binding affinities reveal that the binding of antibody a and s protein will be potentially disrupted. next, we study the bfe changes ∆∆g induced by mutations on the sars-cov- s protein rbd for the antibody fab - (pdb: xey) in figure . most mutations induce small changes in the thee binding free energies, while mutations, g r and s l, have large negative bfe changes. overall, out of mutations on the rbd lead to negative bfe changes, which means % of mutations will potentially weaken the binding between antibody fab - and s protein. particularly, mutation s n on the rbd induces a negative bfe change with a high frequency of . while some mutations leading to positive bfe changes, more mutations induce negative bfe changes with large magnitude. antibody fab - shares a similar binding domain with ace and thus is a potential candidate for the direct neutralization of sars-cov- . however, bfe change predictions indicate that the mutations on s protein weaken the fab - binding with s protein and make it less competitive with ace . in figure , we illustrate antibody b (pdb: c w), which shares the binding domain with ace as well. one can notice that only four mutations, r s, f s, l f, and s l, have the magnitude of bfe changes larger than kcal/mol and all are negative bfe changes. the rest mutations have a small magnitude of changes. mutation v a has a frequency of and small positive bfe changes. interestingly, mutation s l induces large bfe changes for antibodies b and fab - . antibody b will reduce its competitiveness with ace if mutations r s, f s, l f, and s l become dominant. finally, we consider the bfe change predictions for antibody s and s protein complex, whose re- ceptor binding motif (rbm) does not overlap with the rbm of ace . the bfe changes induced by mutations are predicted. among them, changes are positive. similar to the aforementioned antibodies, most of the mutations lead to small changes in their binding affinity magnitude but three mutations, t s, v i, and k n, induce large negative changes. the binding of antibody s might be disrupted, considering that a majority of mutations induce negative bfe changes with large magnitude. while antibodies play a variety of functions in the human immune system such as neutralization of infection, phagocytosis, antibody-dependent cellular cytotoxicity, etc., their binding with antigens is crucial for these functions. our analysis of bfe changes following mutations on s protein suggests that some antibodies will be less affected by mutations, which is important for developing vaccine and antibody therapies. the bfe change analysis of other antibodies is described in the supporting material (s ). in this section, we build a library of mutation-induced bfe changes for all mutations and all antibodies. in principle, we could create a library of all possible mutations for all antibodies, as we did for ace [ ] . here, we limit our effort to all existing mutations. antibody a on the ntd has been discussed above. we consider antibodies on the rbd. based on our earlier analysis, three types of sars-cov- s protein secondary structural residues have f l g d e k v i y h s p s f v f n s d v v i r i r t k r k n i v n k n k l i different mutation rates. among them, the random coils are major components of the rdb and the ntd, as shown in fig. . therefore, mutations on the rbd are split into three categories based on their locations in secondary structures helix, sheet, and coil. in figure , we present the bfe changes for ace and antibodies induced by mutations on helix residues of the s protein rbd. the frequency for each mutation is also presented. most mutations on helix residues lead to positive bfe changes (green squares), whereas some mutations induce negative bfe changes (pink squares). the n k mutation having the largest frequency, , shows mild bfe changes on ace and antibodies. mutations k n and y c induce positive bfe changes on most of ace and antibodies. especially, antibodies c and bd- have larger bfe changes than ace , which indicates that they are stronger competitive than ace . antibody cb may be potentially a good therapeutic candidate as its bfe changes are positive following all mutations, but this needs to be confirmed by other mutations on the coil and sheet residues. in figure , we present the bfe changes for ace and antibodies along with frequencies on mutations of sheet residues of the s protein rbd. the mutation r s has a large variance of the bfe changes such that both positive and negative changes occurred on antibodies and ace . clearly, antibodies bd , bd , cb , mr , and mr -k y lead to negative bfe changes on mutations of rbd sheet residues, which reduce their competitive binding ability with ace after mutations. as for mutations with high frequencies, the mutation r k has negative changes on most antibodies, which poses a danger of disrupting the binding of antibodies and s protein. figure presents the bfe changes for ace and antibodies along with the log of frequencies on each mutation of coil residues on the s protein rbd. overall, most mutations on coil residues lead to negative bfe changes. interestingly, cv has the most positive bfe changes following mutations, which can be a good candidate for potential therapy. for the high-frequency mutation s n, the bfe changes are mild on ace and antibodies. however, mutation l f induces negative bfe changes for all antibodies except for cova - , which is considered as a potentially dangerous mutation for antibody therapies. n d n s n k r k k n t p v i r k r s l m l r y f q l s p s l y h in statistics, most mutations ( of ) occur on residues whose secondary structures are coil, while out of mutations are on the helix, and out of mutations are on the sheet. here, mutations on the random coils and mutations on helix are not calculated due to the far distance to antibodies. moreover, residues on coil have more negative bfe changes ( negative bfe changes vs. positive bfe changes), while residues on the helix or sheet have more positive bfe changes ( and negative bfe changes vs. and positive bfe changes, respectively). lastly, the maximum bfe changes of the helix, sheet, and coils are . kcal/mol, . kcal/mol, and . kcal/mol, while the minimum bfe changes are - . kcal/mol, - . kcal/mol, and - . kcal/mol, respectively. binding affinity changes (kcal/mol) figure : illustration of sars-cov- mutation-induced maximal and minimal binding free energy changes for the complexes of s protein and antibodies or ace . here, the maximal change strengthens the binding while the minimal change weakens the binding for each complex. figure indicates the bfe changes extreme values (maximal in blue and minimal in red) of the complexes of s protein ace or antibodies following mutations. many antibodies, such as cr and cr h -d , are not very sensitive to the current s protein mutations. however, some other antibodies, such as cv , fab - , and ey z nb, can be dramatically affected by sars-cov- mutations. the increasing number of affected and dead individuals, the global spread situation, and the lack of prophylactics and therapeutics give rise to the urgent demand for the prevention of covid- . vaccination is the most effective and economical means to prevent and control pandemics [ ] . currently, vaccines are in various clinical trial stages, as reported in an online covid- treatment and vaccine tracker ( https://covid- tracker.milkeninstitute.org/#vaccines intro). broadly speaking, there are four types of coronavirus vaccines in progress: virus vaccines, viral-vector vaccines, nucleic-acid vaccines, and proteinbased vaccines, as shown in figure . the first type of vaccine is the virus vaccine, which injects weakened or inactivate viruses to the human body. a virus is conventionally weakened by altering its genetic code to reduce its virulence and elicit a stronger immune response. a biotechnology company codagenix is currently working on a "codon optimization" technology to weaken viruses, and it has weakened virus vaccine is in progress [ ] . unlike a weakened virus, the inactivated virus cannot replicate in the host cell. a virus is inactivated by heating or using chemicals, which induces neutralizing antibody titers and has been proven to have its safety [ ] . at this stage, both sinopharm, which works with the beijing institute of biological products and wuhan institute of biological products, and sinovac, which works with institute butantan and bio farma is developing inactivate sars-cov- vaccines that are in phase iii clinical trials. the second type of vaccine is the viral-vector vaccine, which is genetically engineered so that it can produce coronavirus surface proteins in the human body without causing diseases. there are two subtypes of viral-vector vaccines: the non-replicating viral vector and the replicating viral vector. there are non-replicating viral vector vaccines in phase iii trials. astrazeneca and the university of oxford, whose vaccine is in phase iii trials in many countries. it works by taking a chimpanzee virus and coating it with the s proteins of sars-cov- . the chimp virus causes a harmless infection in humans, but the spike proteins will activate the immune system to recognize signs of a future sars-cov- invasion. notably, the booster shots can be needed to keep long-lasting immunity. moreover, at this stage, only one replicating viral-vector vaccine is in phase i. institut pasteur themis, in cooperating with the university of pittsburgh cvr and merck sharp & dohme is developing such a replicating viral vaccine, which tends to be safe and provoke a strong immune response [ ] . the third type of vaccine is nucleic-acid vaccines, including two subtypes: dna-based vaccines and rna based vaccines. at least teams are currently working on nucleic-acid vaccines since they are safe and easy to develop. the dna-based vaccine works by inserting genetically engineered blueprints of the viral gene into small dna molecules such as plasmids for injection. moreover, the electroporation technique is employed to create pores in membranes to increase dna uptake into cells. the injected dna will produce mrna by transcription with the help of the nucleus in human cells. such an mrna will translate viral proteins (mostly spike proteins), which are dutifully produced by cells in response to the genes, alarm the immune system, and should produce immunity. currently, there are four dna-based vaccines in phase ii. similar to dna-based vaccines, the rna-based vaccines provide immunity through the introduction of rna, which is encased in a lipid coat to ensure its entering into cells. two rna-based vaccines are in phase iii, and companies such as moderna, biontec, and pfizer are working on the advanced development of rna-based vaccines. the fourth type of vaccine is the protein-based vaccines, which aims to inject viral proteins directly to human bodies to trigger immune readiness. protein subunits vaccine is one of the subtypes of the proteinbased vaccine. more than teams are working on vaccines with viral protein subunits, such as spike proteins and membrane (m) proteins. another subtype of the protein-based vaccine is the virus-like particle (vlp) vaccine. the vlp vaccines closely resemble viruses. however, they are not infectious since they do not contain viral genetic material. the non-replicating propriety provides a safer alternative to weakened virus vaccines, the hpv vaccine or newer flu vaccines are vlp vaccines. currently, teams are working on the vlp vaccines for the future prevention of covid- . since the structural basis of antibody cdrs, or paratope, is random coils, we hypothesize that cdrs favor antigenic random coils as complementary epitopes, i.e., antigenic determinants [ , ] . figure depicts the d structure of s protein, where the random coils are drawn with green strings, and the other secondary structure is described with the purple surface. it shows that the rbd and the ntd mostly consist of random coils. the rbd is the antigenic determinant of structurally-known sars-cov- antibodies; meanwhile, the ntd is the binding domain of antibody a , which confirms our hypothesis. figure marks the secondary structure of the s protein. the red, blue, and green colors represent helix, sheet, and random coils of s protein. it can be seen that the s protein mostly consists of random coils, which means there are many other potential antigenic epitopes on the s protein for antibody cdrs. we believe that the emphasis of direct binding competition with ace in the past [ , , ] has led to the neglecting of many important antibodies that do not bind to the rbd. therefore, we suggest that researchers pay more attention to antibodies that do not bind to the rbd. vaccine efficacy is an essential issue for the control of the covid- pandemic. s protein is one of the most popular surface proteins for the vaccine development. however, mutations accumulated on the s protein of sars-cov- , which may reduce the vaccine efficacy. as we found in section , mutations are more likely to happen on the random coils of s protein, which may have a devastating effect on vaccines in the development. as shown in figure , mutations could considerably weaken the binding between the s protein and antibodies and thus pose a direct threat to reduce the efficacy of vaccines. however, there are a few obstacles in determining the exact impacts of mutations to covid- vaccines. firstly, the four types of vaccine platforms can produce very different virus peptides, which will result in different immune responses, as well as antibodies. secondly, even for a given vaccine platform, the different peptides may be produced due to different immune responses caused by gender difference, age difference, race difference, etc. therefore, in this work, we proposed to understand the impact of sars-cov- mutations on covid- vaccines by the statistical analysis. by evaluating the binding affinity changes induced by existing sars-cov- antibodies, as shown in figure to figure , we can notice that the k n, y c, f l, and f l mutations enhance the binding of almost all of the antibodies. in contrast, the r k, l f, and p r mutations have weakened the binding of almost all of the existing antibodies. moreover, mutation k n enhances the binding of antibody ey a, whereas mutation v i weakens the binding of antibody s . furthermore, it is noticed that many mutations such as k n, q r, and g r considerably disrupt many antibodies and thus may bring a threat to future vaccines. figure depicts the maximal and minimal binding free energy changes for s protein complexes and antibodies or ace . it can be seen that antibodies cr , cr h -d , bd- , bd- , and bd- - are not very sensitive to the current mutations on the s protein. however, other antibodies, such as cv and ey a, are very sensitive to current mutations. in a nutshell, by setting up a sars-cov- antibody library with the statistical analysis based on the mutation-induced binding free energies changes, we can estimate the impacts of sars-cov- mutations on covid- vaccines, which will provide a way to infer how a specific mutation will pose a threat to vaccines. this approach works better when more antibody structures become available. another important factor in prioritization is mutation frequency. figures , , and have provided frequency information from our snp calling. once a mutation is identified as a potential threat, it can be incorporated into the next generation of vaccines in a cocktail approach. in principle, all four types of vaccine platforms allow the accommodation of new viral strains. coronavirus disease (covid- ) pandemic has gone out of control globally. there is no specific medicine and effective treatment for this viral infection at this point. vaccination is widely anticipated to be the endgame for taming the viral rampant. another promising treatment that is relatively easy to develop is antibody therapies. however, both vaccines and antibody therapies are prone to more than , unique mutations recorded in the mutation tracker. we present a prediction of mutation threats to vaccines and antibody therapies. first, we identify existing mutations on the severe acute respiratory syndrome coronavirus (sars-cov- ) spike (s) protein, which is the man target for both vaccines and antibody therapies. we analyze the mechanism, frequency, and ratio of mutations along with the secondary structures of the s protein. additionally, we build a library of antibodies with structures available from the protein data bank (pdb) and analyze their two-dimensional ( d) and three-dimensional ( d) characteristics by employing computational biophysics. we further predict the mutation-induced binding free energy (bfe) changes of s protein and antibody complexes by a model called topnettree based on deep learning and algebraic topology. from these studies, we infer that some of s protein mutations may disrupt the binding of antibodies and s protein, which will further affect the efficacy and reliability of vaccines. to prioritize mutation threats, we also take into consideration of mutation occurrence frequency. the resulting algorithm indicates that some high-frequency mutations such as m i, s f, and s f with negative bfe changes may potentially disrupt the efficacy and reliability of vaccines and antibody therapies currently in the development. our method can provide the efficient prioritization of mutations to guild the design of the next generation of vaccines and antibody therapies. supporting material is available for: s method; s multiple sequence alignments of the antibodies and pairwise identity scores; and s mutation-induced changes of binding free energies of antibody-sars-cov- spike protein complexes. p s a t a s t s r k r t a s a t s n c s c s d y a v s l f l v l p s p l t a a s g r q e q k q p q r t s d y s f k r k n v i v f v a g s g v n k l f f l r k k q k n s f s y n t i f i v i t s p t n e q e g e d i v a v g s s g s t s i s n s r t a t k t i p s p l n h n d g s v f v a e k e q e d g s g r f l f l f s p r l i h q genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding covid- vaccine development and a potential nanomaterial path forward covid- : four fifths of cases are asymptomatic, china figures indicate clinical and immunological assessment of asymptomatic sars-cov- infections projecting the transmission dynamics of sars-cov- through the postpandemic period deployment of convalescent plasma for the prevention and treatment of covid- immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications the orf , orf and nucleocapsid proteins of sars-cov- inhibit type i interferon signaling pathway covid- , immune system response, 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respiratory syndrome coronavirus vaccine bioinformatic prediction of epitopes in the emy antigen of echinococcus multilocularis. experimental and therapeutic medicine structural analysis of b-cell epitopes in antibody: protein complexes this work was supported in part by nih grant gm , nsf grants dms- , dms- , and iis , michigan economic development corporation, george mason university award pd , bristol-myers squibb, and pfizer. the authors thank the ibm tj watson research center, the covid- high performance computing consortium, nvidia, and msu hpcc for computational assistance. rw thanks dr. changchuan yin for useful discussion. the authors declare no competing interests. key: cord- - wennun authors: altmann, daniel m title: adaptive immunity to sars-cov- date: - - journal: oxf open immunol doi: . /oxfimm/iqaa sha: doc_id: cord_uid: wennun the majority of severe acute respiratory syndrome coronavirus (sars-cov- exposed individuals mount an antibody response within around -weeks and spike antigen-binding responses correlate well with functional virus neutralization. a minority makes little detectable antibody, generally those with either very mild/asymptomatic disease or those with severe/lethal infection. however, in general, antibody titre correlates with viral load and duration of exposure. there is evidence for cross-reactivity with the other human coronaviruses, though the functional impact of this is as yet unclear. therapeutic use of neutralizing monoclonal antibodies offers potential for clinical use. while there is evidence for neutralizing antibody as a correlate of protection, some cases indicate the potential for full recovery in the absence of antibody. studies of t-cell immunity following acute infection show cd and cd responses to epitopes across diverse viral antigens, possible cross-reactivity with epitopes from the common cold human coronaviruses and large-scale activation. however, in severe cases, there is evidence for t-cell lymphopaenia as well as expression of exhaustion markers. analysis of serum biomarkers of disease severity implicates a hyperinflammatory contribution to pathogenesis, though this has not been mechanistically delineated beyond a likely role of raised il- , considered a therapeutic target. despite rapid progress, there remain pressing unknowns. it seems likely that immune memory to sars-cov- may be relatively short lived, but this will need longitudinal investigation. also, this is a disease of highly variable presentation and time course, with some progressing to protracted, chronic symptoms, which are not understood. the contribution of immunopathological mechanisms to tissue damage, whether in the lung, kidney, heart or blood vessels, is unclear. the immunology underlying the differential susceptibility between the very young and the very old is unresolved, a question with ramifications for vaccine roll-out. the greatest challenge relates to rapid generation, testing and manufacture of vaccines that are immunogenic, protective (at least from symptomatic disease) and safe—a challenge that looks achievable. infection, immunology research has been placed in the spotlight, with intense curiosity and scrutiny about many specific aspects of the immune response to this viral infection: when does immunity develop, what are the correlates of protection, what is the temporal relationship between immunity and infectivity, do all develop protective immunity and can reinfection occur, what part is played by immunopathology in pathological damage to the lungs and other organs? on top of this has been impatience for updates on progress in rapid resolution of the translational challenges posed by global roll-out of reliable antibody serodiagnostics and of safe reliable vaccines [ ] [ ] [ ] [ ] . immunology has never had to grapple with questions of this enormity under such time pressure. among the countless manifestations of the 'new normal' has been an overturning of conventions for publishing so as to address the pressure for data updates in real time: the tendency has been for data to emerge as soon as it is generated, on social media, then within days or weeks posted on repositories such as biorxiv as a non-peer-reviewed preprint, then subsequently snapped up for full publications in prestigious journals. there have been many consequences of this publishing revolution. there has been a vibrant, refreshing foreshortening of the publication timeline. this is a field that had developed norms whereby big papers necessitated the pooled work of perhaps a score of scientists over years of funded research, submitting a manuscript for laboured, iterative, peer-review stretching over - months, so that the full cycle from concept, to funding, to research, to publication might be years plus. in the 'new normal', some of the highest profile papers have used standard, pre-existing technologies such as multiparameter flow cytometry panels and rnaseq pipelines to describe and define immune parameters in patients hospitalized in january and february, the papers reporting them appearing in march and april. while there may indeed be a price for reduced rigour in peer review, many might argue that the scrutiny of a scientific peer group via social media has gone some way to substituting a proxy arbiter of quality control. with these points in mind, my aim here has been to present an overview of some of the key knowns and unknowns of sars-cov- adaptive immunity, relying both on preprints and on published findings. my focus has been to some extent informed by the recurrently posed questions that have clearly been of concern, whether in media interviews or policy discussions. few in the immunology research community had prior, handson, experience with the immunology of coronaviruses, and one of the challenges has been to convey the notion that, as for any host defence programme, once you drill down beyond textbook generalities of viral immunity, the devil is in the detail, and that is what current research must resolve. the spread of a fatal respiratory syndrome focused on initial cases who had visited wuhan seafood market in china and was first reported in december [ , ] . within a little over weeks of the initial case, genomic sequence for a novel coronavirus was published in early january [ ] . it was initially termed -ncov and then, sars-cov- . in terms of known human infections, it is phylogenetically close to sars-cov and is believed to be a new zoonotic transfer, probably from bats, although it is still unknown whether there was an intermediate species, such as pangolin [ ] . interestingly, a recently sequenced betacoronavirus from bats in yunnan province, termed rmyn , has % sequence identity with sars-cov- , but with critical changes in receptor-binding domain (rbd) of the spike protein, meaning it probably lacks the key feature of binding to human angiotensin-converting enzyme (ace ) [ ] . the coronaviruses have single-stranded rna genomes encoding non-structural proteins as well as structural proteins: spike, envelope, membrane and nucleocapsid. while host immunity may be revealed against any part of the viral proteome, much initial immunology research has focused on immunity to the spike antigen; this is driven by the knowledge that the interaction between the rbd within the spike antigen and human ace is critical for viral entry and infectivity, and that antibodies against spike can be protective through neutralization. in this regard, many initial clues came from extrapolating from the immunology of the closely related infections caused by sars-cov and mers [ ] [ ] [ ] [ ] . the genomic sequence was rapidly shared and published, facilitating design of pcr and antibody-based diagnostics [ ] . an initial challenge in design of antibody tests, especially in the face of limited supply of positive control serum samples for testing, was to validate binding that would be specific to this virus [ ] . the confounder of cross-reactivity with sars or mers antibodies is a concern, while prior exposure to the common cold hcov viruses is considerably more widespread in human populations, though the genomic sequences show far lower conservation [ ] . most test strategies have relied on recombinant spike antigen. some use the nucleoprotein, though this is more conserved across coronaviruses and therefore more prone to detect crossreactive binding. while lab assays were validated and cohort antibody data accrued [ ] [ ] [ ] [ ] [ ] , there was a considerable public health imperative to scale-up antibody tests for patient screening and for seroprevalence studies [ ] . clearly, there is a range of approaches for antibody tests, covering lateral flow devices optimized to give a yes/no binary answer for antigen-binding, elisa-based approaches, and then functional neutralization assays based either on pseudotype virus or on live virus assayed under bsl conditions. rolling out reliable antibody testing at scale and at speed proved extremely challenging and has only recently been resolved, based largely on elisa approaches. by analogy to studies on sars and mers, most exposed, symptomatic individuals would be predicted to show an antibody response to spike antigen [ ] [ ] [ ] [ ] . furthermore, when tested for functional virus neutralization, this has tended to correlate well with total antigen-binding antibody by elisa [ ] . data sets have now been shared for spike antibody in a large number of sars-cov- patient cohorts. a number of general points emerge from this: patients show a wide range of antibody titres, absence or near absence of antibody is associated with very mild infection, with fatal outcome or with immunodeficiency; the time course for appearance of igm, igg and iga antibodies follows conventional kinetics over the initial - days of infection, the appearance of detectable antibody roughly contemporaneous with the disappearance of infectious virus from nasopharyngeal swabs; despite differential disease susceptibility between children, adults and the elderly, this is not reflected in simple quantitative differences of antibody titre [ ] [ ] [ ] [ ] [ ] ] . some data indicate that cumulative viral load is correlated with antibody titre, raising concerns that those with very mild exposure may have more marginal or undetectable antibody responses, as was seen in sars and mers. however, while initial antibody studies were skewed to analysis of those 'tip of the iceberg' cases severe enough to be hospitalized, more data sets are now available for milder exposures such as healthcare workers, reassuringly showing significant antibody responses [ ] . chung and colleagues used a systems serology approach to investigate functional correlates of antibody responses across age groups and disease profile [ ] . systems serology harnesses the power of machine learning with data sets from multiple assays of antibody functionality including avidity and fc receptor binding to generate correlative signatures [ ] . they identified a convalescence biosignature associated with igg , fccr binding and c q engagement, as well as an influence of hlaii polymorphisms. there has been much speculation as to whether differential immune repertoires help to inform the differential susceptibility of the very young and the very old. this study offers the perhaps counterintuitive suggestion that children may benefit from an igm-dominated signature, unlike the class-switched igg and iga signatures of the elderly. differences across the lifespan may also relate to recent exposure to epitope-cross-reactive hcov common cold viruses, though the functional extent of any such cross-reactivity and associated protection has been a source of controversy. a recent study by ng et al. finds evidence for cross-reactivity between hcov and sars-cov- spike and nucleoprotein, even to the extent of functional neutralization [ ] . since many key questions about durability of the antibody response and about correlates of protection have been hard to address in this short timeframe, there has been value in recourse to the coronavirus immunology literature, especially in relation to sars and mers [ ] [ ] [ ] [ ] . this suggests a consensus of around weeks to igg seroconversion, lower responses in asymptomatic or mild infection, variably poor durability of antibody response beyond or years and neutralizing antibody as a likely correlate of protection. certainly in follow-up of mers patient, a significant minority showed no detectable antibody at months. more studies will be needed to ascertain whether the greater igg repertoire of older individuals, including an hcov crossreactive repertoire, may contribute to enhanced pathogenesis through antibody-dependent enhancement [ ] . many teams have moved rapidly to express neutralizing human monoclonals characterize the binding site structural biology and evaluate potential translational use as therapeutics [ ] [ ] [ ] , following the rationale trialled in recent years for infections including hiv, ebola and c. diff [ ] [ ] [ ] . in some reported cohorts, there has been a minority of patients who have made a full recovery, seemingly without generating any detectable antibody response [ ] . there are also reports of people unable to make any b-cell response at all due to agammaglobulinaemia who can also make a full recovery [ ] . whatever else, this suggests that neutralizing antibodies are not strictly required for recovery and other parts of the specific response, such as t cells, may serve to offer sufficient protection. the nuances of these emerging data sets have significant ramifications for clinical management of patients with inflammatory and autoimmune diseases across many specialties, weighing up the profile of their different disease-modifying therapy protocols to establish which are most likely to be safe. meanwhile, following many false starts, seroprevalence data sets are starting to appear from diverse urban locations around the globe. some care is needed in collating these data as it can sometimes be hard to ascertain the precise sampling location and procedure, sample size, detection assay used or indeed whether derived from actual antibody tests or from predictive models. while results obviously look very different in different affected centres, seroprevalence levels seem typically to be in the - % range, far short of the % plus needed for herd immunity, thus the urgent imperative for effective vaccines [ ] . experience from t cell studies in sars immune donors suggests that strong cd and cd immunity may endure for a number of years [ ] . initial analysis of immune subsets in acute covid- largely focused on hospitalized patients with severe infection, though studies of the response during milder infection are now appearing [ , [ ] [ ] [ ] [ ] [ ] [ ] . analysis of the hospitalized cohorts shows a picture of large scale t-cell activation, especially cd cells, along with t cell lymphopaenia as a correlate of severity, and expression of exhaustion markers such as pd- and tim- [ , ] . cd and cd t-cell immune responses can be detected to diverse regions of the viral proteome including the nucleoprotein, spike/rbd and the main protease [ ] [ ] [ ] . antiviral t-cell immunity correlates with neutralizing antibody titres [ ] . peptide epitope mapping of antiviral t-cell responses has been taken to support a case for the possibility of cross-reactive protection by memory t cells recognizing epitopes shared with hcov sequences [ ] . thieme et al. recently evaluated cd and cd responses against spike, membrane and nucleocapsid antigens, comparing between moderate, severe and critical disease. all three antigens contained epitopes, though the strongest response was to the membrane protein. the strongest responses, often polyfunctional, were seen in severe cases [ ] . should we be devoting extensive attention to the details of t-cell immunity if the data suggest that neutralizing antibody titre is itself a candidate correlate of protection? the answer is resoundingly in the affirmative, for several reasons. as we debate one of the biggest concerns around the fragile durability of coronavirus antibody responses, t-cell memory may be more enduring than b cell. extrapolating from progress in influenza vaccinology, it is likely that the most efficacious vaccine approaches need to look to strong recognition by both b-cell and t-cell receptors [ ] . whether in the context of natural or vaccine-induced t-cell immunity, there is a need for some scrutiny of the elicited cytokine profile. for example, in sars infection, th cytokine responses can be a cause of lung immunopathology [ ] . another recent study reported a deep immune profiling analysis, comparing parameters in patients compared to convalescent individuals or controls with respect to around immune parameters and clinical parameters [ ] . the comprehensiveness of this analysis is useful for its ability to reinforce a number of observations suggested by smaller studies. the study reiterates the observations of others in relation to lymphopaenia, t-cell exhaustion, decreased tregs and cd t cells, and the notion of development of neutralizing antibody as a likely correlate of protection. analysis of the parameters by tsne-enabled delineation of distinct, clinically related, immune phenotypes including an activated immune profile associated with expanded b-cell plasmablasts and activated cd t cells that are positive for ki , hla-dr, cd , cd , pd- , icos and fas. back to back papers from barouch and colleagues using a macaque infection and challenge model show the benefits of relevant animal models for clarifying some of the unknowns, although, with the caveat that short-term rechallenge at weeks is a poor proxy for understanding longer-term protection. the studies nevertheless show that either viral infection or dna vaccination results in non-sterilizing protection from symptomatic disease and that neutralizing antibody may indeed be the best correlate of protection [ , ] . from some of the earliest reports of serum biomarkers for severity of disease in patients who require ventilation, there has been evidence of a signature that includes inflammatory markers such as high ferritin, crp, d-dimer and il- [ ] . consistent reports of raised il- led to a series of clinical trials with the therapeutic monoclonal, tocilizumab, in what appear to be promising trials [ ] . another recent study highlights a biosignature of cxcl , ccl and il- receptor antagonist associated with high viral load, impaired lung function, lung injury and lethal outcome [ ] . we perhaps need some caution in overuse of the rather too simple term, 'cytokine storm', lest it blinds us to considering the aetiology and detail of what looks a rather unusual signature, worthy of investigation. which cells have produced the response and to what specific viral stimulus? certainly, this is not particularly reminiscent of a classic superantigen septic shock, to which it is sometimes related [ ] and, for example, many classic t-cell-derived cytokines are not a feature here. furthermore, there have been a number of reports of a kawasaki-like disease in children, now numbered at several hundred cases [ ] , with a disease now provisionally termed, 'paediatric inflammatory multisystem syndrome temporally associated with sars-cov- ' (pims-ts) [ ] . kawasaki disease itself still lacks a fully defined immunopathogenesis-it is considered an infection-triggered inflammatory cascade associated with the il- b pathway [ ] , and responding to therapeutics including ivig. despite rapid progress, there are rather urgent unknowns. this is an infection that affects different populations, whether in relation to age group or ethnicity, extremely differently, with old age by far the largest risk factor for severe outcome. while immunology has been able to offer hints as to the differential response that may underpin these differences, we still lack clarity. we are only starting to grapple with the detail of the diversity of cell types and tissues that can be infected by sars-cov- . understanding the nuances of infection, immunity and immunopathology in the lung, kidney and heart will be important. note that ace expression is relatively widespread across different cell-types, and we are only at the beginning of understanding the ramifications of this beyond the upper respiratory tract. while much has been learnt about immune correlates of infection, data so far are rather heavily weighted to analysis of severe, hospitalized patients. this leaves a vital knowledge gap to fill about the nature of immune recognition and immune memory of the many millions of individuals infected by the virus, either asymptomatically or symptomatically but managed in the home. such individuals comprise the vast majority of the exposed population, and there is an urgent imperative to build the narrative as to the extent and features of their adaptive immune response. perhaps the most critical aspect of this is the durability of that response, of concern with respect to mitigation of further waves. by analogy to the other human coronaviruses, the worrying prediction is that immunity may not commonly be durable beyond a few years. till now, all resources have necessarily been devoted to the firefighting of the response to the acute pandemic. attention is gradually shifting to the need for a more granular understanding of the interplay between host immunity and damaging immunopathology, whether in lung disease, in the pathway leading to thrombosis, or at other sites. in the future, considerable healthcare resource will need to be allocated to covid- follow-up clinics. the indications already are that, both among those who were severely affected and those that were not, there may a need to treat those living with long-term consequences of the infection, from lung fibrosis or bronchiectasis, to renal and cardiac complications and to chronic systemic, post-viral syndromes. the real test of how far the fast track immunology pipelines have progressed will be whether effective vaccines can be widely delivered in the near future. while there has been much optimism about the likelihood or even, inevitability, of phase i/ii trials shifting promptly into wide-scale production and roll-out during , the ups and downs of many other initiatives over the past decade caution against blithe assertions. there has at times been an unhelpful tendency to flip-flop between extremes of unrealistic optimism ('definitely by september') and unrealistic pessimism ('we must accept then there may never be a vaccine'). as with any vaccine programme in history, there is a multitude of possibilities between these. experience to date with sars-cov- suggests that this may not prove to be an infection that throws up insurmountable confounders to vaccine design-approaches that can safely and durably elicit neutralizing antibody look likely to work. the effort already has been unprecedented, spanning every known vaccinology platform, from adjuvanted conjugate vaccines, to attenuated or inactivated virus, to rna and dna vaccines and recombinant adenovirus approaches. while they are all well-established approaches with strong credentials and track records in a research setting, many have never before crossed the finish line to full manufacture and licensure. each approach comes with its own nuanced strengths and weaknesses in terms of magnitude of response, durability of response, number of boosts likely to be required, ability to stimulate different aspects of mucosal or systemic immunity, bias in terms of b cells, t cells and t-cell subset polarization, safety profile, ease of manufacture and supply chain/storage issues. these are important questions that have thus far received little attention in the discussions framed as a simple race to the finish line. it will also be important to educate policymakers and the public about the nature and limits of clinical trials to avoid erosion of confidence through unrealistic expectations: it seems unlikely that any of the vaccines will elicit sterilizing or lifelong immunity, but ones that significantly mitigate the most severe disease manifestations would be 'good enough'. on the contrary, under this intense scrutiny, any adopted candidates that are seen to be less good than this carry the risk of collateral damage for all other vaccines at a time of global health vulnerability under pressure from vaccine hesitancy. this pandemic merits a response from the immunology community that promotes the 'best answer', which may not be the 'first'. for this purpose, it is invaluable to foster coordinated endeavours through bodies such as cepi, gates foundation and wellcome, as an antidote to parochial 'vaccine nationalism' that risks the piecemeal development of rival, regional vaccines, which may impact both on 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immunology of sepsis an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study kawasaki-like disease: emerging complication during the covid- pandemic dissecting kawasaki disease: a state-of-the-art review key: cord- -cdmoazrl authors: richardson, eve; galson, jacob d.; kellam, paul; kelly, dominic f.; smith, sarah e.; palser, anne; watson, simon; deane, charlotte m. title: a computational method for immune repertoire mining that identifies novel binders from different clonotypes, demonstrated by identifying anti-pertussis toxoid antibodies date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: cdmoazrl due to their shared genetic history, antibodies from the same clonotype often bind to the same epitope. this knowledge is used in immune repertoire mining, where known binders are used to search bulk sequencing repertoires to identify new binders. however current computational methods cannot identify epitope convergence between antibodies from different clonotypes, limiting the sequence diversity of antigen-specific antibodies which can be identified. we describe how the antibody binding site, the paratope, can be used to cluster antibodies with common antigen reactivity from different clonotypes. our method, paratyping, uses the predicted paratope to identify these novel cross clonotype matches. we experimentally validated our predictions on a pertussis toxoid dataset. our results show that even the simplest abstraction of the antibody binding site, using only the length of the loops involved and predicted binding residues, is sufficient to group antigen-specific antibodies and provide additional information to conventional clonotype analysis. next-generation immune repertoire sequencing (ig-seq or rep-seq [ , ] ) is providing us with comprehensive information about adaptive immune repertoires across individuals [ ] and immune states [ ] . progress has been made in the task of interrogating the vast diversity of b-cell receptor (bcr) repertoires, primarily through the analysis of predicted clonal relationships inferred via clonotyping [ ] . ig-seq and associated clonal analysis are finding increasing importance in antibody discovery both as a method of identification of putative antigen-specific antibodies [ ] [ ] [ ] and more recently as a method of lead antibody optimization through repertoire mining [ ] . the identification of antibodies which are predicted to bind to the same site (epitope) is a key component of bcr repertoire analysis and antibody discovery. the starting point for most bcr repertoire analysis is the reduction of thousands or millions of bcrs into orders of magnitude fewer clonotypes [ ] . a clonotype is defined as a group of antibody sequences which derive from a common progenitor b cell [ ] . during b cell development, the variable (v), diversity (d) and joining (j) gene segments encoding the variable domain of the antibody heavy chain undergo recombination [ ] . a requirement for two sequences to be predicted to share the same clonotype is therefore common v-and j-germline gene assignment [ ] . the d gene is not usually included in standard clonotype definitions due to difficulty in its assignment [ ] . the variable domain of the antibody heavy chain consists of the framework regions and hypervariable complementaritydetermining regions (cdrs). cdrhs and are encoded by the v gene while the region spanning the recombined v, d and j segments corresponds to the third and most diverse loop on the antibody heavy chain, the cdrh . the processes of junctional diversification (the insertion of palindromic and random nucleotides at the junction between the v, d and j genes) during recombination as well as somatic hypermutation [ ] during affinity maturation further contribute to the diversity of the cdrh . sequence identity in the cdrh is therefore included as a marker of shared origin in most clonotyping tools [ ] . the nucleotide or amino acid sequence identity across the cdrh required for two sequences to be considered in the same clonotype varies across studies -in studies performing clonotyping with length-normalised amino acid sequence identity thresholds, sequence identity thresholds vary between % and % [ ] . published clonotyping methods use only heavy chain information which is sufficient to capture most clonal relationships [ ] . a number of publicly available, well-supported pipelines have made clonotype analysis standard practice [ , ] . this has permitted large advances in the practical utility of ig-seq data [ , ] . clinically, it has found use in tracking minimal residual disease in blood cancers [ ] , monitoring vaccination responses [ ] [ ] [ ] and providing mechanistic insights into immune-mediated diseases [ , [ ] [ ] [ ] . clonotyping has also proven useful in antibody discovery as a means of selecting candidate sequences for expression as monoclonal antibodies [ ] [ ] [ ] and recently as a method of lead antibody optimisation via repertoire mining [ ] . antibodies within the same clonotype are likely to target a common epitope [ , ] . the majority of antibodies binding to the same epitope in antibody-antigen complex structures in the structural antibody database (sabdab, a database of experimentally-solved antibody and antibody-antigen complex structures) have highly similar cdrh s [ ] . however, it has also been observed that multiple clonotypes may converge on the same epitope. for example, scheid and colleagues identified clonotypes from distinct ighv families converging on the cd binding site in gp [ ] . separate clonotypes have also been observed to bind to overlapping epitopes on the hemagglutinin stem [ ] or globular head, and on multiple epitopes on the ebola virus glycoprotein [ ] . wong and colleagues identified pairs of antibodies with sub- % cdrh amino acid identity binding to the same epitope within sabdab [ , ] . this convergence between clonotypes offers the potential to improve our understanding of the functional landscape of bcr repertoires; large-scale functional convergence between lineages could, for example, explain the apparent scarcity of public clonotypes [ ] . this hypothesis is supported by evidence that while clonotypes are infrequently shared between individuals, the range of antibody structures that these clonotypes generate is more similar between individuals [ ] . in the context of antibody discovery, being able to identify binders to the same epitope from different clonotypes would aid in optimisation of developability or binding affinity, by allowing hopping between germline scaffolds. in antibody discovery, clonotyping is used to search for clonal relatives of lead antibodies in bulk ig-seq data sets in order to identify antibodies which target the same epitope but which have either an increased affinity or a superior developability profile. this process is referred to as "immune repertoire mining". hsiao and colleagues performed clonotyping on a set of bulk repertoires and used the resultant clonotypes for hit expansion against two targets [ ] . they achieved greater than an order of magnitude improvement in affinity for both targets and between % and % of tested heavy chain variants retained target-binding [ ] . this suggests that sampling within a clonotype can be highly effective as a means of repertoire mining. however, the method does not allow the identification of binders to the same epitope which derive from different clonotypes, which currently limits the sequence distinctness of novel binders which can be recovered from immune repertoires. in this paper, we describe a new method to identify functional convergence of antibody sequences that is germline-independent and that considers only the binding site of the antibody sequences, the paratope. we call this approach "paratyping". we show how paratyping allows clustering of antigenspecific sequences from different clonotypes, and is a rapid, structurally intuitive way of clustering functionally-related antibodies. paratyping simplifies the complex phenomenon of antibody-antigen interaction into sets of shared residues. learning the complexities of antibody-antigen interactions as part of a predictive model of antigen binding has been achieved in the case of the antigen-interaction of one therapeutic monoclonal antibody [ ] . however, such approaches rely on a large (on the order of ) library of experimentally validated binding and non-binding variants. paratyping removes the need for large amounts of training data and is generalisable across antigens. we first show the rationale for our method, paratyping, using the structures of a pair of antibodies from different clonotypes that bind to the same epitope. paratyping is then applied to a single-cell data set of sequences raised against pertussis toxoid (ptx) in a transgenic mouse platform where it is as accurate as clonotyping but identifies different binders. we then perform a prospective experimental test of the method by expressing as monoclonal antibodies and experimentally testing predicted ptxbinding and non-binding antibodies mined from a set of non-enriched bulk heavy chain sequencing repertoires. our experimental test demonstrates that paratyping identifies ptx-binding antibodies from different clonotypes to our original hits. this expands the sequence space available through repertoire mining and permits favourable shifts in in-silico developability metrics. of particular advantage is paratyping's ability to prediction common antigen reactivity of antibodies from different v and j gene backgrounds, which has implications for large-scale repertoire analysis. antibodies from different clonotypes have been observed to converge on the same binding site [ ] [ ] [ ] [ ] . we hypothesise that these functionally convergent antibodies may use the same paratope for interaction, and examined the structural antibody database (sabdab) [ ] for evidence of this. we define the epitope and paratope as those residues with any atom within . Å of any residue in the cognate antibody or antigen respectively. : an example of two antibodies that bind to the same epitope but derive from different clonotypes. the murine c (pdb id: do ) and d (pdb id: zpv) anti-mers-cov antibodies target the same residues on the receptor binding domain of the spike protein, despite being derived from different v and j genes and displaying cdrh amino acid identity ( . %) below the standard clonotyping definition ( % - %) [ ] (c). the antibodies use over % of the same paratope residues (b) to achieve this functional convergence. main of the mers-cov spike protein at the same epitope ( . % of epitope residues are shared across the pair of structures). the antibodies come from different clonotypes with both low cdrh identity ( . %) and different germline origins (v - - /j vs v - - /j ). however, c and d use largely the same paratope residues to achieve this epitope convergence, with . % of paratope residues conserved across the structures. another example of epitope convergence between antibodies with differing j genes and sub- % cdrh identity can be seen in the anti-lysozyme antibodies hyhel- and hyhel- (see supplementary figure ). in a standard implementation, antibodies with the same v and j genes, cdrh s of the same length, and above a threshold level of amino acid identity across the cdrh are considered to be in the same clonotype [ ] . in our novel method, paratyping, antibodies with the same length cdrs and above a threshold level of sequence identity across the predicted paratope residues are grouped into the same paratype. to test the ability of paratyping and clonotyping to group antibodies that target the same epitope we performed a test in a single-cell (paired vh/vl) data set of antibodies isolated from genetically engineered mice that have a full set of human immunoglobulin variable region genes [ ] immunised with pertussis toxoid (ptx). although we had pairing information within the single-cell data set, given that the majority of repertoire sequencing data to date is heavy-chain only [ ] , we demonstrate the method using only the heavy chain information. the sequences were annotated with a ptx-binding ( ) or non-binding label ( ) (as per the "single-cell data set" section of methods), and we used paratyping (our new method) and clonotyping (the conventional approach) to identify ptx-binding sequences. for each of the ptx-binders in turn, we mimicked a repertoire mining experiment by using paratyping or clonotyping to identify binders amongst the remaining sequences (one-vs-all crossvalidation). each of the ptx-binders is referred to as a "probe" antibody; sequences that are within the same paratype or clonotype as the probe are predicted to bind ptx. the precision and recall of the two methods (calculated over the aggregate of predictions) for repertoire mining are comparable ( figure , table ). the precision and recall using clonotyping and paratyping with varying cdrh sequence identity or paratope sequence identity thresholds, respectively, are shown in figure a and b. the methods require different sequence identity thresholds for optimum performance but have similar precision-recall profiles. for clonotyping, the optimal heavy-chain only threshold is % cdrh amino acid identity. for paratyping, the optimum occurs at % paratope identity. at these optimal thresholds, clonotyping % % % clonotyping % % % table : precision-recall values for prediction of ptx-binding according to paratyping and clonotyping at the optimal thresholds of % and % respectively. sequence identity is calculated across the predicted paratope for paratyping and across the cdrh for clonotyping. the methods behave comparably over the full precision-recall curve. these thresholds were then used for the prospective repertoire mining experiment. recovers binders with % precision and % recall (meaning that % of the binders in this data set are not related by clonotype to any other). paratyping recovers binders with a precision of % and a recall of % (meaning that % of binders in this data set have distinct paratopes). we expect that this would be an overestimation of performance in the bulk data set due to the enrichment of ptxbinders created through antigen-specific sorting. for each probe, a prediction can be made by both paratyping and clonotyping (hereafter labelled a "both" prediction), paratyping alone (labelled "paratype-only") or clonotyping alone (labelled "clonotypeonly"). out of all of these predictions, . % are made by both methods with "paratype-only" predictions (precision: %) and "clonotype-only" predictions (precision: %). paratyping and clonotyping make a number of method-exclusive predictions, as shown in figure , where the probe antibodies with the largest number of "paratype-only" and "clonotype-only" predictions are shown in dendrograms with these predictions. figure v genes ( ), those with different j genes ( ) and those with cdrh identity below . % ( ) . for example, a pair of binders with just % h sequence identity but % predicted paratope identity were clustered together. paratype predictions can potentially be further validated by building homology models of the sequences [ ] . in this case, modelling of the sequences suggests that the pair of sequences would also be predicted to be highly structurally similar (see supplementary table in order to test how the methods would scale in a larger and non-enriched data set, we performed a prospective repertoire mining experiment in which the heavy chain sequences from ptx-binding antibodies were used to identify novel ptx-binding heavy chains from a set of bulk heavy chain repertoires. we first looked at how the methods scale in terms of number of predictions, and then experimentally validated a number of the predictions of both clonotyping and paratyping. other sequences which are in the same paratype or clonotype are predicted to also bind ptx. these predicted ptx binding sequences are coloured according to whether they are identified by both paratyping and clonotyping ("both"), paratyping but not clonotyping ("paratype-only") or clonotyping but not paratyping ("clonotype-only"). circular leaves represent true ptx binding antibodies (i.e. true positives) while triangular leaves represent sequences that do not bind ptx (false positives). dendrogram a shows the probe which had the most "clonotypeonly" predictions, of which % are true positives; dendrogram b shows the probe with the most "paratype-only" predictions, of which % are true positives; dendrogram c is the probe antibody which is associated with the most false positives ( % true positives) while dendrogram d is the probe antibody associated with the most true positives ( true positives). performance is heterogeneous across probes ranging between % and % precision; precision and recall values reported elsewhere in this manuscript consider the performance in aggregate of all probes. v and j genes are annotated where this changes within a dendrogram. dendrograms are constructed using the full vh sequence with the neighbour-joining algorithm of the r package ape [ ] , plotted using the r package ggtree [ ] . the units of the scale bar are amino acid substitions per residue. using the known ptx binders as probes, , heavy chain sequences from bulk sequencing repertoires were searched for paratype-or clonotype-related sequences. sequences were identified by both clonotyping and paratyping, by paratype-only and by clonotype-only. the paratype-only predictions can be categorised as those with a different v gene ( ), those with a different j gene ( ) and those with sub- % cdrh identity ( ). for the experimental validation of predicted ptx binders and non-binders, we created a category of prediction more stringent than the "paratype-only" or "clonotype-only" categories to show the utility of paratyping in a real antibody discovery experiment context where multiple probe sequences are available. as shown in figure , a particular probe antibody may make predictions via clonotype or paratype alone. as reflected in the similarity in the precision-recall values calculated over the aggregate of probe antibodies in the single-cell one-versus-all cross-validation, these "method-unique" predictions become rarer when considering a larger number of probes -such predictions, which could not be found by another method even when using the full complement of known binders, are referred to as "paratype-unique" or "clonotype-unique" predictions. of the potential predicted ptx binders, were selected for expression (see methods) and ptx-binding assay. an additional antibodies predicted to not bind ptx (due to paratope identity or shared lineage with confirmed non-binders) were also tested. the predictions were split into thirds according to whether they were predicted by both methods (labelled "both"), or were unique across all binders for paratyping ("paratype-unique") or clonotyping ("clonotype-unique"). of the ( %) novel heavy chains predicted to bind ptx by both paratyping and clonotyping were true ptx binders. paratope identity between known and predicted binders ranged between % and %, with cdrh identity ranging between % and %. of the ( %) of the clonotypeunique predictions were true ptx binders, with minimally % predicted paratope identity to a known binder, % cdrh identity and % cdrh identity (amino acid identity calculated over the heavy chain cdrs). of the ( %) of the paratype-unique ptx binders bound ptx. the minimal cdrh identity of a ptx binder to any known binder was % with % paratope identity. the distribution of cdrh , total cdr and total vh amino acid identity of novel ptx-binding heavy chains to known ptx-binding antibodies is shown in figure . none of the predicted ptx non-binders bound ptx. as in the single-cell data set, the success rate in the predictions which were made by both clonotyping and paratyping is higher than either method alone. the success rate of paratype-unique predictions is significantly lower than that of clonotype-unique predictions. however, it may not be appropriate to compare performance across different probe antibodies, some of which may be liable to activity cliffs (a concept from small molecule chemistry where a compound exhibits a large change in activity given only a small change in structure [ ] ). a direct comparison can be made where both clonotype-unique and paratype-unique predictions were made using the same known ptx binder. this occurred for ptx probes, and across these an average precision of % for paratype-unique and % for clonotypeunique was observed. paratyping identified ptx-binding antibodies that could not be found using clonotyping ("paratypeunique"), for example those using a different v gene to any of the known ptx binders. an example is shown in figure ) , where the original antibody used the inherently autoreactive v gene v - [ ] , which may be problematic in development. however, paratyping recovers seven ptx-reactive antibodies which use the v - gene segment instead. heavy chains predicted to bind ptx are coloured as green, blue or purple depending on whether they are predicted to bind via both methods or were clonotype-or paratype-unique. asterisks indicate heavy chains selected for testing, all of which were validated as ptx binding. a shows sequences predicted to bind ptx that use a different v gene (v - ) than the known ptx binder used for prediction (v - ). sequences using a different j gene to the known ptx binder are shown in b. figure : cdrh , total cdr and total vh amino acid identity of novel ptx-binding antibodies to the known ptx-binding antibody by which they were identified, according to method by which they were identified. paratyping enables the discovery of ptx-binding sequences with lower sequence identity across each of these regions with minimally % cdrh identity, % cdrh identity and % total vh identity. paratyping also recovered novel ptx-binding heavy chains which derive from different j genes and examples with cdrh identities well below most clonotyping thresholds (commonly % - %). the minimal cdrh identity of a validated ptx-binding antibody to any known binder was %, suggesting that paratyping can identify antibodies that bind to the same epitope that could not be found by any clonotyping method. one of the limitations of clonotyping as a method for immune repertoire mining is the relatively narrow sequence space it is capable of making predictions within, meaning that the discovered antibodies may have conserved developability problems. we have already seen one example where paratyping's ability to jump between germlines allows us to avoid an autoreactive v gene-derived antibody; other developability problems such as aggregation propensity may also be improved by using paratyping. of the original antibodies used for repertoire mining, antibodies were flagged by the therapeutic antibody profiler (tap) tool as having possible developability issues due to cdr length, high density of charge or hydrophobicity, or charge asymmetry between the heavy and light chains. as paratyping only groups antibodies with the same length cdrs, we consider only the latter four de- figure shows the improvement in patch surface hydrophobicity achieved by immune repertoire mining using cl- as a probe antibody. cl- had an amber flag for this metric. repertoire mining was used to identify a number of predicted ptx-binding antibodies. it can be seen that the more sequence-distinct paratype-only predictions are able to achieve greater changes in psh. these predictions were not assayed as they were within the clonotype of another known binder and therefore not "paratype-unique". characterising the functional relationship between sequence-distinct antibodies is an important step in our understanding of the adaptive immune landscape. mapping antigen preference to antibody repertoires will allow us to identify epitope convergence of antibodies at a large scale. in a test system of transgenic mice immunised with pertussis toxoid (ptx), we show for the first time that prediction and comparison of paratopes can be used to group antigen-specific antibodies in both an enriched, single-cell data set and non-enriched bulk heavy chain repertoires. we demonstrate the utility of the method in the context of an antibody discovery experiment alongside the conventional approach of clonotyping and discover new anti-ptx antibodies from different clonotypes to any known binders. we first developed the method in a single-cell data set, where paratyping and clonotyping were able to group ptx-specific antibody heavy chains with high precision ( % and % respectively). these results may not map to the bulk sequencing data set given that the sequences derived from both plasma-, memory-and antigen-sorted cells (leading to ca. % of sequences being ptx-reactive). to validate the method in a less enriched data set, we performed a prospective experimental test of paratyping in a non-antigen sorted set of bulk heavy chain sequencing repertoires. in the prospective experimental test, paratyping allowed us to discover new ptx-binding antibodies that we could not have found using clonotyping. these include antibodies that use different germline genes as well as antibodies with lower cdrh identity than in common definitions ( - %). in terms of antibody discovery, paratyping allows us to identify sequence-distinct antibodies that bind to the same epitope and which can differ significantly in developability or affinity. in the terms of repertoire analysis, paratyping expands our ability to functionally group antibodies beyond clonotypes, and therefore allows us to detect specific cases of epitope convergence between lineages -we found epitope convergence between v - /j and v - /j lineages, v - /j and v - /j lineages, v - /j and v - /j lineages and v - /j and v - /j lineages in pertussis toxoid binders. we did not observe pairs of antibodies in the same paratype using different v gene families. however, paratope identity across the germline-encoded cdrhs and is equal to or in excess of % across members of v and v , and v and v (see supplementary figure ), so we predict that it should be possible to use paratyping to find binders from different v gene families, should a large enough sequencing data set be mined. the implications of this for immune repertoire clustering are as yet unexplored. if such convergence is widespread, it is possible that clonotyping overestimates functional diversity and this may account for low proportions of clonotypes shared across individuals [ ] . success rate in the prospective experimental validation was variable across the categories of prediction. we found that % of sequences predicted by both clonotyping and paratyping to be binders bound ptx. the success rate was considerably lower in clonotype-unique predictions ( %) and even lower in paratype-unique predictions ( %). it should be noted that these method-unique predictions form the minority of predictions for either method ( - % of predictions). the lower success rate of paratyping versus clonotyping may be attributable to the particularly low cdrh identity of these predictions to known binders -paratyping does not give special weight to the cdrh in the paratope identity calculation despite the particular role it plays in antigen complimentarity. predictions with as little as % cdrh identity to a known binder were assayed but no antibody with an h amino acid identity below % bound to pertussis toxoid, suggesting that paratyping could be further improved by the use of cdrh weighting. paratope prediction only takes around . seconds per sequence (of which . seconds corresponds to cdr extraction) as opposed to . seconds per sequence for vdj annotation using igblast [ ] ) which means it is considerably more tractable for large datasets than homology modelling (on average seconds per sequence, times slower than germline gene annotation [ ] ). it has the advantage that it does not rely on the upkeep of consistent and complete germline databases (a leading cause of disagreement between germline annotation tools [ ] ), but rather on the distribution of a pretrained paratope prediction model [ ] . the paratope prediction step is purely sequence-based and structural modelling is not required, meaning that immune repertoires can be annotated without accurate germline alignment and without access to large compute power. paratyping was shown to identify functional relationships between ptx-binding antibodies that are not related by clonotype; we would expect this to generalise across protein antigens. as an example, we looked at cov-abdab [ ] , a database of antibodies and nanobodies known to bind to betacoron- and c , shown in figure , are another example. deciphering the functional landscape of immune repertoires will greatly improve our understanding of the adaptive immune system. improving our ability to group antibodies binding to the same epitope is a step towards this. our results show here that the simple and computationally rapid abstraction of the antibody binding site used by paratyping is sufficient to group antigen-specific antibodies in a way which provides us with additional information to clonotyping. this additional information is particularly significant in the context of antibody discovery, where it allows us to recover different novel antigenspecific antibodies from immune repertoires. five genetically engineered mice that have a full set of human immunoglobulin variable region genes [ ] were immunised with pertussis toxoid (ptx). paired (v h /v l ) sequences were recovered from antigen-sorted, plasma and memory cells via a previously published method [ ] . antibody sequences with greater than % effect value relative to the positive control were labelled as binders. this resulted in ptx-binders and non-binders. heavy chains from sorted splenic b-cells from the same individual five mice as the single-cell data set were sequenced using standard protocols [ ] and processed using the presto/change-o pipeline [ , ] . this resulted in , heavy chain sequences. for quality control, only sequences with read count (reads with a particular unique molecular identifier (umi)) above or equal to two or a consensus count (reads with different umis but the same nucleotide sequence) above or equal to ten were considered, reducing the size of the data set to , sequences. clonotypes were defined as groups of heavy chain sequences sharing the same v and j genes, with identical cdrh lengths and a number of amino acid mismatches equal to or below a threshold sequence identity. vj annotation was performed with igblast [ ] within change-o [ ] . cdrh s were extracted according to the north definition [ ] with imgt numbering [ ] performed with anarci [ ] . paratypes were defined as heavy chain sequences sharing the same cdr lengths and greater than a threshold sequence identity across the predicted paratope regions. cdrs were extracted according to north definitions [ ] with imgt numbering [ ] performed via anarci [ ] . parapred [ ] was used for paratope prediction using the model as distributed by e. liberis at https://github.com/eliberis/parapred. to convert the output of parapred, binding probabilities, into a binary label, we selected a threshold of . as deemed optimal by the authors of the original paper [ ] , i.e. residues with a predicted probability of being in the paratope of above . were annotated as paratope residues. paratope identity was defined as the number of identical paratope residues (residues which are predicted to be in the paratope in both cases) divided by the smallest number of paratope residues of either sequence being compared (figure ). figure : method of calculating predicted paratope identity. x indicates cdr residues not predicted to constitute the paratope. in the example shown, the paratope identity is . %. figure : graphical illustration of the process of repertoire mining via paratyping. a probe antibody is selected. paratope prediction is performed both on the probe antibody and the bulk data set. heavy chain predicted paratope identity is used to mine the bulk repertoire for new predicted binders. in the repertoire mining experiment, we selected a number of hit antibodies from the single-cell data set to use as probes. spr was carried out on the htrf-positive antibodies and the highest affinity representatives were selected from each clonotype containing only sequences labelled as binders equating to antibodies, in order to align with previous repertoire mining experiments [ ] . we also selected a number of non-ptx binding antibodies to be used as a negative control. the non-ptx binding antibodies were selected as representatives of clonotypes containing only non-ptx binding sequences ( antibodies). the heavy chains from these probe antibodies were used as probes to mine the bulk repertoires via both paratyping and clonotyping, using the optimal sequence identity thresholds from the single-cell data set ( % predicted paratope identity for paratyping and % cdrh sequence identity for clonotyping). the paratyping process is illustrated in figure . predictions were labelled as "paratype-only", "clonotype-only" or "both" as detailed in the results section. if an antibody is within the same paratype as a particular probe but not within its clonotype, it is a "paratype-only" prediction and vice-versa. a prediction is labelled as "both" if it is within both the paratype and clonotype of the same probe. in the "experimental validation of predicted binders" section of methods, the more stringent "paratype-unique" and "clonotype-unique" definitions were used. we selected heavy chain sequences from the bulk data set for expression. of the sequences were predicted to be ptx binders using the ptx-binding probe sequences. we created a new way of categorising predictions in order to fully test the method in the context of an antibody discovery experiment. we define "paratype-unique" predictions as predictions which are not within the paratype of not only the probe in question (paratype-only) but of any of the full complement of probes, and similarly for "clonotype-unique" predictions. paratype-unique predictions are a more stringent subset of paratype-only predictions and present a greater challenge of the method (as such predictions tend to be corroborated by fewer probes). predicted ptx-binding heavy chain sequences were selected from each of the three categories of prediction ("paratype-unique", "clonotype-unique" and "both"). five of the "both" predictions were identical to their probes. the remaining sequences assayed were predicted non-ptx binding heavy chain sequences with sequences selected from "clonotypeunique", "paratype-unique" and "both" categories of prediction. the heavy chain sequences selected for expression from the bulk repertoire were paired with the cognate light chain of the probe sequence by which they were identified. within-clonotype vh/vl pair-ing has been validated [ , ] as a method of reconstituting binding where the cognate light chain is not sequenced. pairings were only made where there was greater than % sequence identity across the residues considered to constitute the v h /v l interface, based on solvent accessibility [ ] , in order to maximise the probability of expression. these positions lie outside of the cdrs and therefore do not enforce any constraint on binding site sequence identity. the predicted binding and non-binding antibodies were expressed in hek cells. the assay is as described in the single-cell data set section of methods with the exception that the anti-ptx antibody b was used as positive control. antibody sequences with an f value exceeding were labelled as ptx-reactive antibodies. to evaluate the performance of either method in grouping ptx-binding sequences, we calculated precision and recall of the method in the single-cell data set according to the following definitions of true positives, false positives and false negatives. the task is to group antibodies that bind pertussis toxoid. however, the method is hypothesised to work by grouping by epitope. there will be multiple epitopes on pertussis toxoid so not all ptx-binding antibodies will be grouped into a single paratype or clonotype. as a result, we do not expect perfect recall. further, we do not classify antibodies as non-binders if they do not group with a particular binder -we only predict that they do not bind at the same epitope as the binder in question. as a result, we do not calculate a "true negative" rate. • true positive (tp): a ptx-binding sequence that was identified by another ptx-binding sequence. • false positive (fp): a non-ptx-binding sequence that was identified by a ptx-binding sequence. • false negative (fn): a ptx-binding sequence that was not identified by any ptx-binding sequence. we calculated the in-silico developability metrics of total cdr length, patches of surface hydrophobicity (psh), patches of positive charge (ppc), patches of negative charge (pnc) and structural fv charge symmetry parameter (sfvcsp) using the therapeutic antibody profiler tool (tap) [ ] . the tool calculates these metrics from homology models built using abodybuilder [ ] and compares them to a database of clinical-stage therapeutics (csts) [ ] . according to the metric in question, values are flagged as "amber" (extreme) if they lie outside the upper or lower, or upper and lower, % of observed values among csts. an antibody is flagged as "red" if the value of a particular metric is outside of the observed range in csts. [ ] funding the code and data associated with this study are available at http://opig.stats.ox.ac.uk/resources. the promise and challenge of high-throughput sequencing of the antibody repertoire. en practical guidelines for b-cell receptor repertoire sequencing analysis commonality despite exceptional diversity in the baseline human antibody repertoire analysis of the b cell receptor repertoire in six immune-mediated diseases. en bioinformatic and statistical analysis of adaptive immune repertoires monoclonal antibodies isolated without screening by analyzing the variablegene repertoire of plasma cells. en mining the antibodyome for hiv- -neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains. en neutralizing antibodies against west nile virus identified directly from human b cells by single-cell analysis and next generation sequencing. en immune repertoire mining for rapid affinity optimization of mouse monoclonal antibodies the analysis of clonal expansions in normal and autoimmune b cell repertoires somatic generation of antibody diversity. en anarci: antigen receptor numbering and receptor classification. en cutting edge: ig h chains are sufficient to determine most b cell clonal relationships. en immunosequencing: applications of immune repertoire deep sequencing. current opinion in immunology sequencing the functional antibody repertoire-diagnostic and therapeutic discovery measurement and clinical monitoring of human lymphocyte clonality by massively parallel v-d-j pyrosequencing. en lineage structure of the human antibody repertoire in response to influenza vaccination. en human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements studying the antibody repertoire after vaccination: practical applications b cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes. en antibody repertoire analysis in polygenic autoimmune diseases igh sequences in common variable immune deficiency reveal altered b cell development and selection ab-ligity: identifying sequence-dissimilar antibodies that bind to the same epitope. en. biorxiv. publisher: cold spring harbor laboratory section: new results sequence and structural convergence of broad and potent hiv antibodies that mimic cd binding. en vaccine-induced antibodies that neutralize group and group influenza a viruses therapeutic monoclonal antibodies for ebola virus infection derived from vaccinated humans. en sabdab: the structural antibody database. en massively scalable genetic analysis of antibody repertoires. en. biorxiv, evidence of antibody repertoire functional convergence through public baseline and shared response structures. en. biorxiv. publisher: cold spring harbor laboratory section: new results deep learning enables therapeutic antibody optimization in mammalian cells by deciphering high-dimensional protein sequence space. en. biorxiv, complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. en observed antibody space: a resource for data mining next-generation sequencing of antibody repertoires. en ape . : an environment for modern phylogenetics and evolutionary analyses in r. en ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. en automated antibody structure prediction with data-driven accuracy estimation advancing the activity cliff concept regulation of inherently autoreactive vh - b cells in the maintenance of human b cell tolerance. en five computational developability guidelines for therapeutic antibody profiling igblast: an immunoglobulin variable domain sequence analysis tool. en benchmarking immunoinformatic tools for the analysis of antibody repertoire sequences. en parapred: antibody paratope prediction using convolutional and recurrent neural networks. en cov-abdab: the coronavirus antibody database. en. biorxiv. publisher: cold spring harbor laboratory section: new results en. us b . library catalog: google patents presto: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires. en. bioinformatics . publisher: oxford academic change-o: a toolkit for analyzing large-scale b cell immunoglobulin repertoire sequencing data. en a new clustering of antibody cdr loop conformations imgt unique numbering for immunoglobulin and t cell receptor variable domains and ig superfamily v-like domains de novo identification of vrc class hiv- -neutralizing antibodies by next-generation sequencing of b-cell transcripts. en thera-sabdab: the therapeutic structural antibody database. en. nucleic acids research . publisher: oxford academic, d -d key: cord- -dujd gg authors: chenoweth, alicia m; wines, bruce d; anania, jessica c; mark hogarth, p title: harnessing the immune system via fcγr function in immune therapy: a pathway to next‐gen mabs date: - - journal: immunol cell biol doi: . /imcb. sha: doc_id: cord_uid: dujd gg the human fragment crystallizable (fc)γ receptor (r) interacts with antigen‐complexed immunoglobulin (ig)g ligands to both activate and modulate a powerful network of inflammatory host‐protective effector functions that are key to the normal physiology of immune resistance to pathogens. more than therapeutic monoclonal antibodies (mabs) are approved or in late stage clinical trials, many of which harness the potent fcγr‐mediated effector systems to varying degrees. this is most evident for antibodies targeting cancer cells inducing antibody‐dependent killing or phagocytosis but is also true to some degree for the mabs that neutralize or remove small macromolecules such as cytokines or other igs. the use of mab therapeutics has also revealed a “scaffolding” role for fcγr which, in different contexts, may either underpin the therapeutic mab action such as immune agonism or trigger catastrophic adverse effects. the still unmet therapeutic need in many cancers, inflammatory diseases or emerging infections such as severe acute respiratory syndrome coronavirus (sars‐cov‐ ) requires increased effort on the development of improved and novel mabs. a more mature appreciation of the immunobiology of individual fcγr function and the complexity of the relationships between fcγrs and antibodies is fueling efforts to develop more potent “next‐gen” therapeutic antibodies. such development strategies now include focused glycan or protein engineering of the fc to increase affinity and/or tailor specificity for selective engagement of individual activating fcγrs or the inhibitory fcγriib or alternatively, for the ablation of fcγr interaction altogether. this review touches on recent aspects of fcγr and igg immunobiology and its relationship with the present and future actions of therapeutic mabs. the regulatory approval of the first therapeutic monoclonal antibodies (mabs) in the s ushered in the modern era of immune therapy. since then, mabs have become one of the most clinically successful therapeutic modalities across a diverse array of diseases. they have revolutionized the treatment of chronic inflammatory diseases and of some cancers including otherwise incurable malignancies. they are commercially important and in , five mabs collectively grossed $ . billion in sales, placing them in the top ten drugs globally. mab development is expanding rapidly with over mabs approved for clinical use or in late-stage clinical trials and over in various stages of clinical development. the therapeutic actions of mabs can take many formsneutralization of the target such as cytokines in autoimmune disease, clearance of the target such as virus in infection or immunoglobulin (ig)e in allergy, induction of innate effector cell activation that leads to target destruction by direct killing or the induction of apoptosis and the induction of adaptive immunity. most therapeutic mabs are igg in origin and the heavy-chain subclass determines many of their biological properties including their long plasma half-life ; complement activation, which is important in the action of some cytotoxic mabs [ ] [ ] [ ] and importantly engagement by their fragment crystallizable (fc) region with specific cell surface receptors, called fccr, the subject of this review. in normal homeostatic immunity, there is a balance between igg immune complex activation of proinflammatory responses through the activating-type fccrs-which leads to the destruction of opsonized pathogens-and of the modulation of these destructive effector responses by the inhibitory-type fccr, thereby avoiding injury to the host. thus, therapeutic mabs powerfully exploit these opposing activities, making them versatile drugs whose therapeutic potency can be improved by specific engineering of fc-fccr interactions. many therapeutic mabs depend, to varying degrees, on fccr function ( figure , table ) for their mechanism of action (moa) and/or their pharmacokinetic properties. for some mabs interaction with fccr is central to their moa, such as the destruction of a target cell by antibody-dependent cell-mediated cytotoxicity (adcc; figure a ) or antibodydependent cell-mediated phagocytosis (phagocytosis or adcp; figure b ). this also includes mabs that may harness the inhibitory action of fccriib to modulate the proinflammatory responses of immunoreceptor tyrosine activation motif (itam)-dependent receptor signaling complexes (figure c ). for other mabs, fccr may play a secondary role, such as the removal or "sweeping" of all immune complexes formed by cytokine or virus-specific neutralizing antibodies or of opsonized fragments of lysed target cells which in antigen-presenting cells may also feed the antigen into the antigen-presentation pathways (figure d ). in addition, fccrs, particularly fccriib (figure e ), are also key participants in the moa of immune-stimulating agonistic mabs or apoptotic mabs by acting as a scaffold for the additional cross-linking of mabs already bound to a cellular target, thereby inducing a signal in the target cell. this review focuses on the cell-based effector functions that arise from the interaction of igg with the classical human leukocyte fccr. although beyond the scope of this review, it should be noted that the igg-fc portion dictates other aspects of an antibody's biology, including its serum half-life mediated by the neonatal fcr (fcrn), the activation of complement c , antiviral protection via the intracellular receptor trim and interactions with the fc receptor-like family. the human leukocyte receptors fall into two functional groups, namely, proinflammatory, activating-type receptors (fccri, fccriia, fccriic, fccriiia and fccriiib, which are also known as cd , cd a, cd c, cd a and cd b, respectively) and the anti-inflammatory, inhibitoryreceptor group (fccriib also called cd b) which was the first immune checkpoint described. these fccrs are high-avidity sensors of immune complexes which initiate, and then modulate, cell responses. in the context of normal immune physiology, opsonized target molecules can engage various fccrs and induce a spectrum of effector responses which can be harnessed by many therapeutic mabs ( figure , table ). these responses are not mutually exclusive and one therapeutic mab may initiate various responses via different fccrs and via different cell types. understanding the importance of cell-based effector functions in the moa of therapeutic mabs requires an appreciation of fccr biology (tables - ) which also underpins future efforts to tailor new mabs for the exploitation-specific effector responses. in this review, we address only key aspects of the extensive knowledge of the human leukocyte fccr family as it relates to effector functions. a number of other reviews more comprehensively explore fccr biology physiology, biochemistry, genetics and structure. , [ ] [ ] [ ] [ ] notwithstanding the recognized differences between the immunobiology of human fccr and of rodents or nonhuman primates, animal models of fcr effector function in vivo have helped shape the strategies for the development of current therapeutic mabs and are well reviewed. , furthermore, humanized fccr models will provide even greater insights into the future. the tissue distribution of the human leukocyte fccr is well documented and reviewed comprehensively elsewhere. , , in the context of effector functions harnessed by therapeutic mabs, several aspects of the cellular distribution (table ) should be emphasized. fccr expression profiles differ between cell lineages but almost all mature human leukocytes, and platelets, express at least one fccr (table ) . it should also be appreciated that the cellular expression levels and receptor diversity as will be described later is also influenced by the activation state of the cells, anatomical location and the cytokine environment which modulates fccr expression, particularly for fccri and fccriib. for example, resting monocyte subpopulations may express only fccriia but activated macrophages express fccri, fccriia and fccriiia and/or fccriib. thus, specific characteristics of leukocyte fccr expression are summarized as follows: fccri is not usually expressed until induced by cytokines such as interferon-c on monocytes, neutrophils, macrophages, microglial cells in the brain, dendritic cells and mast cells. the sensitivity of fccri to interferon-c suggests that its in vivo activity is closely tied to immune activation events, and mouse studies have suggested that it has a critical role early in immune responses. , its role in the moa of antibodies may vary with anatomical location. fccriia is expressed only in primates and shows the broadest expression of all fccrs, being present on all innate (b) antibody-dependent cell-mediated phagocytosis, and/or trogocytosis of large immune complexes, by professional phagocytes via activating fccr such as fccriiia and fccriia. biological sequelae include the destruction of the ingested complexes which may also feed antigen into antigenpresentation pathways of antigen-presenting cells (apcs). (c) inhibition of cell activation by fccriib. the immunoreceptor tyrosine activation motif (itam)-mediated signaling of b-cell antigen receptors (left) or of activating fccr (right) on innate immune cells such as macrophages and basophils is inhibited by igg fc-mediated co-cross-linking of these activating receptors with the inhibitory fccriib. this leads to phosphorylation of the fccriib immunoreceptor tyrosine-based inhibitory motif (itim) and consequently recruits the phosphatases that modulate the itam-driven signaling responses leading to diminished cell responses. (d) sweeping or internalization of small immune complexes leading to their removal and, in apc, to enhanced immune responses. (e) scaffolding in which the fccrs play a passive role. typically involving fccriib, no signal is generated in the effector cell but "super-cross-linking" of the opsonizing antibody by the fccr on one cell generates a signal in the conjugated target cell, for example, induction of apoptosis or activation in agonistic expansion of cells and/or their secretion of cytokines. in extreme cases, this leads to life-threatening cytokine storm. adcc, antibody-dependent cell-mediated cytotoxicity; ag, antigen; bcr, b-cell receptor; ig, immunoglobulin; nk, natural killer. leukocytes. it is also present on platelets but its role in effector functions is not established but it is important in certain immune thrombocytopenias. a polymorphic form of this receptor is the only human receptor for human igg . this, together with its limited species expression and unique itam-containing cytoplasmic tail (reviewed by anania et al. ), suggests a unique function in human leukocytes. interestingly, polymorphism of the receptor is associated with systemic lupus erythematosus and resistance to gram-negative organisms. a rare, hyper-responsive form is a risk factor for neutrophil-driven anaphylaxis in ig replacement therapy. fccriic is an activating fccr whose expression is regulated single nucleotide polymorphism that permits expression in approximately % of humans and in whom it is present at low levels on natural killer (nk) and b cells. it has arisen by gene duplication/ recombination resulting in an extracellular region derived from fccriib, which binds igg , and with an itam-containing cytoplasmic tail related to the activating receptor fccriia. thus fccriic provides igg with an activation receptor pathway and confers a new biology of igg in these individuals. its low frequency in the population may also confound in vivo mab clinical testing or use, but as yet there is no evidence for this. fccriii forms are two highly related gene products, fccriiia and fccriiib. the fccriiia is expressed on nk cells and professional phagocytes, particularly macrophages. it is only recently apparent that fccriiia is expressed on neutrophils, albeit at low levels, but plays a role in their function. fccriiib is unique to humans and unlike other fccrs it is attached to cell membrane via a glycophosphatidyl anchor. it is expressed, predominantly and abundantly, on human neutrophils. its effector function depends in part on its coexpression with fccriia. the lack of fccriiib on macaque neutrophils appears to be compensated for by an increase in fccriia expression. fccriibs are the inhibitory-type fccr and arise from a single gene. they lack intrinsic proinflammatory signaling and are instead immune checkpoints. they provide feedback regulation by antibodies, in the form of immune complexes, to inhibit b-cell activation by specific antigen. they also control activating-type fccr function on innate cells. two major splice variant forms of fccriib exist with differential tissue expression profiles. fccriib preferentially expressed on b lymphocytes contains a -amino acid cytoplasmic insertion necessary for membrane retention and cocapping with the b-cell antigen receptor (bcr). fccriib is the predominant inhibitory receptor found on basophils and neutrophils, as well as on subpopulations of mast cells, dendritic cells and some monocytes/macrophages. fccriib lacks the cytoplasmic insertion of fccriib and consequently can internalize rapidly including with the activating fcr when they are co-cross-linked. it is not clear which form is present on human t cells. one additional comment on tissue distribution is that fccr expression on t cells has been difficult to establish unequivocally. however, there is increasing evidence that tlymphocyte populations express fccr. some cd t cells express fccriiia and ab t cells reportedly express fccriia, fccriib or fccriiia but the significance with respect to effector function mediated by antibody is presently unclear. [ ] [ ] [ ] [ ] [ ] expression on nonhemopoietic cells the immunobiology of fccr is studied and understood almost exclusively in the context of hematopoietic cell function but relatively recent investigations have identified and explored fccr expression on nonhemopoietic cells. these studies suggest important roles in normal immune function and in the moa of some therapeutic mabs. the most extensively characterized receptor expression is fccriib which is expressed on follicular dendritic cell, airway smooth muscle and liver endothelium. its abundant expression on liver sinusoidal endothelial cells (lsecs) is estimated to represent the majority of in vivo fccriib expression. , - as fccriib lacks intrinsic proinflammatory signaling function, its role on these nonhemopoietic cells involves immune complex handling without the danger of, or the need for, induction of local tissue destructive inflammatory responses. on lsec its major role appears to be immune complex sweeping, a process of removal of small immune complexes such as opsonized virus or macromolecules. this scavenging role by fccriib on lsec can be exploited in principle by mabs forming small soluble complexes with their targets such as antiviral, anticytokine or similar antibodies. effector functions that are initiated via the activating-type fccr occur by signaling via the itam pathway of immune receptors. this well-characterized pathway is used by bcr and t-cell antigen receptors, the ige receptor fceri and iga receptor fcari (reviewed extensively by hogarth and pietersz anania et al. and getahun and cambier ) induction of an activating signal requires the aggregation of activating fccr by immune complexes, or by antigen in the case of the bcr. this aggregation at the cell membrane results in specific tyrosine phosphorylation of the itam by src kinases, thus initiating the activation cascade. - the inhibitory-type fccrs, fccriib and fccriib , whose expression is cell lineage restricted, modulate the itam signaling of the bcr or the activating-type fccrs, respectively. their function is dependent on the immunoreceptor tyrosine inhibition motif in their cytoplasmic tail. , this checkpoint action requires that fccriibs are coaggregated with the tyrosinephosphorylated itam-signaling receptor complex which results also in immunoreceptor tyrosine inhibition motif tyrosine phosphorylation and consequential recruitment of lipid or protein tyrosine phosphatases that powerfully dampen the itam-induced cell activation. not all opsonized targets are equal: size, distance, valency and fc geometry affect potency to understand the immunobiology of fccr effector responses particularly in the therapeutic mab context, it is important to appreciate that the quality and potency of such effector responses is greatly affected by the nature of the igg immune complex and/or the state of potential effector cells. first, opsonization, per se, of a target is not necessarily sufficient to ensure fccr interaction in a way that initiates an effector response. although it is the igg fc that interacts with and clusters the fccr to induce a response, the nature of the fab interaction with its epitope can strongly influence the likelihood or potency of fccr effector responses by influencing the density of appropriately presented fc portions. and also the size of the immune complex. furthermore, the display/ orientation and geometry of the fc portions, as a consequence of the fragment antigen-binding (fab) interaction with the target epitope, can result in effector responses such as adcc that differ substantially in potency, presumably because the orientation of the fc makes fccr engagement more, or less, accessible. , second, in innate effector cells at rest, the largely linear actin cytoskeleton and the extracellular glycosaminoglycan glycocalyx regulate function by interacting with large glycoproteins, such as cd , arranging these into ordered "picket" fences. , these corral receptors, including the fccrs, and sterically inhibit their interaction with ligands. upon cell activation, cytoskeletal remodeling is associated with the loss of the receptor corrals, allowing fccrs and other receptors to freely diffuse, engage ligand, cluster and signal. the influence of such surface constraints on receptors and effector cell function helps explain some of the observed epitope distance requirements for optimal mab function, , which were apparent in a comparative study of adcc and adcp. adcc was optimal when the epitope was displayed close, . nm "flush" or . nm, to the target membrane where close conjugation of effector and target by the mab presumably facilitates the delivery of pore-forming proteins to the target membrane as required by adcc. interestingly, complement-dependent cytotoxicity which also utilizes poreforming proteins for its cytotoxicity has similar distance constraints. by contrast, adcp was poor when targeting epitopes displayed close or "flush" to the target cell membrane (within~ . nm) but adcp activity was restored when the epitope was displayed . nm off the membrane, demonstrating different optimal epitope distance requirements for adcc and adcp. although the action of agonistic/antagonistic mabs is mechanistically distinct to those eliciting cytotoxicity and adcp, the distance segregation between target and fccr + cells is also important. indeed, the membrane proximal epitopes of cd and cd are important for the fccr function in the complex moa of these mabs. , clearly, the effects of immune complex valency, fc density, presentation and geometry together with fccr organization in the cell membrane suggest that the development of mabs to certain targets will be heavily influenced by the context of use. thus, improved mab potency may not necessarily be achieved by engineering of the fc polypeptide or its glycan alone. a more function-oriented approach early in mab selection and development by, for example, application of rapid screening technologies that select for effector potency, followed by fc engineering may be more productive. adcc and adcp are the most widely appreciated fccrdependent effector functions (figure a, b) and are, respectively, mediated primarily via fccriiia on nk cells and professional phagocytes such as macrophages. these effector functions, particularly nk cell adcc, are believed to be major components of the moa of cytotoxic therapeutic mabs used in cancer therapy. in addition, adcp can also occur via fccriia and fccri, but the extent to which cytotoxic anticancer therapeutic mabs depend on these for their moa in patients is unclear. the improvement in clinical utility of mabs engineered for selectively increased fccriii binding suggests that fccriia and fccri may be less important in vivo in cell killing effects but perhaps are more important in other aspects of therapeutic efficacydiscussed later. fccriib is an immune checkpoint , and its splice variants are potent modulators of itam-dependent signaling ( figure c ). this modulatory function occurs only when fccriib is coaggregated with an itam signaling receptor. thus, b-cell activation by the binding of the antigen in the immune complex to the bcr is regulated by the simultaneous binding of the fcs of the immune complex to fccriib on the same cell. in innate leukocytes, the activating-type fcr (i.e. fccri, fccriia, fccriic, fccriii) and the high-affinity ige receptor, fceri, and the iga receptor, fcari, are all modulated by immune complex co-engagement with fccriib . the inhibitory function contributes to the moa of therapeutic antibodies that target cell-activating molecules where the target cells also express the inhibitory fccriibs such as the bcr (discussed later). thus, b-cell activation is modulated by the simulatenous binding of the antigen in the immune complex to the bcr and the binding of the fcs, also in the immune complex, to fccriib on the same cell. the removal of immune complexes in humans depends primarily on the complement receptor pathway and to a lesser degree the fccr. among the fccrs, it has been widely believed that immune complex removal only occurs by phagocytosis/endocytosis of activating-type fccr. surprisingly, the inhibitory fccriib, which lacks intrinsic activating function, plays a major role in clearance, and rapidly "sweeps" away small complexes from the circulation (figure d) . , a major tissue involved in the clearance is likely to be the lsec, where fccriib is expressed abundantly in mice and humans. this role is potentially important in resistance to viruses and toxins but may also be key to optimal performance of therapeutic igg mabs whose primary moa is believed to be only neutralization of soluble macromolecules, for example, cytokines or ige. monoclonal antibody therapy is a form of passive immunization. indeed, longer-term vaccine-like or vaccinal immunity has been demonstrated in anti-cd treated mice via fccriia , and in vitro recall memory responses from cd -treated patients. although this is dependent on fccr and anti-cd , the mechanism by which long-term anti-tumor response is established remains unclear. nonetheless, the active involvement of fccr in the enhancement of antigen-specific immunity by uptake of immune complexes through fccr is historically well documented in experimental systems where fccrs bind immune complexes and thereby feed antigens into the antigen-presentation pathways. this has been demonstrated in vivo for small immune complexes via human fccri on human antigen-presenting cells and in mice. similarly, the capacity of fccriibs to bind and rapidly internalize antigen-antibody complexes suggests that it too may significantly influence feeding antigens into professional antigen-presenting cells of hematopoietic origin such as dendritic cells and possibly b lymphocytes. although not a classical major histocompatibility complex-dependent antigen presentation, fccriib on the stroma-derived follicular dendritic cells influences antibody immunity by recycling antigen-antibody complexes to the cell surface for presentation of intact antigen to b cells. although somewhat speculative, fccriib's rapid internalization/sweeping of complexes by the abundant lsec, which interact with lymphocytes and can present antigen, may have a significant role in shaping immune responses. fccr-expressing cells can be critical, but passive, participants in the moa of some mabs (figure e ). in fccr scaffolding, igg mab molecules that have opsonized the cell surface of a target cell are additionally cross-linked by their fc portions engaging the fccrs that are arrayed on the surface of a second cell. this "supercross-linking" of the target-bound mab by the fccr lattice or "scaffold" on the adjacent cell greatly exceeds the target cross-linking by the mab alone, thereby inducing a response in the target cell. scaffolding was originally identified as the basis of t-cell mitogenesis induced by anti-cd mab. , the cd mabs alone were poor mitogens but the "super-cross-linking" of the tcell-bound cd mab by the membrane fccr on adjacent cells, particularly by monocytes, induced rapid t-cell expansion and cytokine secretion but did not require activation of fccr-expressing cells. regrettably, fccr scaffolding came to prominence and clinical relevance because of its causal role in the catastrophic adverse events resulting from the administration of anti-cd and anti-cd (tgn ) mabs. nonetheless, fccr scaffold-based induction of intracellular responses in a target cell can also lead to beneficial therapeutic effects. such examples are the induction of apoptotic death in a target cell, which is likely part of the moa of daratumumab in multiple myeloma or the controlled agonistic expansion of cells, for example, via cd mab agonism. igg subclasses: specificity and affinity for fccr most fccrs (table ) are weak, low-affinity receptors (affinities in the micromolar range) for igg-fc, irrespective of whether the igg is uncomplexed, monomeric or when it is complexed with antigen (i.e. an immune complex). the very avid binding of immune complexes to an effector cell surface that displays an array of fccr molecules is the result of the collective contributions of the low-affinity interactions of each fc of the iggs in the complex with an fccr. this avidity effect is necessary as the fccrs operate in vivo in environments of high concentrations of uncomplexed monomeric igg (normally - g l - ). thus, the avid multivalent binding of the complex out competes uncomplexed, monomeric igg. the notable exception to this is the enigmatic fccri. this receptor shows high, nanomolar affinity for uncomplexed monomeric igg and thus would be expected to be constantly occupied in vivo by the normal circulating monomeric igg. however, igg dissociation permits engagement with immune complexes. furthermore, fccri is not expressed or expressed poorly on resting cells, requiring interferon-c for induction of its expression, presumably at sites of inflammation. although the human igg heavy-chain constant domains have greater than % identity, key amino acid differences confer each subclass with unique structural and functional properties. igg and igg are "universal" ligands, that is, they bind to all fccrs. formal measurement of the weak, micromolar k d interactions of the low-affinity receptors with monomeric igg also revealed differing affinities between the low-affinity fccrs, with inhibitory fccriib generally having the lowest affinity and fccriii the higher, sometimes referred to as a "moderate" affinity receptor. , the strength of igg interaction can also be affected by fccr polymorphism and in the context of therapeutic mabs, variation in fccriiia is particularly important. the most common and possibly clinically significant polymorphism is phenylalanine/valine variation at position in the igg-binding site, wherein fccriiia-f binds igg less well than the fccriiia-v form. igg and igg have more restricted fccr specificity. igg has low affinity (k a =~ m - ) for the inhibitory fccriib, but is also a high-affinity ligand for fccri (k a =~ m - ). igg exhibits a highly restricted specificity, showing functional activity with only one polymorphic form of fccriia (binding affinity k a =~ . m - ) which is permitted by the presence of histidine at position of its igg-binding site. this fccriia-h form is expressed in approximately % of the population, whereas igg has no functional activity on the other common allelic form, fccriia-r , which contains arginine at position . , igg subclasses: structure and properties the extracellular regions of the fccr are structurally similar. each low-affinity fccr has two ectodomains, whereas the high-affinity fccri has a third domain but this is not directly involved in igg binding. the interaction between the igg subclasses and the fccr is most comprehensively defined for human igg by both x-ray crystallographic , , and mutagenesis structure/function analyses. [ ] [ ] [ ] these studies defined key regions of the igg sequence required for interaction with their fccrs. crystallographic analyses of the human igg -fc complexed with fccri, fccrii or fccriii show that these interactions are similar in topology, and asymmetric in nature. the second extracellular domain of the fccr inserts between the two heavy chains. here it makes contacts with the lower hinge of both h chains and with residues of the adjacent bc loop of one ch domain and the fg loop of the other. the n-linked glycan at asparagine (n ) of the heavy chain is essential for the structural integrity of the igg-fc by affecting the spacing and conformation of the ch domains. indeed, its removal ablates fccr binding. of particular relevance to therapeutic mab development is that the normal low-affinity igg interaction with fccriiia is profoundly increased by the removal of the core fucose from the n fc oligosaccharide. no crystallographic data are available for igg or igg fc in complex with fccr, but mutagenesis studies of the fc and the fccr revealed general similarity, but with critical differences, in the interaction of these subclasses with their cognate fccr. in igg , the stable interaction of the two heavy chains results from the combined effects of stable covalent inter h-chain disulfide bonds and strong noncovalent interaction of the two ch domains (table ). in stark contrast, in igg and igg the interaction of the ch domains of each h-chain is weak. residues , and (eu numbering) profoundly affect the stability of these interactions. the difference at position (r in igg and k in igg ) confers a -fold decrease in stability of the interface between the two ch domains of igg compared with that of igg (table ) . furthermore, the core hinge of igg differs from igg at position (p in igg and s in igg ), resulting in unstable inter-heavy-chain disulfide bonds. this, together with the destabilizing amino acids in the ch , confers the unique property of half-antibody (fab arm) exchange between different igg antibodies, thereby creating monovalent, bispecific igg antibodies in vivo. , the similarly unstable interactions between the ch domains in igg are conferred by the interface residue m ; however, the stable inter-h-chain disulfide bonds of the core and upper hinge prevent half-molecule exchange (table ) . in addition, igg uniquely has three disulfide bond conformers ( table ). the distinct conformers are formed when ( ) each light chain is attached to the cys residue of ch in the heavy chain (igg -a conformer), ( ) both light chains attach to the upper hinge (igg -b) or ( ) one light chain is attached to the ch cys and one to the upper hinge of the other heavy chain (igg -ab). this results in distinct positioning of the fabs relative to the fc portions in the different conformers, which has implications for the interaction with antigen and the capacity of igg to cross-link target molecules in the absence of fccr binding, for example, in an agonistic mab setting. it should also be noted that igg has not been used in therapeutic mabs despite its unique biology. the main impediment to its use are its physicochemical properties such as susceptibility to proteolysis and propensity to aggregate that present challenges to industry-scale production and stability but protein engineering is attempting to overcome these hurdles. therapeutic antibody design: improving mab potency many factors affecting fccr-dependent responses in vivo come into play during mab therapy. the experience of three decades of clinical use of mabs taken together with our extensive, albeit incomplete, knowledge of igg and fccr structure and immunobiology provides a war chest for the innovative development of new and highly potent mabs through the manipulation of their interaction with the fccr. therapeutic mab engineering strategies are directed by many factors including the biology of the target, the nature of the antigen, the desired moa and possibly the anatomical location of the therapeutic effect, and thus to optimize potency for a desired response, the context of use is critical. the nature of the igg isotype different capabilities for the recruitment and activation of the different immune effector functions are naturally found in the fc regions of the human igg subclasses. thus, to achieve a desired moa, the different igg subclasses are important starting points for the selection and engineering of the optimal mab fc. igg is, in many ways, a proinflammatory or "effector-active" subclass, as it can initiate the complement cascade and is a "universal" fccr ligand. notwithstanding it is also a ligand for the inhibitory fccriib, igg elicits proinflammatory responses through all activating-type fccrs, including adcc, adcp and cytokine release. because of their more restricted fccr-binding profile, igg and igg have offered some choice in potentially avoiding fcr effector function without the need for fc engineering. they have been used as the backbone for therapeutic mabs either because recruitment of patients' effector functions was unlikely to be necessary for the primary moa of the mab or is possibly detrimental to the desired therapeutic effect. however, the use of these unmodified "inert" subclasses is not without consequences and underscores the need for fc engineering to modify fccr interactions-see the "attenuating and ablating fccr related functions of igg" section. thus, the choice of igg subclass for therapeutic mab engineering is an important first step for engineering of novel mabs of improved specificity, potency and safety. igg is the predominant subclass used in the development of cytotoxic mabs where induction of an activation-type response, adcc or phagocytosis, is considered desirable. , , cytotoxic mab cancer therapeutics can control disease progression by one or more mechanisms. their moas include direct induction of apoptotic cell death of the cancer cell (anti-cd , anti-cd ) or blocking receptor signaling (anti-her , anti-egfr). they may also harness fccr effector functions, including adcc in the tumor microenvironment. the approved mabs, rituximab (anti-cd ), trastuzumab (anti-her ) and cetuximab (anti-egfr), are formatted on a human igg backbone and all require activating-type fccr engagement for optimal therapeutic activity. , this presents an example where context of therapeutic use is critical for therapeutic mab design. igg antibodies bind both the activating fccr (e.g. fccriiia) and the inhibitory fccriib. in some environments effector cells will coexpress fccriib together with fccri, fccriia and fcciiia, as may occur on a tumor-infiltrating macrophage. therapy with an igg anti-cancer cell mab may then be compromised by the inhibitory action of fccriib upon the itam signaling of the activating fccr as both types of receptor would be coengaged on such an effector cell by the mab bound to the target cell. this leads to reduced therapeutic mab potency. thus, the relative contributions of the activating (a) and inhibitory (i) fccr to the response by an effector cell, the a-to-i ratio, may be an important determinant in clinical outcome of therapeutic mab activity, , , that is, the higher the a-to-i ratio, the greater the proinflammatory response induced by the therapeutic mab or conversely the lower the a-to-i ratio, the greater the inhibition or dampening of the proinflammatory response. thus, the challenge for the development of more potent fccr effector mabs is to overcome three major obstacles. first, improving activation potency by selectively enhancing interaction with activating-type fccr, particularly fccriiia owing to its predominant role in adcc-mediated killing of tumor cells. second, reducing binding interactions with the inhibitory fccriib. these two approaches improve the fccr a-to-i ratio of cytotoxic igg mabs. third, overcoming the significant affinity difference in the interaction with the main fccriii allelic forms of fccriiia-v and fccriiia-f , , which appears to be an important source of patient variability in responses to therapeutic mab treatment of cancer. at the time of writing, some mabs with improved potency are coming into clinical use. their improved action has been achieved by modifying the n-linked glycan or the amino acid sequence of the heavy-chain fc ( table ) . the typical complex n-linked glycan attached to n of the heavy chain includes a core fucose. antibodies that lack this fucose have approximately -fold improved binding to fccriiia and fccriiib and importantly retain the weak, low-affinity binding to the inhibitory fccriib. furthermore, this glycoengineering increased binding affinity of the modified igg mab for both fccriiia v and f allelotypes. [ ] [ ] [ ] afucosyl versions of the tumor targeting mabs such as anti-her , anti-egfr and anti-cd had greater antitumor effects and increased survival, , , which is a reflection of the greatly increased, and selective, fccriii binding. compared with their unmodified counterparts, the afucosyl mabs showed dramatic improvement of fccriii-related effector responses such as stronger nk cell-mediated adcc, or enhanced neutrophil-mediated phagocytosis through fccriiib and/or fccriiia. however, certain neutrophil functions via fccriia may be compromised. , there are six afucosylated antibodies in late-stage clinical trials or approved for treatment (table ) . notable is obinutuzumab, an afucosyl anti-cd mab which nearly doubles progression-free survival in chronic lymphocytic leukemia patients as compared with the fucose-containing rituximab. this dramatic improvement in clinical utility reinforces the value of glycan engineering specifically and of fc engineering generally in anticancer treatments. alteration of the amino acids in the heavy-chain fc can alter igg specificity and affinity for activating fccrs. the anti-cd antibody mor (xmab ) is currently in phase iii trials for the treatment of chronic lymphocytic leukemia. it contains two mutations in its igg fc, s d and i e, which increases affinity to fccriiia, particularly the "lower-affinity" fccriiia f allele. the mab shows increased fccriii-mediated adcc and phagocytosis in vitro, and reduced lymphoma growth in mouse models. margetuximab is an adcc-enhanced igg fcengineered variant of the approved anti-her mab trastuzumab in phase iii for her -expressing cancers. , alteration of five amino acids (l v, f l, r p, y l and p l) enhanced binding to fccriiia which also had the additional effect of decreasing binding to the inhibitory fccriib, and thereby increased its a-to-i fccr ratio. this was apparent when compared with unmodified trastuzumab the margetuximab showed enhanced adcc against her + cells in vitro and demonstrated superior antitumor effects in an her expressing tumor model in mice. the anti-cd ocaratuzumab is an fc-engineered igg mab in late-stage clinical trials for the treatment of a range of cancers, including non-hodgkin lymphoma and chronic lymphocytic leukemia. two fc mutations, p i and a q, conferred about -fold increase in binding to both major allelic variants of fccriiia and elicited sixfold greater adcc than unmodified igg . thus, the engineering of the fc domain or glycan for improved fccriiia binding is a powerful tool to create more potent and clinically effective anticancer mabs. there are circumstances where binding to fccr is unnecessary or undesirable in the moa of a therapeutic mab. unmodified igg irrespective of its subclass or intended therapeutic effect has the potential to engage an fccr which may lead to suboptimal therapeutic performance or to unexpected and catastrophic consequences. , clearly reducing or eliminating fccr interactions, when they are not required for therapeutic effect, may be desirable. indeed, this had been addressed by the choice of igg subclass or by modifying the fc region. indeed, most efforts in fc engineering mabs that have translated to an approved drug have focused on the reduction or elimination of fccr interactions (table ) . one approach to minimize interactions with the activating fccr has been the use of igg or igg backbones, which show a more restricted specificity for the activating fccr and consequently have been traditionally, and simplistically, viewed as "inert" igg subclasses. notwithstanding the unexpected, and fccr-dependent, severe adverse reaction induced by the igg tgn mab, the igg or igg backbones have been successfully used in many settings. indeed, checkpoint inhibitors, such as mabs targeting ctla- or the pd-l /pd- interaction for the suppression of inhibitory signals that contribute to immune tolerance in the tumor microenvironment, are formatted on an igg backbone. pembrolizumab, nivolumab and cemiplimab are all anti-pd- antibodies currently used for cancer therapy and have been formatted on an igg backbone - with a stabilized core hinge (s p) to prevent half-igg exchange. similarly, the checkpoint inhibitor tremelimumab is an anti-ctla- antibody formatted on an igg backbone to avoid potential adcc killing of target cells. however, the use of igg and igg as "inert" subclasses is problematic. both bind to the activating receptors fccriia-h and fccri, respectively (table ) , and initiate effector functions such as neutrophil activation and apoptosis induction. , interestingly, in experimental systems, cross-linking of anti-pd- igg based mab by fccri switched its activity from blocking to activatory. moreover, igg binds to fccriib, which may scaffold the therapeutic mab. although scaffolding may be beneficial in some contexts, for example, in immune agonism, it can be disastrous and unexpected in others as it was for the anti-cd tgn mab. thus, the igg and igg subclasses are not the optimum choice for "fcr-inactive" mabs, and so modifying the fc is a more direct approach. the complete removal of the heavy-chain n-linked glycan is well known to ablate all fccr binding by dramatically altering the fc conformation. , , , atezolizumab, an igg anti-pd-l checkpoint inhibitor mab, utilizes this strategy and eliminates fccr and also complement activation. modification to the fc amino acid sequence of the fccrcontact regions can also be used to reduce fccr binding. a widely used modification of igg is the substitution of leucine and in the lower hinge sequence (l l g g ) with alanine (l a l a). it is often referred to as the "lala mutation" and effectively eliminates fccr binding by more than fold , and is used in teplizumab and spesolimab (table ) . a separate strategy has used combinations of amino acid residues from the fccr-binding regions of igg and igg , which have restricted fccr specificity, together with other binding-inactivating mutations. the lower hinge amino acids of the igg mabs durvalumab (anti-pd-l ) and anifrolumab (anti-interferon-a receptor; table ) were modified to mimic the lower hinge of igg (l f). they additionally incorporated l e in the lower hinge and p s in the f/g loop of the ch domain to ablate fccr binding by disrupting two major fccr contact sites and also coincidently decreasing c q activation. igg mabs have been similarly engineered to eliminate their interaction with fccri and fccriib. the igg anti-pd- antibody tislelizumab has had its fccr contact residues in the lower hinge e , f , l substituted with the equivalent residues of igg p, v, a (e p, f v, l a) as well as the additional d a mutation which disrupts a major fccr contact in ch . it also has substitutions in the core hinge s p and the ch l v and r k to stabilize the h-chain disulfides and ch interactions, respectively, thereby preventing half-ig exchange characteristic of natural igg . collectively, these modifications create a stable igg with no fccr binding nor complement activation. thus, fc engineering is an effective way to remove fccr effector functions and may be preferable to using unmodified igg or igg backbones that have a more restricted repertoire of fccr interactions but which are still able to induce certain effector functions. preferential or specific fc engagement of fccriib over the activating fccr offers several potential therapeutic advantages for new mabs in distinct therapeutic settings. harnessing the physiological inhibitory function of fccriib by mabs that target itam receptors has the potential to shut down itam-dependent signaling pathways of major importance in antibody pathologies. , such itam signaling receptors include the bcr complex on b cells which is active in systemic lupus erythematosus, the fceri on basophils and mast cell subsets in allergies or the activating-type fccr on a variety of innate leukocytes in antibody-mediated tissue destruction. in such scenarios, the itam signaling receptor complex that is targeted by the therapeutic mab must be co-expressed on the cell surface with the inhibitory fccriib. this permits coengagement with itam signaling receptor by the fab of the mab and inhibitory fccriib by its fc which is the critical requirement in the inhibitory moa for such therapeutic mabs ( figure ) . obexelimab (also known as xmab ; table ), currently in early clinical testing in inflammatory autoimmune disease, is an igg mab that targets cd of the bcr complex. it contains two fc modifications, s e and l f (also known as "self" mutations), that selectively increased fccriib binding by -fold to about nm, which results in powerful suppression of bcr signaling and the proliferation of primary b cells. the anti-ige mab omalizumab is an igg mab approved for the treatment of allergic disorders. , a similar but fc-engineered igg mab xmab , currently in early clinical testing, contains the affinityenhancing self modifications. both mabs sterically neutralize the interaction between ige and its highaffinity receptor fceri to prevent basophil and mast cell activation. , however, xmab exhibited more efficient removal (sweeping; discussed later) of circulating ige and also inhibited b-cell ige production, presumably by binding to the ige bcr on the b-cell surface and coclustering with fccriib via its affinity-enhanced fc domain. thus, xmab 's selective modulation of ige production by ige + b cells in addition to its enhanced clearance of ige may offer significantly improved therapeutic benefits in allergy therapy beyond simple ige neutralization. the "self" mutations have also been used in agonistic mabs (discussed later). one cautionary note is that the arginine (r ) of the igg-binding site in fccriib is critical for the enhanced affinity binding of "self"-mutated fcs but it is also present in the activating-type "high responder" fccriia-r . thus, antibodies modified with "self" have very-high-affinity binding to fccriia-r with a potentially increased risk of fccriia-dependent complications in patients expressing this allelic form, although, so far, none have been reported in clinical trials. however, an alternative set of six fc mutations, termed "v " (p d, e d, g d, h d, p g and a r), potently enhanced fccriib binding without increasing fccriia-r interaction. enhancing the sweeping of small immune complexes the expression of fccriib on lsec and its action in the "sweeping" or removal of small immune complexes has opened up new possibilities for the application of fccriib-enhancing modifications. antibodies or fc fusion proteins, whose primary moa is the neutralization of soluble molecules such as ige or cytokines, are particularly attractive candidates for this approach. proof-of-concept for this strategy has been demonstrated in experimental models. indeed, this may be a significant component of the rapid disappearance of ige from the circulation of patients treated with the anti-ige xmab containing the fccriib enhancing "self" modifications, as described previously. agonistic mabs induce responses in target cells by stimulating signaling of their molecular target. typically, this is to either enhance antitumor immunity by engaging costimulatory molecules on antigen-presenting cells or t cells (i.e. cd , - bb, ox ) or promote apoptosis by engaging death receptors on cancer cells (i.e. dr , dr , fas). the role of fccr in the action of these types of mabs appears to be primarily as a scaffold. fccriib is often the predominate receptor involved and the extent of its involvement is complex. in the case of cd , the degree of fccriib scaffolding potency is linked to the epitope location of the targeting mab with greater potency seen for membrane proximal epitopes. , it is also noteworthy that depending on the epitope location, the scaffolding of anti-cd mabs may convert antagonist mabs to agonistic. engineering of the igg fc region for enhanced and/or specific binding to fccriib can greatly improve agonistic function. , [ ] [ ] [ ] such mutations induced significantly greater agonistic activity in an anti-dr model through increased induction of apoptotic death and decreased tumor growth compared with unmodified igg . the "self" modifications that dramatically and selectively increase affinity for fccriib have also been used to enhance immune agonism in an anti-ox model. the incorporation of the "v " fc mutations into igg specifically enhance fccriib interaction -fold, conferring the enhanced agonistic activity of an anti-cd antibody and an anti-ox mab. , monoclonal antibodies are potent therapeutics in a number of chronic or once incurable diseases. however, there is still extensive unmet clinical need as well as considerable room for improvement in many existing therapeutics. further understanding of how antibody structure affects fccr function is essential for future development of more potent and effective mabs. already, engineering of the igg fc and its glycan has proved a potent and effective approach for increasing the clinical effectiveness, functional specificity and safety of therapeutic mabs and is an emerging pathway to the development of the "next-gen" therapeutics. future directions in the development and use of therapeutic antibodies should increasingly mimic normal protective antibody responses, which are polyclonal and elicited in the context of innate receptor engagement which includes the fcr as well as other powerfully responsive systems including the toll-like receptors and complement receptors. furthermore, the mixed subclass nature of these normal antibody responses suggests that circumstances may arise in therapeutic strategies where there is value in having distinctly modified fcs for the nuanced engagement of different fccr family members. treatments comprising multiple mabs and immune stimulants are under investigation in infectious disease for neutralization coverage of variant strains. indeed, such an approach may be most effective in emerging infectious disease such as severe acute respiratory syndrome coronavirus (sars-cov- ) infection. the use of multiple mabs tailored for distinct effector functions and targeting different epitopes will maximize the opportunity for cocktailing of effector functions in different types of diseases. indeed, in a small but contemporary example outside of infectious disease, the fda-approved combination in adenocarcinoma therapy uses a cocktail of two mabs, pertuzumab and trastuzumab, against her . rather than one type of fc to conquer all, the combined use of appropriately selected mabs whose individual components are enhanced for the engagement of different fccr members may utilize multiple components of the spectrum of effector responses on offer by the immune system. such "next-gen" biologics will begin to realize the full potential of fccr-mediated antibody immune therapeutics and offer transformational change for the treatment of intractable and incurable diseases. 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approaches to enhance the agonism and effector functions of an anti-ox antibody pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer we thank halina trist for assistance with the manuscript. we also thank nhmrc, janina and bill amiet trust, margaret walkom trust and nancy e pendergast trust, genmab, for their support. none. key: cord- -j ywrl authors: barlough, j. e. title: cats, coronaviruses and coronavirus antibody tests date: - - journal: j small anim pract doi: . /j. - . .tb .x sha: doc_id: cord_uid: j ywrl feline infectious peritonitis and other coronavirus infections of cats are briefly reviewed. interpretation and applications of feline coronavirus antibody tests are described, and general recommendations are provided for practitioners. some of the major unresolved questions regarding coronavirus infections of cats are delineated. the coronaviruses are a large and widely distributed family of single-stranded ribonucleic acid viruses, and are important causes of respiratory and enteric disease, vasculitis, serositis, hepatitis, and encephalomyelitis in several avian and mammalian species (siddell et al., ) . feline infectious peritonitis virus (fipv), transmissible gastroenteritis virus (tgev) of swine. canine coronavirus (ccv), and human respiratory-tract coronaviruses of the e group together comprise an antigenic cluster of closely-related viruses within the coronaviridae family (pedersen, ward & mengeling, ) . in fact, the major structural polypeptides of fipv, tgev, and ccv are so similar antigenically that some regard these three agents as host-range mutants rather than as individual viral species (horzinek, lutz & pedersen, ) . in domestic and exotic cats, fipv is the aetiologic agent of a lethal disease-feline infectious peritonitis (fip)-characterized by fibrinous serositis, vasculitis, and formation of disseminated pyogranulomas (wolfe & griesemer. : montali & strandberg, hayashi et al., ) . serosal membranes, liver, kidneys, omentum, lungs, eyes, and central nervous system are commonly affected. the pathology is the result of complex immunologically mediated phenomena involving arthus-like antigen-antibody-complement interactions across vessel walls (jacobse-geels, daha & horzinek, , pedersen & boyle, ; weiss, dodds & scott, ; weiss & scott, a-c; hayashi, lshida & fujiwara, ) . recent studies have shown that some cats with either naturally or experimentally-acquired serum coronavirus antibody experience a more rapid, fulminating disease course following fipv exposure than do coronavirus antibody-negative cats receiving the same challenge dose (pedersen & boyle, ; weiss, dodds & scott, ; weiss & scott, a-c) . a potential state of antibody-mediated hypersensitivity thus exists in certain coronavirus antibodypositive cats challenged with fipv, with this antibody perhaps ( ) accelerating the uptake of fipv (in the form of immune complexes) into reticuloendothelial and polymorphonuclear cells, and ( ) promoting widespread destructive inflammatory reactions in blood vessel walls and tissues through immune complex deposition and complement activation (jacobse-geels, daha & horzinek, pedersen & boyle, ; weiss, dodds & scott, ; weiss & scott, a-c; hayashi et al., a hayashi et al., , . uptake of fipv into macrophages appears to be enhanced by impaired t lymphocyte function (hayashi et al., b) . in addition to fipv, cats are susceptible to natural infection with certain enteric coronaviruses which may or may not be variants of fipv (pedersen et al., ; mckiernan et al., ; dea, roy & elazhary, ) . these feline enteric coronaviruses (fecvs) can produce a range of effects from asymptomatic infection of the gastrointestinal tract to severe enteritis in kittens and adult cats. the nature of the relationship between fecvs and fipv is perhaps illuminated by the observation that certain fipv strains are capable of producing either fip or enteritis, or both (hayashi et al., , a . intestinal lesions can also be produced in newborn pigs by oral inoculation with virulent fipv (woods, cheville & gallagher, ) . it is thus possible that fecvs and fipv represent pathogenetic (rather than host-range) variants of a single coronavirus typevariants possessing, however, a relatively broad spectrum of virulence from asymptomatic infection to enteritis to lethal, disseminated fip (barlough, b) . reports indicate that at least two other coronaviruses in the fipv antigenic cluster can infect cats under experimental conditions: tgev, which produces an asymptomatic infection and is excreted in faeces for as long as three weeks post-exposure (reynolds & garwes, ) , and ccv, which also produces an asymptomatic infection and is excreted from the oropharynx for at least one week stoddart, baldwin & scott, ) . at present the frequency of infection of cats in nature with these two coronaviruses is not known. coronavirus e of human beings does not appear to replicate to any extent in experimentally-inoculated cats (barlough, a) . in fip, hypersensitization by coronavirus antibody is dependent upon the identity of the coronavirus(es) that originally incited the antibody response. thus, antibody arising from exposure to fipv or fecvs can hypersensitize (weiss, dodds & scott, ; pedersen & boyle, ; pedersen et al., ; weiss & scott, a-c) , while antibody resulting from exposure to tgev, ccv, or coronavirus e usually does not (witte et al., ; woods & pedersen, ; barlough, a; barlough et af., b; stoddart, baldwin & scott, ) . however, some sensitization due to a tgev strain apparently occurred in one report (toma et al., ) . it should be emphasized at this point that the mere presence of coronavirus antibody in an animal's serum does not mean that fip will ever develop in that animal in the future, even after repeated fipv exposure. fip is a relatively uncommon disease in nature, even in crowded cattery situations; the vast majority of coronavirus antibody-positive cats will never develop it. the factors that determine whether fip does develop following fipv exposure are multiple, probably including: dose and virulence of infecting virus strain; route of exposure; age and immune status at time of exposure; possibly genetic predisposition; concurrent viral infections (e.g., feline leukaemia virus); and adverse environmental influences, such as stress and overcrowding (scott, weiss & hoshino, ; barlough & weiss, ) . the route by which fipv is spread in nature is still unknown. however, it is most likely that initial infection results from ingestion and/or inhalation of the virus. virus is probably excreted into the environment by a number of routes: in oral and respiratory secretions, faeces, and possibly urine. close contact between cats usually is required for effective transmission of fipv, although the possibility of virus transmission in excreta and by other indirect methods (on clothing, bedding, feeding bowls, etc.) also exists. the potential for transmission by haematophagous arthropods is unknown. transmission across the placenta to the developing foetus, although suggested in the past (flagstad & larsen, ; pastoret & henroteaux, ) , has not yet been conclusively proven to occur. in common with many other enveloped viruses, fipv is quite unstable once outside its host, and is rapidly inactivated by many common detergents and disinfecting agents, such as sodium hypochlorite (bleach) (pedersen, ; barlough & weiss, ) . the clinical diagnosis of fip is made by evaluation of history and presenting signs and the results of supportive laboratory procedures . clinicopathologic and serologic procedures important in diagnosis include: analysis of thoracic and abdominal effusion (viscous, opaque, straw-coloured to yellow, specific gravity . to . , protein to g/dl, variable cell numbers, high fibrin content), haemogram, clinical chemistry profile, serum protein electrophoresis (hypergammaglobulinaemia), serum coronavirus antibody titre, and biopsy (when possible). it is important to remember that a biopsy is the only test procedure that can be used to definitively diagnosis fip in the living animal. explortory laparotomy with organ punch biopsy of affected tissues (especially liver, spleen, omentum, and mesenteric lymph node) is the preferred technique for collection of biopsy samples (percutaneous needle biopsy cannot be recommended owing to the friability of diseased organs and the potential for serious haemorrhage). similarly, complete necropsy examination with histologic evaluation of suitable tissues will provide a reliable diagnosis after death. any diagnosis of fip made in the absence of biopsy or necropsy examination must be considered presumptive. this is because of the large number of potential 'fip look-alike' diseases that can affect cats. these can include: lymphosarcoma and other tumours (especially those involving the liver, biliary tract, kidneys, or lungs), cardiomyopathy, pyothorax, chylothorax, septic peritonitis, hepatitis, internal abscessation, diaphragmatic hernia, pansteatitis, toxoplasmosis, cryptococcosis, and tuberculosis (barlough & weiss, ) . thus, in individual cases, clinicopathologic and serologic procedures will assist in ruling out possible diagnoses, but only biopsy or necropsy examination will definitively identify the fip disease process. laboratory test procedures for detection of coronavirus antibody in feline sera include biological assays such as virus neutralization (vn); non-biological, immunochemical techniques such as indirect immunofluorescence assay (ifa), enzyme-linked immunosorbent assay (elisa), and kinetics-based elisa (kela); and other methods such as agar gel immunodiffusion and passive haemagglutination , though the availability of such tests varies worldwide. either fipv itself or one of the other coronaviruses in the fipv antigenic cluster (usually tgev or ccv) can be used as the target antigen in most of these assays. the use of non-fipv coronaviruses in antibody-testing procedures has become popular in recent years because of long-standing difficulties in routinely propagating fipv in the laboratory. in general, the immunochemical tests (especially the ifa) have gained the greatest popularity among veterinary diagnostic laboratories, in part because of their relative ease of performance and widespread availability of the pertinent immunotechnologies. it has been proposed on the basis of serosurvey data that most fipv infections in nature result only in seroconversion without progression to lethal, disseminated fip ). this is because serum coronavirus antibody can be found not only in cats with fip but also in many healthy cats and in many cats with other diseases, indicating that exposure of cats to coronavirus(es) is much more widespread than was once believed. in the general healthy feline population-excluding cats in catteries and multiple-cat householdsapproximately to per cent of cats will have positive coronavirus antibody titres (note: 'positive' refers only to the presence of antibody, not to the presence of the fip disease process). a special situation is encountered when cats are clustered together in catteries, in which case positive titres are either completely absent (i.e., there has been no coronavirus exposure), or are present in to per cent of the cats within a household (indicating efficient spread of virus once it has been introduced). the occurrence of coronavirus antibody in a cattery does not necessarily correlate with its fip history; e.g., antibody has been found in healthy cats in catteries that have experienced death losses to fip as well as in catteries that have never lost a cat to fip. most cats with histopathologically confirmed fip have serum coronavirus antibody, often of high titre . because many cats with undiagnosed illnesses also have elevated titres (indicating previous coronavirus exposure), interpretation of their titres may be difficult. added complexity has been contributed to interpretation of coronavirus antibody titres in healthy cats and in cats with undiagnosed illnesses by the finding that other coronaviruses (e.g., fecv, and experimentally tegv and ccv) can also infect cats and generate coronavirus antibodv in their sera. because these viruses are all serologically cross-reactive with each other and with fipv, and because several of them are used relatively interchangeably in commercially available coronavirus antibody tests, the non-specificity of these tests is readily apparent. the serodiagnostic potential of these assays (i.e., their ability to identify cats with active fip and/or potential virus carriers/excretors) is thus limited not only by the widespread distribution of serum coronavirus antibody in the feline population, but also by the possibility that non-fipv coronaviruses may be responsible for some of the seroconversions that they detect. the actual distribution of antibodies in the general feline population to each of these coronaviruses is therefore unknown, and will remain unknown until highly specific assays capable of differentiating antibody against one coronavirus (e.g. fipv) from antibody against another (e.g., tgev) are developed. these difficulties are compounded further by the plethora of test procedures (i.e., ifa, vn, elisa, kela) employed by different laboratories, and by the absence of standardization of testing protocols. results are therefore best interpreted in the light of specific information provided by the testing laboratory utilized, on the significance of titre levels generated by the individual test that it performs. effect of recent vaccination. recent research has shown that antibody against bovine serum components can be found in the serum of certain cats-antibody capable of reacting with antigenically similar bovine serum components present in cell cultures used to propagate target viruses for immunochemical assays such as the ifa, elisa, and kela . because such serum components can adhere tightly to both cells and virus (johansson, bergquist & grandien, ; kraaijeveld, madge & macnaughton, ; , reactivity against them can be mistaken for a coronavirus antibody response unless feline serum samples are tested in parallel against uninfected cell culture control preparations . one possible explanation for the presence of this reactivity is routine vaccination. owing to the frequent presence of extraneous cell culture material in many partially purified commercial biologicals, routine parenteral vaccination would seem to provide an ideal opportunity for vaccinees to respond immunologically not only to vaccine virus but also to immunogenic elements of cell culture medium, such as bovine serum proteins (bonin, schmidt & schmidt, ; johansson, bergquist & grandien, ; tizard, ; barlough et al., , a barlough et al., , snyder, eernisse & erickson, ) . kela studies have shown that this reactivity dissipates with time, and that the probability of encountering it can be minimized if serum samples for elective serotesting are drawn no sooner than three to four months following the most recent parenteral vaccination . the presence of serum coronavirus antibody in any cat, whether healthy or diseased, is indicative of only one thing: previous exposure to a coronavirus in the fipv antigenic group. a positive coronavirus antibody titre, while consistent with a clinical diagnosis of fip, does not indicate that a cat actually has fip, because many healthy cats and many cats with other diseases are also coronavirus antibody-positive. neither, however, does a positive titre indicate that a cat is protected against fip, because most cats with fip also are coronavirus antibody-positive. considering that fip occurs only sporadically in the general feline population, and that most cats in fip-problem households are coronavirus antibody-positive and yet do not contract fip, it would appear that many cats (perhaps most cats) with coronavirus antibody are protected against the natural disease. the question remains whether it is coronavirus antibody that actually confers this protection or whether unrecognized cellular immunologic factors are involved. it is especially important to realize that present-day coronavirus antibody tests have absolutely no predictive value; i.e., a positive titre in no way indicates that a cat is doomed to develop fip at some uncertain future date. despite all the problems with current feline coronavirus antibody testing methods, there are still some select situations in which determination of antibody titres can be of benefit to the veterinary surgeon and to the cat owner ): ( i ) as a screening test, to determine the presence or absence of antibody in a previously untested household, and to detect potential virus carriers/excretors when introducing new cats into coronavirus antibody-negative households. based on our current understanding of feline coronaviral serology, screening would appear to be the major use for coronavirus antibody testing today. screening of cats in a household experiencing undiagnosed disease problems may be especially useful. only about to per cent of the cats (a minimum of three) in such a household need to be tested, because antibody will be either totally absent or present in to per cent of the animals (scott, weiss & hoshino, ) . while the discovery of coronavirus antibody-positive cats in such households will not diagnose the problem, knowledge that coronavirus antibody is absent may be helpful in ruling out an fipv-group coronavirus as the aetiologic culprit. ( ) as an aid (and nothing more than an aid) in the clinical diagnosis of a diseased cat with signs suggestive of fip. a coronavirus antibody titre determination should be given no more weight than any of the other routine procedures used in arriving at a clinical diagnosis. a positive titre will not diagnose fip, but a negative titre will usually rule it out. coronavirus antibody-negative fip cases. a very small percentage of cats with fip do not have detectable coronavirus antibody in their sera. several explanations for this phenomenon are possible: ( ) detectable antibody may sometimes disappear from the circulation during the terminal stages of the disease. submission of serum from some moribund cats thus may result in a negative titre determination despite the presence of disseminated fip (barlough, ) . ( ) immune-complexing is an important immunopathologic feature of fip. in certain cases, if extensive immune complexing is present at the time of testing, it is conceivable that there may be little free, unbound coronavirus antibody available to be detected. this 'cloaking effect' may in fact be the explanation, at least in part, for the absence of detectable antibody in the serum of some moribund cats. ( ) the swiftness of the fip disease process is an important factor, especially in animals without previous coronavirus exposure. cats experiencing a peracute disease course (such as some young kittens) may display a rather sluggish antibody response that can be more difficult to detect in the earlier stages, especially if a non-fipv coronavirus (tgev, ccv) is used by the laboratory for antibody detected. although serologically cross-reactive with fipv, these viruses nevertheless are different from fipv and thus are not as sensitive as fipv at detecting lower levels of specific anti-fipv antibody. a test-and-removal programme for coronavirus antibody-positive cats similar to that utilized for feline leukaemia virus-positive cats, based upon current scientific information, cannot be recommended (scott, ; barlough & weiss, ) . because there is no available serodiagnostic .test that can differentiate between antibody-positive cats with fip, antibody-positive cats with diseases other than f p, or 'fip-immune' seropositive cats, that can specifically identify antibodypositive cats that are excreting fipv, or that can even identify the exact coronavirus(es) against which the antibodies in seropositive cats were raised, there is no known medical reason for destroying these animals. there is no recognized environmental reservoir of fipv; the natural reservoir is assumed to be infected cats. how, then, does the virus maintain itself in these animals? for how long do infected cats harbour the virus? for how long do they excrete the virus, and by what route(s)? what route is most important for effective virus transmission to other cats? is excretion continuous or intermittent? is it possibly stress-related? what percentage of cats infected with fipv actually become chronic carriers? to what extent is a coronavirus antibody-positive cat a potential disease threat to other cats with which it may come into contact? can an infected queen infect her kittens in utero? if so, does in utero infection result in disease? clearly, further research will be required before these questions and others can be satisfactorily resolved. importantly, an antigen detection test for identifying carrier animals that are excreting fipv, similar to those currently available for feline leukaemia virus infection, is urgently needed so that rational flpv control procedures can be devised. until then, control must be based on isolation of cats with suspected fip and maintenance of coronavirus antibody-negative catteries, when possible. euthanasia of coronavirus antibody-positive cats to achieve this latter purpose, however, cannot be justijied. this work was supported by unrestricted contributions to the cornell feline health center. the author wishes to thank mr h. e. carter and an anonymous scrutineer for their helpful suggestions. unpublished data experimental studies with four coronaviruses in cats serodiagnostic aids and management practice for feline retrovirus and coronavirus infections feline infectious peritonitis feline coronaviral serology evaluation of a computer-assisted, kinetics-based enzyme-linked immunosorbent assay for detection of coronavirus antibodies in cats experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus effect of recent vaccination on feline coronavirus antibody test results calf serum content of various viral vaccines and possibilities for its reduction coronavirus-like particles in the feces of a cat with diarrhea the occurrence of feline infectious peritonitis in denmark glomerulonephritis associated with feline infectious peritonitis systemic vascular lesions in feline infectious peritonitis enteritis due to feline infectious peritonitis virus role of thymus-dependent lymphocytes and antibodies in feline infectious peritonitis after oral infection role of neutrophils in production of early enteritis after oral infection with feline infectious peritonitis virus in passively sensitized kittens antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses isolation and characterization of feline c and evidence for the immune complex pathogenesis of feline infectious peritonitis antibody, immune complexes and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis antibodies to calf serum as a cause of unwanted reaction in immunofluorescence tests isolation of feline coronaviruses from two cats with diverse disease manifestations ) extraperitoneal lesions in feline infectious peritonitis ) epigenetic transmission of feline infectious peritonitis morphologic and physical characteristics of feline infectious peritonitis virus and its growth in autochthonous peritoneal cell cultures antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species ( ) virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus fip antibody test-interpretation and recommendations feline infectious peritonitis ) a preliminary report on two sources of nonspecificity in the enzyme-linked immunosorbent assay (elisa) unpublished data an introduction to veterinary immunology echec de l'immunisation contre la peritonite infectieuse feline par injection de virus de la gastro-enterite transmissible du porc laboratory diagnosis of feline infectious peritonitis pathogenesis of feline infectious peritonitis: nature and development of viremia i lb) antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence disseminated intravascular coagulation in experimentally induced feline infectious peritonitis untersuchungen uber die antigenverwandtschaft der wren der felinen infektiosen peritonitis (fip) und der transmissiblen gastroenteritis (tge) des schweines lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses key: cord- -px fe mn authors: infantino, maria; grossi, valentina; lari, barbara; bambi, riccardo; perri, alessandro; manneschi, matteo; terenzi, giovanni; liotti, irene; ciotta, giovanni; taddei, cristina; benucci, maurizio; casprini, patrizia; veneziani, francesca; fabbri, sergio; pompetti, adolfo; manfredi, mariangela title: diagnostic accuracy of an automated chemiluminescent immunoassay for anti‐sars‐cov‐ igm and igg antibodies: an italian experience date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: px fe mn a pandemic of coronavirus disease (covid‐ ) caused by severe acute respiratory syndrome coronavirus (sars‐cov‐ ) has been spreading throughout the world. though molecular diagnostic tests are the gold standard for covid‐ , serological testing is emerging as a potential surveillance tool, in addition to its complementary role in covid‐ diagnostics. indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. sixty‐one covid‐ patients and patients from a control group were tested by iflash clia analyzer for anti‐sars cov‐ antibodies igm and igg. all covid‐ patients were hospitalized in san giovanni di dio hospital (florence, italy) and had a positive oro/nasopharyngeal swab reverse‐transcription polymerase chain reaction result. the highest sensitivity with a very good specificity performance was reached at a cutoff value of . au/ml for igm and of . for igg antibodies, hence near to the manufacturer's cutoff values of au/ml for both isotypes. the receiver operating characteristic curves showed area under the curve values of . and . for anti‐sars cov‐ antibodies igm and igg, respectively. iflash clia analyzer has shown highly accurate results for the anti‐sars‐cov‐ antibodies profile and can be considered an excellent tool for covid‐ diagnostics. the need to accelerate progress in diagnostics, serological tests have been developed. more than different assays have been proposed so far but almost all have poor regulatory status and lack clinical and analytical performance review. in fact the speed with which they are released in the market and the versatility of immunoassays such as source of antigen and secondary antibody conjugate, make them poorly evaluated tests. given that during the outbreak test validation is not a priority and given that nonlaboratory specialists are allowed to handle these tests because of limited staff resources has meant that unregulated testing has spread widely. in particular, since rapid tests do not require any instruments or laboratory personnel they could be set up anywhere and at any time, especially in developing nations with limited healthcare resources and in remote settings. the more relaxed rules of the fda's "policy for diagnostic tests for coronavirus disease- during the public health emergency" issued on march , has allowed the market easier access to these tests as well as easier and faster diagnostics, but the lack of control in the production process is also dangerous making these tests potentially less reliable. along with chromatographic rapid immunoassays as qualitative tests, quantitative antibodies detection tests, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay (clia), have spread often by fully automated analyzers. these technologies characterized by high-throughput and low complexity have helped us to use serological testing more accurately during both antibody development and monitoring the different phases of the disease. indeed, being able to receive information about the antibody concentration and time kinetics of humoral response is very important for diagnostic, prognostic, and therapeutic applications. the aim of the this study was to assess the diagnostic performance of a novel fully automated clia for the quantitative detection of anti-sars-cov- igm and igg antibodies. [ ] [ ] [ ] [ ] however, it is unclear which antibodies are optimally effective in the scenario of covid- and which of them are neutralizing. there is also uncertainty as to which antibody isotype (igm, igg or iga) (single or combined) is the best choice in these different contexts. as with most existing studies on the diagnostic performance of the sars-cov- antibodies, our preliminary data showed that most covid- patients have both igm and igg, and only few of them have isolated igg or igm antibodies. on the one hand, in reference to igm and igg combination, the overall sensitivity of % may reflect that some patients may not yet develop antibodies or will never develop (the length of time from the symptoms onset to serological test ranged from to days); on the other hand, the % specificity performance of igg antibodies makes them an appropriate test for the different immunization protocols. with regard to igm false positive results, it's important to underline that we designed a disease control group made up of (a) donors from last winter when other coronaviruses were active who had all negative results; (b) autoimmune and infectious diseases dating back at least year in which we found four reactive sera. this means that we had no cross reaction with other coronaviruses but two cmv infections and two rheumatic diseases interfered with the test, even if with a low titer. this data can be added to the known issues concerning igm by rapid tests such as the lack of specificity together with the low sensitivity our experience highlights the importance of a clia method, not only to overcome the problems of the subjective reading of the band (especially weak) in the rapid tests, but for the wide range of potentials inherent to a quantitative method, such as assisting with diagnosis and evaluating the disease through antibodies profiles. furthermore, selection of igg antibodies at high level concentrations may be helpful in developing vaccines and treating sars-cov- by convalescent plasma therapy. abbreviations: clia, chemiluminescence immunoassay; lr + , positive likelihood ratio; lr−, negative likelihood ratio; npv, negative predictive value; or, odds ratio; ppv, positive predictive value. genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia laboratory testing for coronavirus disease (covid- ) in suspected human cases: interim guidance serological assays for sars-cov- infectious disease: benefits, limitations and perspectives sars-cov- specific antibody responses in covid- patients centers for disease control and prevention. coronavirus disease (covid- )-information for laboratories immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic performance of vivadiag covid- igm/igg rapid test is inadequate for diagnosis of covid- in acute patients referring to emergency room department performance characteristics (with % confidence intervals) of anti-sars-cov- antibodies igm and igg at different cutoff values as determined by clia method anti-sars-cov- igm antibodies food & drug administration, policy for diagnostic tests for coronavirus disease- during the public health emergency development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis viral kinetics and antibody responses in patients with covid- . medrxiv a comparison study of sars-cov- igg antibody between male and female covid- patients: a possible reason underlying different outcome between gender analytical performances of a chemiluminescence immunoassay for -ncov igm/igg and antibody kinetics diagnostic value and dynamic variance of serum antibody in coronavirus disease assessment of immune response to sars-cov- with fully-automated maglumi -ncov igg and igm chemiluminescence immunoassays covid- ) and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak key: cord- - o agjiq authors: yuan, tom z; lujan hernandez, ana g; keane, erica; liu, qiang; axelrod, fumiko; kailasan, shweta; noonan-shueh, madeleine; aman, m javad; sato, aaron k; abdiche, yasmina n title: rapid exploration of the epitope coverage produced by an ebola survivor to guide the discovery of therapeutic antibody cocktails date: - - journal: antib ther doi: . /abt/tbaa sha: doc_id: cord_uid: o agjiq background: development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to ebola virus glycoprotein with only published sequences. methods and results: a rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. a focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of ebov gp from allosteric effectors reported from literature. conclusions: critically, this workflow allows us to probe the epitope landscape of ebov gp without any prior structural knowledge of the antigen or structural benchmark clones. incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. the use of high-throughput epitope binning during new outbreaks such as the current covid- pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment. . furthermore, since viral antigens have evolved a remarkable propensity to mutate rapidly as a strategy to defy human immune responses, antibody cocktails with broad epitope coverage lower the risk of mutagenic escape which will otherwise render antibodies ineffective, as observed in non-human primates following treatment with a cocktail comprised of antibodies targeting highly similar epitopes on the ebov gp . although characterizing the antigenic surface of viral glycoproteins is advantageous in developing therapies, detailed information on their structure and the roles of particular binding epitopes in protection are often lacking which poses a critical bottleneck in responding to new outbreaks or viral isolates. furthermore, discrete epitopes of the viral antigen may play distinct mechanistic roles that are unknown, cooperative , and have varying levels of risk for mutagenic escape . these factors confound the ability to design effective cocktail therapeutics. epitope binning is a useful empirical method for organizing antibodies into epitope families by assessing the blocking relationships that emerge from a pairwise and combinatorial competition of antibodies against their specific target antigen . however, when studying large panels of antibodies comprising hundreds of clones, exploring an exhaustive pairwise competition matrix of the entire set by standard technologies such as facs, elisa, and label-free biosensors is tedious and resource intensive, so for practical reasons, the scope of these assays is often limited to the blockade against a small set of benchmark 'reagent' antibodies of known specificity or function. antibodies that are binned in competition with a handful of controls constitute a 'few-on-many' approach and rely upon the existence of such standards. in contrast, high-throughput epitope binning assays that expand the number of pairwise permutations that can be addressed in a single experiment not only provide practical advantages of speed and minimal sample consumption, but also provide exquisite resolution revealing small differences in epitope specificity and nuanced binding modes such as allosteric modulation that may be relevant for functional activity [ ] [ ] [ ] . additionally, such assays are self-referencing and do not require the use of controls, so are universally applicable to any antibody library as soon as sufficient protein is expressed and provided that the target antigen is available . improvements in the throughput of label-free biosensor technologies such as octet-htx (fortebio), ibis-mx (ibis technologies), and lsa (carterra) enables the use of epitope binning as a high-throughput screening process rather than being reserved for small panels of antibodies , . previous studies have employed this method to determine fine epitope differences among antibodies to human progranulin figure ). synthetic biology advances enabled the rapid, high-throughput dna synthesis, expression, and purification of antibodies with only the variable domains' amino acid sequences as an input. the ebov gp target was chosen as it is wellstudied, biologically relevant and available in recombinant purified form commercially. this set of antibodies was chosen due to the availability of in-depth characterization reported in the literature, allowing us to benchmark our binning assignments to published bins assigned by facs. following a non-optimized, 'first pass' ht-spr binning that revealed the epitope landscape including rare epitopes, representative antibodies were selected from each bin. these 'pathfinder' antibodies were further investigated in an independently conducted 'second pass' binning along with benchmark anti-ebov gp antibodies supplemented with orthogonal data such as antibody sequence, in vitro cell-based neutralization of live virus and in vivo survival to lethal challenge in mouse models to provide a comprehensive analysis that can aid in the rational design of therapeutic cocktails. variable heavy and light domains of anti-ebov gp antibodies were sourced from bornholdt et al. thirteen antibodies with defined structural epitopes from the literature were cells as described previously . the ebov gp sample used in all epitope binning experiments appeared to be homogeneous in analytical size exclusion chromatography while migrating at an apparent molecular mass of ~ kda, indicating substantial glycosylation (supplemental figure s ). epitope binning was performed in a premix format using a carterra lsa spr a premix assay format (figure a ) was chosen for the epitope binning study because ebov gp is a multivalent antigen comprising of a trimer of gp +gp heterodimers . figure b shows an overlay plot of the sensorgrams obtained for a ligand that gave clear binding responses in the premix binning assay, due to its facile regeneration, giving reproducible antigen binding responses, and full self-block. in this rapid, non-optimized set-up, no attempt was made to optimize the concentration of the premixed antibodies and they were used in a 'batch dilution' mode as a -fold dilution of the supplied stock (corresponding to final antibody binding site concentrations of - nm, with a mean of nm). clearly, this would not have achieved a molar excess of premixed antibody to antigen for some of the low-expressing antibodies, so we limited the premixed analytes to only those clones that showed clearly detectable ligand binding and above-mean expression (> µg from . ml culture). since each analyte injection consumed μl, we needed around μg per ebov gp per injection and since we had a limited supply of our purified antigen ( . - mg), we would have nearly exhausted it if we injected ebov gp premixed individually with all antibodies (> injections, including antigen alone injections). shows a heat map of the results from all active ligands (rows) and fully saturated premixes (columns) in the binning assay, revealing seven distinct epitope clusters or 'communities' without the inclusion of the benchmark antibodies. due to the low expression of some clones and the requirement for their saturation of the antigen as premixed analyte, as judged by premixes giving a full block somewhere (self or elsewhere) in the competition matrix, the number of antibodies that were successful as premixes and gave clearly interpretable responses was rather low. despite ligand attrition and the stringent requirement for premixed antibodies to achieve antigen saturation, which reduced our heat map to analytes x ligands, we were able in this non-optimized 'first pass' binning to assign antibodies to one of seven epitope communities without the use of benchmark antibodies (supplemental table s ). ht-spr binning heat maps including limited embedded standards, or only bidirectional ligand/analyte pairs were also generated (supplemental table s , s ). antibodies representative of the epitope coverage of the full library were used as 'pathfinder' reagents to assign missing clones in a second pass, focused binning study the results from our first pass binning led us to select a set of 'pathfinder' antibodies that were representative of each epitope community and re-express them on a larger scale for use as high quality reagents that recapitulated the entire epitope diversity of the library in a few key clones. the pathfinders represented antibodies with good expression and performance in the binning assay as both analyte and ligand to facilitate their use in future binning experiments. we also scaled up the production of seven randomly selected clones from those that we had failed to assign to a community in our first pass binning, due to their poor performance as ligand, to see if we could assign these "unknowns" in a more focused 'second pass' binning experiment. also included were a set of literature controls of known specificity from a collaborator that served as structural benchmarks to assess whether our spr-derived epitope communities (represented by the aggregate coverage of our 'pathfinders') overlapped or extended beyond known epitopes. the pathfinders, unknowns, and structural benchmarks were merged into a single binning experiment that was performed in a completely independent manner than the first-pass binning. in this so-called "second-pass" binning, conducted by a different operator working on a different lsa unit in a separate lab with a fresh batch of antigen and scaled-up antibodies, we successfully recapitulated the expected epitope coverage of the pathfinders (supplemental table s ) and used them to assign the structural benchmarks to one of our identified communities as well as assign the unknowns to a community. even with only the pathfinder antibodies, all epitopes were recapitulated (supplemental table s ). consistent with our first pass results, most of the previously unassigned clones failed to perform as ligand, making us reliant on their performance in the role of premixed analyte to determine their epitope specificities. this was now made possible due to the larger yield available for those clones upon scale-up. the results from the second-pass binning are summarized as a heat map ( figure a ) and as list of assignments ( figure b ). next, we compared our spr binning results obtained from first and second pass experiments to literature assignments made by facs . up to this point, antibody production had been blinded using internal clone names rather than adi names, to test the reliability of our methods. figure a shows a dendrogram of the antibody sequences for the clones assigned to an epitope community by spr. clones are colored by their spr-derived epitope community (inner circle) and compared to their facs-derived epitope bin (outer circle). antibody sequence does not appear to be fully predictive of epitope as unrelated sequence lineages converge upon the same epitope community (figure , supplemental figure s ), while highly related antibodies can bind to distinct, non-overlapping epitopes, as shown by adi- (comm ) and adi- (comm ), both germline vh - antibodies (supplemental table s ). overall, we found excellent agreement between the spr and facs epitope binning determinations from independent studies, with only a few ( out of ) discordant assignments. we were able to align our communities with those determined table s ). the high-throughput nature of the ht-spr binning assay facilitates the identification of rare epitopes as antigen blockade is tested across so many antibodies that those showing unique blocking profiles are readily apparent. of the antibodies that we assigned to epitope communities, two or fewer were assigned to communities - to obtain a clean result (block or not block) in a premix assay format, two caveats must be satisfied. first, the ligands must maintain their antigen-binding activity upon multiple rounds of regeneration. second, the premixed antibody analyte must bind up and saturate (or nearly saturate) its recognized epitopes in the antigen sample to effectively diminish the antigen's free concentration to a barely detectable level. a useful test to verify empirically that the premixed antibodies have achieved saturation of the antigen sample is a 'self-block' where the same antibody is used in the role of both analyte (premixed) and ligand (coupled to chip). thus, a premixed antibody (analyte) that reduces antigen binding to a barely detectable level when probed by its coupled counterpart (as ligand) verifies that the premixed antibody is capable of saturating all epitopes in the antigen sample and therefore can be used reliably to assess the epitope-based competition of other ligands. since we had no 'a priori' knowledge of the true binding affinities of any of the antibodies to our ebov gp antigen (published kinetic data are avidity-influenced measurements on bli ) we elected to use a large molar excess of each premixed antibody relative to the antigen concentration used, while balancing the need to conserve sample (both antigen and small-scale purified antibodies). while we intended to explore the entire x 'analyte x ligand' competitive matrix, we observed about % ligand attrition due to ligands that showed poor antigen binding responses due to their low activity/affinity or being damaged (or conversely, unaffected) upon regeneration, so were excluded from our analysis. to conserve precious antigen, we elected to use as premixed analyte only those antibodies that had shown good ligand binding and good expression. even if the antigen had not been precious, the yields of some antibodies in the small-scale expression would not have been sufficient to produce full saturation of the antigen, which reduced the number of premixed analytes that fulfilled this caveat. conversely, epitope binning with a well behaved, monomeric antigen can be performed as a classical sandwich assay format which requires significantly less analyte because it does not depend on fully saturating the antigen. unfortunately, for ebov gp such a monomeric construct with biological relevance is not available. benchmark antibodies can add tremendous value to binning assays in providing 'mapping' information, since cross-blocking of such standards would infer overlapping epitopes. however, the binning assay itself is not reliant on them, but enhanced by them. four interesting observations that emerged from our high throughput binning analysis that would have likely been overlooked in a simple 'few-on-many' approach are, ( ) cross-talking antibodies, ( ) asymmetric blockade, ( ) apparent antigen heterogeneity, and ( ) rare epitopes ( figure ). while most of the antibodies fell neatly into one of seven discrete communities, a few antibodies were able to 'crosstalk' and block members of more than one community. an example of this behavior is adi- , a structural benchmark clone, which blocked antibodies in comm and comm ( figure a ). this contrasts mab , a comm member that only blocks other comm members and shows significant sandwiching to antibodies from other communities. most antibodies (like mab ) in our study only blocked antibodies in their own community. in addition to adi- , its genetic sibling, adi- also showed crosstalk of communities and (supplemental figure s ) . , . in a 'few-on-many' binning paradigm, adi- (or adi- ) blocking is only known in context relative to the tested benchmarks. from our binning analysis, these two clones were clearly differentiated from the rest without examining their sequences -see supplemental table s showing first pass binning merged with a limited set of embedded controls. when antibody competition is tested in both orders of addition, in rare cases, asymmetric or order-dependent competition is observed. in our study fvm- showed markedly asymmetric blockade of comm members, only blocking them when presented first to the antigen (as premixed analyte), but not blocking any other antibody (except for itself) when presented second (as ligand) ( figure b) . indeed, fvm- has been reported to act cooperatively in cocktails due to it possibly triggering an 'induced' epitope, an epitope that is either formed or exposed upon binding of gp by another antibody . therefore, high throughput binning can reveal possible cases of allosteric modulation that may enable nuanced mechanisms of action (supplemental table s ). visual inspection of the spr sensorgrams suggests the presence of antigen heterogeneity. comm and comm antibodies appeared to bind a subpopulation of the antigen that was distinct from that recognized by other communities. this manifests in the sensorgrams as premixes of comm and comm antibodies showing 'no effect' on the binding of some ligands, while completely blocking others . an example is shown in figure c where adi- (comm ) and kz (comm ) fully self-block but appear to have no effect on ebov binding to the opposing antibody when premixed in solution. the antigen appeared conformationally intact as judged by its high degree of homogeneity when tested by standard analytical sizing methods (supplemental figure s ), so the existence of functional subpopulations that are differentially recognized by antibodies in the panel would not have been obvious otherwise. despite possible antigen heterogeneity, each antibody still exhibits reliable binding to ebov gp, full selfblock and thus did not affect our ability to assign antibodies to epitope communities. high-throughput epitope binning of large panels of antibodies is uniquely positioned to identify rare epitopes. as the blockade matrix size increases, these rare communities are repeatedly assayed and confirmed to only block within their small communities (comm , comm ) or only to self-block (comm ) ( figure d) . mapping the epitope footprint of structural benchmarks show that epitope communities belonging to large, exposed epitopes generate large numbers of community members (comm ) while smaller number of antibodies bind to more occluded epitopes such as the gp core (comm ) (figure ) . assembling antibody cocktails inclusive of these rare epitopes allows further probing of antibody combinations to uncover synergistic effects and avoid biasing therapies to an immunodominant epitope that may be cleaved as a decoy or be tolerant of high rates of mutation. in our binning study, in addition to the trimeric ebov gp we also tested a cleaved form of gp (gpcl), the product of cathepsin cleavage in the endosome and loss of glycan cap. we observed that antibodies in communities - bound to gpcl, while communities - did not, suggesting they targeted the sequence that was cleaved away from the viral membrane (supplemental table s ). one notable exception was fvm- (comm ) which did not bind gpcl, consistent with its known epitope specificity . antibody cocktails that comprise antibodies targeting disparate non-overlapping epitopes enhance neutralization potency by combining multiple mechanisms of action. cocktails that combine several antibodies targeting disparate epitopes can unlock unexpected synergistic effects that enhance neutralization activity beyond the additive contribution of each antibody, as has been demonstrated in treatments for botulinum neurotoxins , sudan virus , ebola virus and hiv- . furthermore, antibodies that individually do not display neutralizing activity as a monotherapy may exert a synergistic neutralizing effect when combined in a cocktail . understanding the epitope landscape of antibodies generated by the human immune response to authentic viral infection is necessary to rationally assemble therapeutic antibody cocktails that condense the entire immune response to a handful of key clones that confer protection. however, the human immune system responds differently to different pathogens. staphylococcus aureus, a commensal pathogen, are strongly germline-biased recent study of the human immune response to vaccination by yellow fever virus d also reported a germline-encoded neutralization . conversely, by merging our empirically determined epitope communities with functional data from the literature, we found that anti-ebov gp antibodies from multiple epitope communities were able to neutralize in a cell-based assay ( figure c) and confer protection in a mouse challenge model (supplemental table s ). these findings reinforce the need to evaluate the epitope landscape of the human immune repertoire to each pathogen empirically. while antibody affinity and developability can be engineered, the binding epitope of an antibody is an innate property that cannot be engineered downstream. forms of their glycoproteins that subvert the immune response towards production of non-neutralizing antibodies. this escape mechanism is present in ebola virus in the form of secreted glycoprotein isoform (sgp) . cocktails that carefully combine antibodies targeting disparate epitopes by utilizing knowledge of the epitope landscape early in the drug discovery process, as enabled by high throughput binning, may be better equipped to overcome mutagenic escape and decoy epitopes. molecular mechanisms of ebola virus pathogenesis: focus on cell death isolation of potent neutralizing antibodies from a survivor of the ebola virus outbreak. science ( -. ) analysis of a therapeutic antibody cocktail reveals determinants for cooperative and broad ebolavirus article analysis of a therapeutic antibody cocktail reveals determinants for cooperative and broad ebolavirus neutralization therapeutic monoclonal antibodies for ebola virus infection derived from vaccinated humans development of clinical-stage human monoclonal antibodies that treat 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human survivor key: cord- - k re y authors: daniell, henry; streatfield, stephen j; wycoff, keith title: medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants date: - - journal: trends in plant science doi: . /s - ( ) - sha: doc_id: cord_uid: k re y abstract the use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. as the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. currently, the cost of biopharmaceuticals limits their availability. plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. here, we discuss recent developments in this field and possible environmental concerns. research in the past few decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments. because most genes can be expressed in many different systems, it is essential to determine which system offers the most advantages for the production of the recombinant protein. the ideal expression system would be the one that produces the most safe, biologically active material at the lowest cost. the use of modified mammalian cells with recombinant dna techniques has the advantage of resulting in products that are identical to those of natural origin; however, culturing these cells is expensive and can only be carried out on a limited scale. the use of microorganisms such as bacteria permits manufacture on a larger scale, but introduces the disadvantage of producing products that differ appreciably from the products of natural origin. for example, proteins that are usually glycosylated in humans are not glycosylated by bacteria. furthermore, human proteins that are expressed at high levels in e. coli frequently acquire an unnatural conformation accompanied by intracellular precipitation, owing to lack of proper folding and disulfide bridges. the production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant to clinical medicine. first, plant systems are more economical than industrial facilities using fermentation or bioreactor systems. second, the technology is already available for harvesting and processing plants and plant products on a large scale. third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as a food (edible vaccines). fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. last, health risks arising from contamination with potential human pathogens or toxins are minimized. in the decade since the expression and assembly of immunoglobulin (ig) heavy and light chains into functional antibodies was first shown in transgenic tobacco, plants have proven to be versatile production systems for many forms of antibodies. these include full-sized igg and iga, chimeric igg and iga, secretory igg and iga, single-chain fv fragments (scfv), fab fragments and heavy-chain variable domains. recently, this list has been extended to include bispecific antibodies, which are made by the genetic fusion of two different scfvs via a flexible peptide linker . plants have great potential as a virtually unlimited source of inexpensive monoclonal antibodies (dubbed 'plantibodies') for human and animal therapeutics (table ) . there is not yet a consensus as to the best plant species or tissue for commercial antibody production. most antibodies expressed to date have been in tobacco, although recently potatoes, soybean, alfalfa, rice and wheat have also been used successfully - . the major advantage of using green tissue (tobacco, alfalfa, soybean) is sheer productivity. both alfalfa and tobacco can support several crops (cuttings) per year, with potential annual biomass yields of tonne ha − and > tonne ha − , respectively. by contrast, the maximum yields of wheat, rice and corn seed are ~ tonne ha − , tonne ha − and tonne ha − , respectively. other advantages of tobacco include its relative ease of genetic manipulation, production of large numbers of seeds (up to a million per plant) and an impending need to explore alternate uses for this hazardous crop. however, seeds are likely to have fewer phenolic compounds and a less complex mixture of proteins and lipids than green leaves, which might be an advantage in purification. another advantage of seeds or tubers is their ability to be stored for long periods. levels of scfv in rice seeds did not show a significant decline after storage at room temperature for six months . potato tubers in cold storage for months lost only % of functional antibody . for short periods of time (five to seven days), dried tobacco and alfalfa leaves can also be stored with little loss of scfv (ref. ) or igg antibody . purification of antibody from stored plant material has the advantages that the processing facility need not be near the field and can be used continually all year, rather than for just a few large batches. to date, only four antibodies have been made in plants that are potentially useful as human therapeutics. only one of these has been tested in humans: a chimeric secretory igg-iga antibody against a surface antigen of streptococcus mutans, the primary causal agent of tooth decay. this tobaccoproduced antibody was applied topically to teeth and found to be as effective as an igg produced in a murine hybridoma at preventing recolonization by s. mutans . the second antibody, a humanized anti-herpes-simplex virus (hsv) antibody made in soybean, was effective in the prevention of vaginal hsv- transmission in a mouse model . its activity was indistinguishable both in vitro and in vivo from the monoclonal antibody produced in cell culture. a third antibody, against carcinoembryonic antigen (cea), has recently been expressed in rice and wheat . cea, a cell-surface glycoprotein, is one of the best-characterized tumor-associated antigens. antibodies against cea are used for in vivo tumor imaging, as well as in antibody-based cancer therapy. levels of scfv in seeds did not show a significant decline after storage at room temperature for six months. this same antibody has been expressed in a rice cell culture . the fourth antibody is an example of both a novel use of plant-produced antibodies and an alternative production system. a plant virus vector has been used to produce a tumor-specific vaccine transiently in tobacco for the treatment of lymphoma . the antibody genes for expression of an scfv were derived from a mouse b-cell lymphoma. the plantproduced scfv was used to immunize mice, which generated anti-idiotypic antibodies (antibodies against the binding portion of the antibody). these mice were protected against infection by the lymphoma that produced the original antibody. other groups have used modified plant viral vectors to produce therapeutically useful antibodies in plants, including an antibody against the colorectalcancer-associated antigen ga - (ref. ). although these vectors might find limited usefulness if the rapid production of an antibody is necessary (perhaps in greenhouse production), their acceptability to regulatory agencies (e.g. the us food and drug administration, dept of agriculture and environmental protection agency) has not been tested. there are no plantibodies yet in commercial production, therefore estimates of cost are difficult to find and involve many assumptions. the costs of producing an igg from alfalfa grown in a m greenhouse are estimated to be us$ - g − , compared with us$ g − for the hybridomaproduced antibody . planet biotechnology (mountain view, ca, usa) has compared the cost per gram of purified iga made by cell culture, transgenic goats, grain ( . tonne ha − ) and green biomass ( . tonne ha − ) (fig. ) . expression levels will have a significant impact on the costs but, at the best expression level reported [ µg g − leaf for a secretory iga (ref. )], the final cost should be well below us$ g − . this significantly undercuts the costs of cell culture (us$ g − ) or transgenic animal production systems (us$ g − ). the biggest component of cost with plantibodies will be purification. however, expression in seeds of rice and wheat opens up the possibility of oral administration of some therapeutic antibodies without the need for expensive purification. some of the properties of igs depend on their glycosylation (e.g. binding to monocyte fc receptors). there is one conserved n-glycosylation site in the ch domain of igg. the structures of n-linked glycans on plant-and murine-produced guy's (an igg ) have been determined and compared . the plantibody n-glycans were more structurally diverse, with % being of the high-mannose type. the other % of the plantibody oligosaccharides had β-( , )-xylose and α-( , )-fucose linked to the man glcnac core. these linkages are typical of plants but are not found in mammalian n-glycans. the plantibody also lacked sialic acid, which represented ~ % of the sugar content of the mouse monoclonal antibody. these differences in glycan structure appear to have no effect on antigen binding or affinity in vitro , , , and might not be significant in vivo either. an igg produced in alfalfa had a serum half-life in balb/c mice that was indistinguishable from that of the hybridoma-produced antibody . however, there is some concern about the potential immunogenicity and allergenicity of plantibodies used as human therapeutics. for mucosal applications, this is not likely to present problems for most people because plant glycoproteins are ubiquitous in the human diet. there has been no evidence of allergic reaction or of a human antimouse antibody (hama) response in patients receiving topical oral application of a secretory iga specific to s. mutans . proteins of microbial and viral pathogens were some of the earliest examples chosen to show the feasibility of transgenic plant expression systems - . the rationale was that key immunogenic proteins of major pathogens could be synthesized in plant tissues and then fed as edible . costs for plants compare green biomass ( . tonne ha − ) and seed production ( . tonne ha − ). cost differences are based primarily on production costs, and it was assumed that purification costs and losses during purification will be the same for all systems. subunit vaccines to humans or commercially important animals. the proof of this concept has since been shown using several bacterial and viral proteins ( table ). the practical aspects of choosing particular foodstuffs in which to deliver defined doses of a vaccine are being explored, and efforts are under way to establish clear regulatory paths for the development of edible vaccines. oral delivery of vaccines is an attractive alternative to injection, largely for reasons of low cost and easy administration. the chances of acquiring mucosal immunity against infectious agents that enter the body across a mucosal surface are also increased with oral vaccines. however, a major concern with oral vaccines is the degradation of protein components in the stomach and gut before they can elicit an immune response. to guard against degradation, several delivery vehicles have been developed to ferry intact proteins to the gut. these include recombinant strains of attenuated microorganisms, bioencapsulation vehicles such as liposomes and transgenic plant tissues. early work with plant-based subunit vaccines used the readily transformed species tobacco, potato and tomato - . however, the most attractive species for expressing subunit vaccine components should have high levels of soluble protein that is stable during storage; seed crops such as cereals are particularly suitable. the embryo fraction is rich in soluble protein and can easily be separated from other seed tissue to increase the concentration of antigen and thus decrease the dose size. the choice of crop defines the type of material to be fed. many plant tissues can be consumed raw but others must be processed. processing facilitates the creation of a homogeneous sample, enabling a defined dose size, but it is important that any heat or pressure treatments involved do not destroy the antigen. alternative processing steps have been applied to a candidate vaccine component against enterotoxigenic strains of e. coli that consists of the b subunit of the heat-labile toxin (lt-b) expressed in corn. a typical mg dose of lt-b could be delivered in an embryo fraction, to decrease the volume of the dose, or in a 'cooked' whole corn snack, to increase palatability and enhance stable storage (fig. ) . in this case, neither treatment degrades the antigen. for commercial animal vaccines, the relevant protein can be expressed in a plant tissue that constitutes a major proportion of the diet, and heat and pressure treatments are not necessary. some key examples that illustrate the range of candidate proteins under investigation and plant expression systems being used are given in table . plant-expressed antigens have been shown to able to induce mucosal and serum immune responses when administered parenterally or orally to experimental animals and, in some test cases, they have offered protection against a subsequent pathogen challenge or challenge model , , [ ] [ ] [ ] [ ] [ ] [ ] . a few of these vaccine candidates have been successfully tested in clinical trials or, where appropriate, in commercial or native animal trials [ ] [ ] [ ] [ ] [ ] [ ] . thus, edible vaccines delivered in plant tissues or processed plant products show great potential for efficacy in target organisms. the bioencapsulation of lt-b in transgenic corn material results in an increased mucosal immune response compared with that achieved with naked antigen when fed to mice . presumably, this is because the antigen is protected from degradation in the gut, and it augurs well for the development of plant-based edible vaccines. the quantity of plant tissue constituting a vaccine dose must be of a practical size for consumption. thus, achieving a high level of expression is crucial. the expression of vaccine components in plants has been increased by using a range of leader and polyadenylation signals and by optimizing codon usage for plants , , . expression could also be raised through crosses of transformed lines to various genetic backgrounds, an approach that has been successfully applied to boost protein production in corn. it is also important that any vaccine component should be present in its native form in the transgenic plant tissue. this has been assessed in several cases by examining the size of the synthesized protein, its ability to form higher-order complexes that mirror microbial or viral structures and, where relevant, by showing an enzymatic or receptor-binding activity , - , , , . the stability of heterologous proteins and the assembly of multisubunit structures depend on the cellular environment and therefore on the subcellular location. favored locations for the expression of selected subunit vaccine components are the cell surface and the endoplasmic reticulum and golgi body , , , , . as with antibodies, transient expression systems (in which candidate vaccine sequences are incorporated into plant viral surface proteins) have also been investigated extensively and high levels of expression have been achieved. a related strategy to that of edible vaccines uses transgenic plants expressing autoantigens, whereby a large oral dose of an autoantigen can inhibit the development of an autoimmune disease through the mechanism of oral tolerance. this approach has been successful in a mouse model for diabetes . generally, levels of pharmaceutical proteins produced in transgenic plants have been less than the % of total soluble protein that is needed for commercial feasibility if the protein must be purified . plantderived recombinant hepatitis-b surface antigen induced only a low level serum antibody response in a small human study, probably reflecting the low level of expression ( - ng g − fresh weight) in transgenic lettuce . in spite of recent improvements in expression levels in potato with a view to clinical trials , expression levels should be increased further for practical purposes. also, even though norwalk virus capsid protein expressed in potatoes caused oral immunization when consumed as food, expression levels are too low for large-scale oral administration ( . % of total soluble protein) , . expression of genes encoding other human proteins in transgenic plants has been disappointingly low: human serum albumin, . % total soluble protein; human protein c, . % total soluble protein; erythropoietin,~ . % total soluble protein; and human interferon-β, < . % fresh weight (table ) . a synthetic gene coding for the human epidermal growth factor was expressed only up to . % of total soluble protein in transgenic tobacco , . in spite of several successful reports of high-level expression of non-human proteins (e.g. phytase, glucanase) via the nuclear genome, there is a great need to increase expression levels of human blood proteins to enable the commercial production of pharmacologically important proteins in plants. one alternative approach is to express foreign proteins in chloroplasts of higher plants. foreign genes have been integrated into the tobacco chloroplast genome, giving up to copies per cell and resulting in the accumulation of recombinant proteins at up to % of the total soluble protein . chloroplast transformation uses two flanking sequences that, through homologous recombination, insert foreign dna into the spacer region between the functional genes of the chloroplast genome, thus targeting the foreign genes to a precise location. this eliminates the 'position effect' upon expression that is frequently observed in transgenic plants with genes inserted into the nuclear genome. in addition, gene silencing has not been observed with chloroplast transformation, whereas it is a common phenomenon with nuclear transformation. chloroplast genetic engineering is an environmentally friendly approach, minimizing several environmental concerns , . importantly, chloroplasts can process eukaryotic proteins, including enabling correct folding and the formation of disulfide bridges. chaperonin proteins are present in chloroplasts and might function in the folding and assembly of non-native proteins of both prokaryotic and eukaryotic origins. also, chloroplast proteins are activated by disulfide bond oxidation-reduction cycles using the plastid thioredoxin system or protein disulfide isomerase . accumulation of large quantities of a fully assembled form of human somatotropin with the correct disulfide bonds ( % total soluble protein) provides strong evidence for hyperexpression and assembly of pharmaceutical proteins using this approach. such folding and assembly of foreign proteins should eliminate the need for expensive in vitro processing of pharmaceutical proteins produced in recombinant organisms. for example, % of the total operating cost for the commercial production of human insulin in e. coli is associated with in vitro processing (formation of disufide bridges and cleavage of methionine) . purification is likely to represent most of the cost of biopharmaceutical production in plants. for the commercial production of insulin in e. coli, chromatography accounts for % of operating expenses and % of equipment costs . therefore, new approaches are necessary to minimize or eliminate chromatography in the production of pharmaceutical proteins. one successful recent approach is targeting pharmaceutical proteins to seed oil bodies. this was shown with hirudin, an anticoagulant first isolated from the leech hirudo medicinalis. an oleosin-hirudin fusion protein has been targeted to oil bodies of brassica napus seeds and purified by flotation centrifugation for commercial production in canada . another novel approach is the use of gvgvp as a fusion protein to facilitate single-step purification without the use of chromatography. gvgvp is a protein-based polymer encoded by synthetic genes. at low temperatures, it exists as an extended molecule but, upon raising the temperature above the transition range, the polymer hydrophobically folds into dynamic structures called β-spirals that further aggregate by hydrophobic association to form twisted filaments . using this approach, single-step purification of an insulin-polymer fusion has recently been shown. inverse temperature transition offers several advantages, including facilitating the scale-up of purification from grams to kilograms (o. carmona-sanchez and h. daniell, unpublished) . yet another recent approach is the use of a chaperonin protein to fold foreign proteins into cuboidal crystals, allowing their purification in a single step by centrifugation . one additional advantage of this method is the protection of foreign proteins from cellular proteases. plant-derived biopharmaceuticals should meet the same standards of safety and performance as other production systems. however, many herbal medicines are now exempt from such close scrutiny and are not required to meet the same standards because of their classification as nutritional supplements. because several environmental concerns have been raised by interest groups to confuse public perception, it is of paramount importance that regulating agencies distinguish between real and perceived public concerns (scientific versus non-scientific). if biopharmaceuticals that are potentially harmful are capable of persisting in the environment and might accumulate in non-target organisms, precautionary measures should be taken. induction of biopharmaceutical production after harvesting (as was done in the case of glucocerebrosidase ) might be one approach to minimize environmental exposure, provided that the use of viral vectors does not introduce additional environmental or regulatory concerns. expression of potentially harmful proteins in a form that must be treated for activation might minimize the risk of exposure. for example, hirudin is produced as a fusion protein and is inactive in this form; it is activated only after it is purified from seeds . another hotly debated environmental concern has been the outcrossing of transgenic pollen to weeds or related crops , . expression of harmful pharmaceutical proteins in non-target plants resulting from such outcrosses might create public concern and negative perception. several gene containment methods are currently being investigated, including apomixis, incompatible genomes, transgenic mitigation, control of seed dormancy or shattering, suicide genes, infertility barriers, male sterility and maternal inheritance. engineering foreign genes via the chloroplast genome has been shown to contain transgenes effectively, although there are a few exceptions in which the chloroplast genome shows biparental inheritance (e.g. pines) . as an example of an alternative strategy, rnase genes have been expressed under the control of a tissue-specific promoter to destroy the tapetum selectively during anther development, resulting in male sterile plants . there is also concern over the expression of harmful proteins in transgenic pollen. for example, the controversial observation of the toxic effect of bacillus thuringiensis (bt) corn pollen on milkweeds (asclepias spp.) fed to monarch butterfly larvae had a significant impact on public perception, even though the validity of this study has been repeatedly questioned. engineering biopharmaceuticals via the chloroplast genome might be a solution. although the cry protein of bt was expressed at high levels in leaves (up to % of total soluble protein), no toxicity was observed when milkweeds dusted with transgenic pollen were fed to monarch butterfly larvae . however, to date, chloroplast genetic engineering has been shown only in tobacco and potato. more recently, several academic and industrial laboratories have initiated projects to extend this technology to other useful crops. also, there are no reports of the production of glycoproteins in transgenic chloroplasts. another public concern is the presence of antibiotic resistance genes or their products (which are used as selective markers) in edible parts of genetically modified crops. however, several approaches are now available to generate plants with transgenes in their nuclear or chloroplast genomes without the use of antibiotic selection. practical considerations will dictate the choice of biopharmaceutical proteins and the crop in which they are to be produced. these include yield, storage conditions, containment properties, initial set up and running costs, purification strategies, size of the market, environmental concerns, public perception and competing technologies. access to several alternative approaches to optimize protein synthesis in plants in an environmentally sound manner augurs well for the safe production of biopharmaceuticals in transgenic plants and for greater availability of these proteins to populations requiring them. toxin b subunit oligomers in transgenic potato plants efficacy of a food plantbased oral cholera toxin b subunit vaccine protective immune response to foot-and-mouth disease virus with vp expressed in transgenic plants expression of immunogenic glycoprotein s polypeptides from transmissible gastroenteritis coronavirus in transgenic plants edible vaccine protects mice against escherichia coli heat-labile enterotoxin (lt): potatoes expressing a synthetic lt-b gene immunogenicity of transgenic plant-derived hepatitis b surface antigen induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein vp immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes a plant-derived edible vaccine against hepatitis b virus immunization with potato plants expressing vp protein protects against rabbit hemorrhagic disease virus immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants plant-based vaccines: unique advantages production of hepatitis b surface antigen in transgenic plants for oral immunization development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein b (ul ) in seeds of transgenic tobacco transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance production of recombinant proteins in transgenic plants: practical considerations application of transgenic plants as production systems for pharmaceuticals transgenic plants for therapeutic proteins: linking upstream and downstream technologies hyper-expression of the bt cry aa operon in chloroplasts leads to formation of insecticidal crystals gm crops: public perception and scientific solutions environmentally friendly approaches to genetic engineering regulation of chloroplast enzyme activities by thioredoxins: activation or relief from inhibition protein disulfide isomerase as a regulator of chloroplast translational activation high-yield production of a human therapeutic protein in tobacco chloroplasts computer-aided process analysis and economic evaluation for biosynthetic human insulin production: a case study molecular farming in plants: oil seeds as vehicles for production of pharmaceutical proteins hyperexpression of an environmentally friendly synthetic polymer gene containment of herbicide resistance through genetic engineering of the chloroplast genome induction of male sterility in plants by a chimaeric ribonuclease gene removing selectable marker genes: taking the shortcut engineering chloroplast genome without the use of antibiotic resistance genes transgenic plants as factories for biopharmaceuticals triple helix assembly and processing of human collagen produced in transgenic tobacco plants expression of full length bioactive antimicrobial human lactoferrin in potato plants key: cord- -asjvf authors: lee, yu-ching; leu, sy-jye c.; hu, chaur-jong; shih, neng-yao; huang, i-jen; wu, hsueh-hsia; hsieh, wen-shyang; chiang, bor-luen; chiu, wen-ta; yang, yi-yuan title: chicken single-chain variable fragments against the sars-cov spike protein date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: asjvf the major concern for severe acute respiratory syndrome (sars), caused by the sars-associated coronavirus (sars-cov), is the lack of diagnostic and therapeutic agents. using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the sars-cov spike protein were characterized. ten truncated spike protein gene fragments were expressed in escherichia coli cells. following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scfv) antibody libraries were established with short or long linkers to contain × ( ) and × ( ) transformants, respectively. after four rounds of panning selection, the scfv antibodies of randomly chosen clones were demonstrated by coomassie blue staining, and verified by western blot analysis. in a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scfv antibodies reacted significantly with sars-cov-infected vero cells, and that those two specific scfv antibodies recognized the same region of the spike protein spanning amino acid residues – . in conclusion, the results suggest that the chicken scfv phage display system can be a potential model for mass production of high-affinity antibodies against the sars-cov spike protein. the severe acute respiratory syndrome (sars) is a newly emerging disease caused by a sars-associated coronavirus (sars-cov) ksiazek et al., ; peiris abbreviations: sars-cov, severe acute respiratory syndrome-associated coronavirus; s, spike; scfv, single-chain variable fragment; e. coli, escherichia coli; rt-pcr, reverse-transcription polymerase chain reaction; cdr, complementarity-determining region; fr, framework region; v h , heavychain variable region; v l , light-chain variable region * corresponding author. tel.: + x ; fax: + . e-mail address: yangyuan@tmu.edu.tw (y.-y. yang) . et al., ) . the virion consists of the following four major structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) (marra et al., ; rota et al., ) . the s protein has two functional domains (s and s ) based on the predicted localization of their amino acid residues: - and - , respectively (he et al., ) . a region located between amino acids and on the s domain serves as a receptor-binding site (dimitrov, ; li et al., ; wang et al., ) . the c-terminal s domain has been shown to mediate membrane fusion during sars-cov infection (tripet et al., ) . further, immunization of mice with recombinant s protein can protect them from sars-cov infection (bisht et al., ; yang et al., ) . the results suggested that the s pro- tein is a good candidate for developing vaccines and antiviral drugs, and that generating monoclonal antibodies to recognize specifically the s protein would be valuable. although monoclonal antibodies with high specificities have been favored for both research and clinical applications in recent years, using the traditional hybridoma approach to generate human monoclonal antibodies for therapeutic purposes is still difficult because it is a tedious and expensive process (groves and morris, ; lillehoj and malik, ) . in contrast, the phage display system is a safe and effective procedure because the process involves the in vitro cloning of antibody repertoires, and subsequent isolation of monoclonal antibodies from combinatorial antibody libraries (barbas et al., ; winter et al., ) . of many recombinant antibody forms, the single-chain variable fragment (scfv) is a small protein entity retaining the variable regions of both heavy and light chains of an entire antibody molecule (bird et al., ; huston et al., ) which can be efficiently generated in a phage display system (chi et al., ; pavoni et al., ; wang et al., ) . antibody production in the chicken is efficient (abouzid et al., ; leclaire et al., ) . it has been reported that constructing chicken antibody libraries using the phage display technology can generate high-affinity scfvs for diagnostic applications (fehrsen et al., ; finlay et al., ; park et al., ) . performing reverse-transcription polymerase chain reaction (rt-pcr) to amplify the entire v region repertoire using one set of primers is simple and convenient because all avian immunoglobulin genes are derived from single light-and heavy-chain variable (v l and v h ) germline sequences (andris-widhopf et al., ; mccormack et al., ; yamanaka et al., ) . using the phage display technology, monospecific scfv and fab antibodies neutralizing the sars-cov infection have been generated from non-immunized individuals and convalescent sars patients (kang et al., ; sui et al., ) . the current study aimed to show that monoclonal igy scfv antibodies which bind specifically to the s protein and sars-cov-infected vero cells can be isolated from chickens immunized with escherichia coli-derived s proteins. ten sets of primers were synthesized to amplify s gene fragments using the sars-cov rna genome as a template (genbank accession no. nc ). the entire procedure was performed using a one-step rt-pcr kit as described by the manufacturer (qiagen, valencia, ca, usa). the amplified s gene products of - bp in length were individually digested with bamhi and xbai restriction enzymes and ligated into the pet- expression vector (novagen, darmstadt, germany). the resultant plasmids were transformed into the e. coli bl- (de ) strain for protein expression. clones were grown in ml lb medium containing ampicillin ( g/ml) at • c overnight. the bacterial culture was then diluted fold in the same lb medium and further grown until the od reached . . for his-fused s protein expression, iso-propyl-␤-d-thiogalactopyranoside (iptg) was added to a final concentration of . mm in the culture for induction. the cell pellet was resuspended in ml of binding buffer ( mm nacl, mm tris-cl, and m urea; ph . ) and lysed by three cycles of freezing (− • c) and thawing ( • c). after centrifugation, the resulting cellular lysate was loaded onto a ni + -charged resin column for protein purification according to the manufacturer (amersham biosciences, uppsala, sweden). female white leghorn (gallus domesticus) chickens were immunized with mixed s protein fragments ( g/each) in an equal volume of freund's complete adjuvant by intramuscular injection. three additional immunizations were carried out in intervals of days. after each immunization, blood was obtained and titrated by an enzyme-linked immunosorbent assay (elisa) to determine the presence of an antigen-specific immune response. the spleens of these chickens were harvested for rna isolation. phage libraries displaying scfv antibodies were constructed according to published protocols with minor modifications (andris-widhopf et al., ; barbas et al., ) . after pcr amplification with chicken-specific primers, gene products of heavy-chain (v h ) and light-chain variable (v l ) regions were subjected to a second round of pcr with a short or long linker to form full-length scfvs, which were cloned further into the pcomb h vector. to increase the cloning efficiency, g of pcomb h and . g of the full-length scfv product were applied in the ligation reaction. the recombinant dnas were transformed into the e. coli xl- blue strain by electroporation, recombinant phage production was initiated by the addition of the helper phage, vcs-m , and were precipitated with % polyethylglycol and % nacl (w/v) and finally resuspended in phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) and stored at • c. microtiter plates precoated with mixed s protein fragments ( . g/well) at • c overnight were blocked with % bsa for h at • c. then, plaque-forming units (pfu) of recombinant phages from the library preparation was added to each well, and the plates were incubated at • c for h. unbound phages were removed, and the wells were washed vigorously with tbst (tbs with . % tween ) buffer. bound phages were eluted with . m hcl/glycine (ph . )/ . % bsa and neutralized with m tris-base buffer. eluted phages were used to infect the e. coli xl- strain in the log phase for amplification, and they were recovered with % polyethylglycol and % nacl for the next round of selection. the panning procedure was repeated in the wells four times. phagemid dna from the final enriched phage was prepared and digested with nhei and spei to remove the phage protein iii gene. the digested dna with compatible cohesive ends was self-ligated, and the resultant phagemid was electroporated into e. coli. individual clones were grown in the presence of . mm iptg for protein induction. cells were pelleted and lysed by three cycles of freezing and thawing. after the final thawing, the supernatants were harvested by centrifugation for subsequent assays. the scfv-expressing lysates were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page). the proteins were transferred onto nitrocellulose membranes (amersham biosciences, little chalfont, uk), which were then blocked with % skim milk in tbst for h. polyclonal goat anti-chicken igy antibodies (bethyl laboratories, montgomery, tx, usa) were added and incubated for an additional hour. the membranes were washed with tbst three times for min each. the bound antibodies were detected by adding horseradish peroxidase (hrp)-conjugated donkey anti-goat immunoglobulin (ig) antibodies (sigma, st. louis, mo, usa). after three washings, the membranes were developed with diaminobenzidine (dab) substrate until the desired intensity was achieved. to examine the binding ability of the scfv-expressing lysate against the s protein, microtiter plates precoated with sars-cov-infected vero cell lysates (euroimmun, lueberk, germany) were used for the elisa. after rinsing with pbst (pbs with . % tween ), test lysates were distributed to wells in duplicate and incubated at • c for h. the bound scfvs were detected with enzyme-labeled antibodies as described above. the nucleotide sequences of heavy-and light-chain genes from chosen clones were determined by an auto-sequencer machine (abi prism ; perkin-elmer, national health research institute). the sequencing primer, ompseq ( -aagacagctatcgcgattgcagtg- ) as described by andris-widhopf et al. ( ) , was used for the v l and v h gene analyses. biochip slides (euroimmun) were used to test the binding reactivities of antibodies in sera or scfv-expressing lysates to native s protein in sars-cov-infected vero cells. slides were incubated with either sera or lysates at room temperature (rt) for h. after washing with tbst, the bound antibodies or scfvs were incubated with mouse anti-human (sigma) or goat antichicken antibodies (bethyl laboratories) at rt for h. after washings, slides were incubated for h at rt with corresponding fluorescein isothiocyanate (fitc)-labeled antibodies (sigma). finally, slides were coated with mounting oil and examined with an immunofluorescent microscope. epitopes were mapped with specific scfvs by detecting their reactivities with truncated s protein fragments by western blot analysis. ten purified his-fused s fragments were transferred onto a nitrocellulose membrane after electrophoresis. following blocking with % skim milk, membranes were incubated with scfv-expressing lysates for h at rt. then, the bound scfv antibodies were detected as described above. the binding affinities of specific scfvs were determined by competitive inhibition assays. briefly, l of each scfvexpressing lysate were first incubated with equal volumes of a series of two-fold-diluted soluble s fragments ( . - g/ml) at rt for h. the reactivities of the mixtures with each fragment individually precoated on a microtiter plate ( . g/well) were measured as described above. the dissociation constant (k d ) of the scfv binders was calculated according to the klotz method (friguet et al., ) . ten truncated fragments of the sars-cov s gene were amplified by pcr, cloned into the pet- vector, and expressed as his-fused recombinant proteins. table lists the locations and predicted gene lengths of these fragments. after transformation and induction, these proteins were expressed successfully in e. coli bl- cells and purified using ni + -charged resin. fig. a shows that these s protein fragments, s -s , had the predicted molecular weights of about - kda after being analyzed by sds-page. to construct the scfv libraries, two female white leghorn chickens were chosen for immunization. after the final immunization step, the chickens were killed, and the rna was extracted from their spleen cells. one set of specific primers were designed to amplify the v l and v h regions of the scfv fragment genes. after a second and consecutive pcr step, the v l and v h genes were joined with a short (ggssrss) or long linker (ggssrssssggggsgggg) to form a full-length scfv gene conformation (data not shown). two libraries, ssc (with the short linker) and lsc (with the long linker), containing × and × transformants, respectively, were constructed in this study. fig . a shows that scfv antibodies were well amplified and expressed. as predicted, the molecular weights of these expressed scfv antibodies with short or long linkers were and kda, respectively. their identities were verified further using specific anti-chicken igy antibodies in the western blot analysis (fig. b) . the binding abilities of clones against the s protein from each library were analyzed using elisa. fig. a shows five clones obtained from the ssc library to be strongly positive (with ods of . - . ) for binding activity, while others showed weak or no reactivity. fig. b illustrates four clones selected from the lsc library to have higher activity (with ods of . - . ). the nucleotide sequences of the v l and v h regions of clones (seven each from the ssc and lsc libraries) with different binding abilities were analyzed. the cdrs of the v l region of five of the clones showed more than % variation. from among those clones, ssc showed the lowest and ssc the highest mutation rates, of % and %, respectively. on the other hand, the v h region analysis showed that ten of the clones showed more than % cdr variations. among those clones, ssc showed the lowest and ssc the highest mutation rates, of % and %, respectively (fig. ) . the binding activities against the s protein expressed by sars-cov-infected cells of clones with od values greater fig. . binding activity of randomly selected clones analyzed by elisa. cellular lysates containing single-chain variable fragment (scfv) antibodies from various ssc (a) and lsc (b) library clones were examined for their binding to sars-cov-infected cell lysates using a commercially available kit. negative control (n) is a cellular lysate lacking scfv expression. bound scfv was detected using anti-chicken light-chain antibodies and was measured at nm. the elisa data were represented as mean of the duplicated wells ± s.e.m. using sigmaplot statistical analysis software. (table ). as represented in fig. , the immunocytochemical staining results showed that clones ssc (c), ssc (d), and lsc (e) had better or similar reactivities compared with that of convalescent serum (a), while clone lsc (f) exhibited no significant binding signal. two negative controls including normal human serum (b) and bacterial cell lysate without scfv expression (g) exhibited no reactivity at all. panels h-n were used to demonstrate the total cell numbers, morphology, and distribution under light microscopy. clones ssc and lsc were used to identify possible antigenic sites on the s protein with western blot analysis. the purity of the s protein fragments (s to s ) was examined and are shown in fig. a. fig. b and c shows that ssc and lsc scfv antibodies recognized mainly the s protein, suggesting that a potential antigenic epitope is located in the region of amino acid residues - of the intact s protein. fig. illustrates the results of the competitive inhibition assays of two representative scfv antibodies against the s fragment. eighy-three percent and % inhibitory effects were found on the binding reactivity of ssc and lsc antibodies, respectively, against the s fragment in the presence of a concentration of g/ml of the free s fragment. the k d values calculated using the klotz plot for scfvs ssc and lsc were . × − and . × − m, respectively. to face a possible sars outbreak in the future, it is necessary to have better diagnostic and therapeutic agents. to overcome the problems which occur frequently during traditional antibody production, such as obtaining low quantities of monoclonal antibodies (mabs) and the occasional loss of their efficiency due to persistent culturing, a phage display system was used to develop avian anti-sars-cov antibodies. the highly conserved and regions of fr and fr facilitate the use of a set of oligonucleotide primers to amplify the v region repertoire for library fig. . competitive inhibition assay of two representative single-chain variable fragment (scfv) antibodies against the s fragment. the amount of bound ssc and ssc scfv in the presence of free s inhibitor was measured and expressed as a percentage of the binding of scfv in the absence of an inhibitor. b and b are the amounts of bound scfv in the presence and absence of the inhibitor, respectively. construction (davies et al., ; mccormack and thompson, ) . two scfv antibody libraries were established which contained × and × clones showing that construction of the chicken scfv antibody library was easier compared to the construction of antibody libraries for humans or other mammals. thus, the clinical and scientific aspects of chicken scfv generation have received recently much attention (fehrsen et al., ; finlay et al., ; park et al., ) . as shown in fig. a and b, the expression and presence of scfv antibodies were examined by coomassie blue staining and western blot analysis. using elisa, it was observed that more than half of the selected clones in both the ssc and lsc libraries could react significantly with sars-cov-infected cell lysates (fig. ) . interestingly, the ssc and lsc scfv antibodies showed strong reactivity in the elisa, but weak binding signals in the subsequent immunocytochemical staining analyses. this discrepant result may have been due to the conformational modification of the s protein in the different preparation processes. some chosen enriched binders against target antigens immobilized on elisa wells have been found to react poorly against the same antigens located on the surface of cells (parren et al., ) . despite this potential problem in our study, three clones (ssc , ssc , and lsc ) were found to bind equally or more efficiently to the s protein compared with the patients' sera by immunocytochemical staining (fig. ) . as shown in fig. , sequence comparison of the clones (seven each from the ssc and lsc libraries) with the chicken germline revealed that all of the cdrs were dissimilar. somatic hypermutations to increase antibody affinity have been found to occur more frequently in cdrs than frs of the rearranged v gene (gearhart and bogenhagen, ) . in fact, the ig genes of the selected clones from hyperimmunized chickens had more mutations in the cdrs as a result of the affinity selection of b cells. therefore, this conclusion is in agreement with that obtained by other researches (finlay et al., (finlay et al., , sapats et al., ) and suggests that high mutation rates in these variable genes indicate an antigen-driven response in the chicken induced by the s proteins. the result of the present study showed that three clones (ssc , ssc , and lsc ) had significant binding signals to intact sars-cov-infected vero cells (fig. ) . to map the potential antigenic epitopes with western blotting, it was found that clones ssc and lsc recognized the purified s fragment ( fig. b and c) , but that clone ssc showed no reactivity (data not shown). conformational folding of the s protein present on the surface of sars-cov-infected cells differs from that of the truncated s proteins immobilized on nitrocellulose membranes, thus leading to differential recognition of clone ssc 's scfv antibody. the western blotting results in the study indicated further an antigenic epitope that spanned amino acids - . previous studies have shown an antigenic domain that spanned amino acid residues - can produce antibodies against sars-cov infection wang et al., ) . based on these results, it is concluded that the ssc and lsc scfv antibodies have specific binding abilities against the s protein and may be able to neutralize the infectivity of sars-cov in susceptible host cells. in conclusion, the identified epitope is a potential candidate for vaccine development, and these specific monoclonal scfv antibodies can help in developing valuable reagents for both scientific and clinical applications. genetic vaccination for production of dna-designed antibodies specific to hepadnavirus 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antibodies using chicken scfvs in inhibition elisas exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins generation of highaffinity chicken single-chain fv antibody fragments for measurement of the pseudonitzschia pungens toxin domoic acid measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay clusters of point mutations are found exclusively around rearranged antibody variable genes veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli human neutralizing fab molecules against severe acute respiratory syndrome coronavirus generated by phage display a novel coronavirus associated with severe acute respiratory syndrome protection against bacterial superantigen staphylococcal enterotoxin b by passive vaccination angiotensin-converting enzyme is a functional receptor for the sars coronavirus the new antibody technologies the genome sequence of the sars-associated coronavirus chicken igl variable region gene conversions display pseudogene donor preference and - polarity immunoglobulin gene diversification by gene conversion development and characterization of a recombinant chicken single-chain fv antibody detecting eimeria acervulina sporozoite antigen in vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type envelope selection, affinity maturation, and characterization of a human scfv antibody against cea protein coronavirus as a possible cause of severe acute respiratory syndrome chicken recombinant antibodies specific for very virulent infectious bursal disease virus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association structural characterization of the sars-coronavirus spike s fusion protein core expression cloning of functional receptor used by sars coronavirus identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system construction of single chain variable fragment (scfv) and biscfv-alkaline phosphatase fusion protein for detection of bacillus anthracis making antibodies by phage display technology chicken monoclonal antibody isolated by a phage display system a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this study was supported by a grant (nsc - -b- - -y) from the national science council (nsc) of taiwan. prof. winston w. shen made editing comments on a previous version of this manuscript. key: cord- -hfa w dg authors: jin, junyeong; park, changhoon; cho, sun-hee; chung, junho title: the level of decoy epitope in pcv vaccine affects the neutralizing activity of sera in the immunized animals date: - - journal: biochemical and biophysical research communications doi: . /j.bbrc. . . sha: doc_id: cord_uid: hfa w dg abstract viral pathogens have evolved a wide range of tactics to evade host immune responses and thus propagate effectively. one efficient tactic is to divert host immune responses toward an immunodominant decoy epitope and to induce non-neutralizing antibodies toward this epitope. therefore, it is expected that the amount of decoy epitope in a subunit vaccine can affect the level of neutralizing antibody in an immunized animal. in this study, we tested this hypothesis by generating an antibody specific to the decoy epitope on the capsid protein of porcine circovirus type (pcv ). using this antibody, we found that two commercial vaccines contained statistically different amounts of the decoy epitope. the vaccine with lower levels of decoy epitope induced a significantly higher level of neutralizing antibody after immunization. this antibody can be used as an analytical tool to monitor the quality of a vaccine from batch to batch. viral pathogens have evolved a wide range of tactics to evade host immune responses and propagate effectively [ ] . one such tactic is to divert the host immune response with a decoy. decoy epitopes have been reported in a wide variety of viruses, including human immunodeficiency virus (hiv) [ ] , feline immunodeficiency virus (fiv) [ ] , hepatitis c [ ] , foot and mouth disease [ ] , middleeast respiratory syndrome coronavirus [ ] , severe fever with thrombocytopenia syndrome virus [ ] , porcine reproductive and respiratory syndrome virus (prrsv) [ ] , murine gammaherpesvirus- (mhv- ) [ ] , and porcine circovirus type (pcv ) [ ] . because a recombinant mutant capsid protein (cp) with a deleted decoy epitope induced a higher level of virusneutralizing antibody than wild type proteins [ ] , it was expected that higher levels of decoy epitope in a prepared batch of subunit vaccine would decrease its efficacy. however, it was not possible to test this hypothesis by measuring levels of decoy epitope in a vaccine because an antibody specific to the decoy epitope was not readily available. pcv has a spherical structure with icosahedral symmetry [ ] . the only structural protein of this virus is a -kd cp, which includes major antigenic determinants that can be used in subunit vaccines [ ] . a previous study showed that an epitope composed of amino acid residues e (cp e ) is the most immunodominant among these antigenic determinants [ ] . because this epitope is buried inside the virus-like particle (vlp) structure (fig. a) , antibodies to this epitope cannot react with the intact virus, and therefore are non-neutralizing [ ] . as expected, pcv infection elicited a humoral response to the cp e epitope and drove the production of non-neutralizing antibodies [ ] . therefore, previous infection with pcv decreased the vaccines prevention of the future infections. since the first vaccine against pcv was introduced to the global market in , nine pcv subunit vaccines have become available [ ] . however, they were expected to contain differing amounts of incomplete vlps, which are non-uniformly aggregated cps with an exposed cp e epitope [ ] . incomplete vlps can induce production of non-neutralizing antibodies and reduce vaccine efficacy [ , ] . as there was no analytical tool to measure the amount of cp e epitope in a prepared batch of vaccine, it was not possible to correlate epitope levels with neutralizing activity after vaccination. in this study, we developed an antibody specific to the cp e epitope, determined the relative amounts of cp e epitope in two commercially available vaccines, and analyzed the influence of the epitope level on the induction of neutralizing antibody. . . preparation of vlp, pcv recombinant proteins, and peptide conjugates pcv cp (amino acids [aa] e ) was expressed in baculovirusinfected sf insect cells, as previously described [ ] . the pcv orf gene (genbank no. eu ) was cloned into the pfastbac expression vector (invitrogen, carlsbad, ca, usa) and transfected into sf cells. the culture supernatant containing the recombinant baculovirus was harvested and used to infect a separate batch of sf cells. after days, vlps were purified from culture supernatant by sucrose gradient ultracentrifugation. recombinant cp with a deletion of the n-terminal region of the protein (deln cp, aa e ) was expressed in escherichia coli and purified, as described previously [ , , ] . a synthetic cp e peptide (stidyfqpnnkrc) was chemically synthesized using fmoc solid-phase peptide synthesis (peptron, inc., daejeon, south korea) and conjugated to bovine serum albumin (bsa) or keyhole limpet hemocyanin (klh) [ ] . white leghorn chickens were immunized with klh-conjugated cp e peptides. a phage-displayed chicken single-chain variable fragment (scfv) library was constructed using total rna isolated from the bone marrow, spleen, and bursa of fabricius of immunized chickens, as previously described [ ] . a positive clone was enriched by biopanning and screened in a phage enzyme immunoassay, as described previously [ , ] . the gene encoding the selected scfv clone was subcloned into a modified mammalian expression vector encoding the c k domain of human igg at the region [ ] . the scfv-c k fusion protein (anti-cp e antibody) was purified from the culture supernatants of transiently transfected hek f cells using kappaselect resin (ge fig. . a novel pcv antibody is specific to the cp e epitope. (a) the three-dimensional structure of pcv (pdb: jci) was visualized by pymol . . one capsid protein (cp) and the immunodominant cp e decoy epitope are marked in green and red, respectively. (b) enzyme immunoassay using the anti-cp e antibody. the amount of antibody bound to antigens coated on microtiter plate was detected by hrp-conjugated goat anti-human c k antibody. the results are the means ± standard deviations from experiments conducted in triplicate; **p < . , ***p < . by two-tailed unpaired student's t-test. (c) immunoblot analysis using the anti-cp e antibody. after the gel electrophoresis, antigens were transfected to nitrocellulose membrane. the membrane was probed with anti-cp e antibody. healthcare, london, uk), according to the manufacturer's instructions. the wells of a -well microtiter plate (corning) were coated with bsa-conjugated cp e peptides, deln cp, and purified vlp in coating buffer ( . m nahco , ph . ) and then blocked with % (w/v) bsa in phosphate-buffered saline (pbs). after incubation with mg/ml of anti-cp e antibody, horseradish peroxidase (hrp)conjugated goat anti-human c k antibodies (chemicon-millipore, billerica, ma, usa) were added to each well. after washing with . % (v/v) tween in pbs (pbst), , , , -tetramethyl benzidine (tmb) (gendepot, barker, tx, usa) substrate solution was added and the absorbance was measured at nm with a multiskan ascent microplate reader (labsystems, helsinki, finland) [ ] . dissolved proteins and bsa-conjugated cp e peptides were boiled for min in lithium dodecyl sulfate sample buffer with reducing agents (invitrogen). the samples were then electrophoretically separated on a nupage e % bis-tris gel (invitrogen) and transferred to a nitrocellulose membrane [ ] . after blocking with % (w/v) skim milk in tris-buffered saline, the membrane was incubated with mg/ml of anti-cp e antibody and probed with hrp-conjugated goat anti-human c k antibodies (chemicon-millipore). the blot was visualized using supersignal west pico chemiluminescent substrate (thermo fisher scientific, il, usa). all animal experiments were performed under the approval of the institutional animal care and use committee of the biopoa (permit no. bp- - - ). after the collection of sera from pcv infected pigs and pigs with no infection history, the competition enzyme immunoassay was performed as described previously with the following appropriate modifications [ ] . after pre-incubating non-infected and infected porcine sera (n ¼ /group) with serially diluted anti-cp e antibody, the mixtures were added to microtiter plate coated with bsa-conjugated cp e peptide. an irrelevant scfv fused with a c k domain was used as an isotypematched control. the amount of bound porcine igg was determined by hrp-conjugated goat anti-porcine igg antibodies (santa cruz biotechnology, tx, usa) and tmb was used for the substrate of hrp. we randomly designated two of out nine commercial vaccines ( the level of total pcv antigen in prepared vlp, deln cp, and the two commercial vaccines was measured by enzyme immunoassay using an anti-pcv polyclonal antibody (veterinary diagnostic laboratory, iowa state university, iowa, usa) [ ] and hrp-conjugated goat anti-rabbit igg antibodies (bethyl laboratories, montgomery, tx, usa), as described previously. to monitor the cp e epitope levels, we performed the enzyme immunoassay using the anti-cp e antibody and hrpconjugated goat anti-human c k antibodies (chemicon-millipore), as previously described. twenty guinea pigs were randomly divided into four groups (n ¼ /group). each guinea pig was immunized intradermally with ml of vlp, deln cp, or commercial vaccine. four weeks after immunization, whole blood was collected and sera were prepared. an indirect fluorescence assay (ifa) was performed using the sera and pcv -infected pk- cells, as previously described [ , ] . to monitor the reactivity of sera to the cp e epitope, enzyme immunoassays, using bsa-conjugated cp e peptides and hrpconjugated anti-guinea pig igg antibodies (bethyl laboratories), were performed as described previously. we performed serum virus neutralization assays using pk- cells, as described previously [ , ] . statistical analysis was conducted using graphpad prism (v . ; graphpad software inc., san diego, ca, usa). results are expressed as means and standard deviations of the indicated number of independent measurements. statistical significance was determined using two-tailed unpaired student's t tests and non-parametric mann-whitney's u test. two-way analysis of variance (anova) was used to analyze the relationship between the two independent factors. for all statistical analyses, p values of < . were considered statistically significant. a phage display library of combinatorial scfv was prepared using chickens immunized with klh-conjugated cp e peptides. the complexity of the library exceeded . Â . to enrich for scfv clones with specific reactivity to the cp e epitope, six total rounds of biopanning were performed. for each round of biopanning, monomeric deln cp and cp e peptides were alternately used as antigens. the n terminus of cp plays a key role in the assembly of the vlp structure. thus, deletion of the n-terminal region exposes the cp e epitope (del cp). after biopanning, the reactivity of individual clones to cp e peptide, deln cp, and pcv vlps was tested by phage enzyme immunoassay. a clone was selected, and its recombinant scfv-c k fusion protein prepared using a mammalian expression system. the recombinant scfv-c k fusion protein reacted to cp e peptide and deln cp, but not to vlps (fig. b) . in immunoblot analysis, the recombinant scfv-c k fusion protein was reactive not only to the cp e peptide and deln cp, but also to vlps, because the cp e epitope on vlp was exposed by the denaturation that occurs during sample preparation (fig. c) . although the cp e segment is too short to harbor more than one antigenic determinant, we further confirmed a number of antigenic determinants using the polyclonal sera of pigs infected with pcv . in a competition enzyme immunoassay employing a microtiter plate coated with bsa-conjugated cp e peptide and hrp-conjugated anti-porcine igg antibody, the recombinant anti-cp e antibody almost completely inhibited the binding of naturally occurring anti-cp e antibody to the peptide in a pcv -infected pig's sera (fig. ) . from these results, we concluded that there is only one antigenic determinant in cp e residues. the relative amount of pcv antigen in two commercially available vaccines was determined in an enzyme immunoassay, using purified vlp and the recombinant deln cp protein. the amount of coated antigen was then determined by anti-pcv polyclonal antibody. because the quantity of bound antibody was not statistically different between the vaccines, we concluded that they contained similar amounts of the recombinant cp (fig. a) . we then determined the relative amount of cp e epitope in these two vaccines with an enzyme immunoassay employing anti-cp e antibody (fig. b) . because we found a higher amount of anti-cp e antibody bound to vaccine a-coated wells than in vaccine b-coated wells, we concluded that vaccine a contained a higher amount of cp e epitope. to monitor the humoral response, we collected sera from guinea pigs (n ¼ /group) that were immunized with vaccines. for control sera, we also immunized with vlps and the recombinant deln cp in parallel. to confirm successful vaccination, we performed ifa as described previously using pcv -infected pk- cells [ , ] . there was no statistical difference between the antibody titer of sera obtained from animals immunized with the two vaccines. as expected, the sera of animals immunized with deln cp showed a statistically lower antibody titer compared to other groups (p < . ) (fig. c) . in an enzyme immunoassay employing a microtiter plate coated with bsa-conjugated cp e peptide, sera from animals immunized with vaccine a had a significantly higher antibody titer than vaccine b-vaccinated animals (p < . ) (fig. d) . additionally, the sera of deln cp-immunized animals had a significantly higher antibody titer than those of vlp-immunized animals (p < . ). then we measured and compared the neutralizing activity of sera in an in vitro infection experiment employing pcv virus and pk- cells. the sera from vaccine b-immunized animals had a significantly higher neutralizing antibody titer than sera from vaccine a-immunized animals (p < . ) (fig. e) . also, the sera of vlp-immunized animals showed significantly higher neutralizing activity than sera of vlp-immunized animals (p < . ). since the emergence of pcv , there has been considerable effort to produce an effective vaccine [ ] . for some time, the main hurdle was efficient overexpression of cp [ , ] . later, it was found that the immunodominant cp e epitope of pcv is buried during viral capsid assembly and that the antibodies targeting this epitope are non-neutralizing, which is a common viral immune-evasion mechanism [ ] . pcv infection inevitably provokes exposure to the decoy epitope and therefore is less effective at generating a protective humoral response than immunization of the subunit vaccine with a minimal amount of exposed decoy epitope [ ] . researchers had predicted that incomplete vlps with an exposed immunodominant cp e epitope can be contaminated during the preparation of subunit vaccine and drive the humoral response to produce non-neutralizing antibodies, but could not quantify the exposed immunodominant cp e epitope in the subunit vaccines because of the lack of proper analytical tools [ e ]. to rectify this, we generated a monoclonal antibody specific to the immunodominant cp e epitope from an antibody library generated using chickens immunized with cp e peptide. this recombinant anti-cp e antibody preferentially reacted to deln cp with an exposed cp e epitope compared to vlp in an enzyme immunoassay (fig. b) . because the length of linear b-cell epitopes can vary widely from to amino acids [ ] , we confirmed that cp e peptides behave as a single epitope in a competition enzyme immunoassay. the recombinant cp e antibody almost completely inhibited the binding of pcv -infected pig sera to cp e peptide. we then used this antibody to quantify the relative amount of cp e epitope in two commercially available vaccines and found that vaccine a contained significantly more decoy epitope than vaccine b. after immunization, vaccine a induced a significantly higher antibody response to decoy epitope than vaccine b, which supported the finding that vaccine b induced a higher amount of neutralizing antibody. in summary, our research demonstrated that the amount of immunodominant decoy epitope present in pcv vaccine can be measured by an epitope-specific antibody. from the measured level of decoy epitope, we could predict the efficacy of a vaccine for the induction of neutralizing antibody titer. we believe that anti-cp e antibody can be used to monitor the varying quality of the subunit vaccine from batch to batch. fig. . recombinant anti-cp e antibody inhibited the binding of naturally occurring antibody. after pre-incubation with sera from the non-infected (a) and infected pigs (b) with anti-cp e antibody (n ¼ /group), the mixtures were added to a cp e peptide-coated microtiter plate. the amount of bound porcine antibody was measured by hrp-conjugated anti-porcine igg antibody. absorbance was measured at nm. the results are the means ± standard deviations from the experiments, which were conducted in triplicate using the sera from three pigs (p , p , and p in each group). the authors have declared that there is no conflict of interest. anti-immunology: evasion of the host immune system by bacterial and viral pathogens neutralizing antibodies generated during natural hiv- infection: good news for an hiv- vaccine? feline immunodeficiency virus (fiv) neutralization: a review immune control and failure in hcv infectionetipping the balance xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines critical epitopes in the nucleocapsid protein of sfts virus recognized by a panel of sfts patients derived human monoclonal antibodies signal peptide cleavage from gp of prrsv: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence the murine gammaherpesvirus- gp acts as an immunogenic decoy to limit virion neutralization antibody recognition of porcine circovirus type capsid protein epitopes after vaccination, infection, and disease replacing the decoy epitope of pcv b capsid protein with a protective epitope enhances efficacy of pcv b vaccine structural insights into the 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(c) anti-pcv antibody in the immunized sera was determined by an indirect fluorescence assay using pcv -infected pk- cells (n ¼ /group). (d) the amount of anti-cp e antibody in the immunized sera was measured in an enzyme immunoassay using a cp e peptide-coated microtiter plate. (e) neutralizing activity of the immunized sera was measured with a virus neutralization assay. results are the means ± standard deviations virus-like particles in vaccine development antibody responses following vaccination versus infection in a porcine circovirus-type (pcv ) disease model show distinct differences in virus neutralization and epitope recognition efficient production of type porcine circovirus-like particles by a recombinant baculovirus characterization of porcine circovirus type (pcv ) capsid particle assembly and its application to virus-like particle vaccine development expression of porcine circovirus orf gene requires codon optimized e. coli cells barbas rd, methods for the generation of chicken monoclonal antibody fragments by phage display phage display: a laboratory manual a point mutation in the heavy chain complementarity-determining region (hcdr ) significantly enhances the specificity of an anti-ros antibody an antibody reactive to the gly -lys epitope of nt-probnp exhibits o-glycosylation-independent binding chicken scfvs with an artificial cysteine for site-directed conjugation western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfateepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein a evaluation of commercial polyclonal-and monoclonal-antibody-based immunohistochemical tests for genotypes of porcine circovirus type and comparison with in-situ hybridization assays comparative efficacy of experimental inactivated and live-attenuated chimeric porcine circovirus (pcv) - b vaccines derived from pcv and pcv b isolates originated in china production, preliminary characterisation and applications of monoclonal antibodies to porcine circovirus characterization of immune response of young pigs to porcine circovirus type infection global status of porcine circovirus type and its associated diseases in sub-saharan africa enhanced production of porcine circovirus type (pcv ) virus-like particles in sf cells by translational enhancers protection of swine against post-weaning multisystemic wasting syndrome (pmws) by porcine circovirus type (pcv ) proteins immunogenicity of empty capsids of porcine circovius type produced in insect cells immunity conferred by an experimental vaccine based on the recombinant pcv cap protein expressed in trichoplusia ni-larvae predicting flexible length linear b-cell epitopes we would like to thank tae-eun kim for the technical assistance and taeyeub lee for structural modeling of pcv using pymol . . transparency document related to this article can be found online at https://doi.org/ . /j.bbrc. . . . key: cord- -sr j z c authors: mersmann, sophia f.; johns, emma; yong, tracer; mcewan, will a.; james, leo c.; cohen, edward a.k.; grove, joe title: learning to count: determining the stoichiometry of bio-molecular complexes using fluorescence microscopy and statistical modelling date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: sr j z c cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. various techniques can provide a qualitative survey of which components are found in a given complex. however, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. we have devised a system in which a given biomolecule is differentially labelled with spectrally distinct fluorescent dyes (label a or b), which are then mixed such that b-labelled molecules are vastly outnumbered by those with label a. complexes, containing this component, are then simply scored as either being positive or negative for label b. the frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. we demonstrate this method using complexes of adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology. the statistical models used in our analysis are available here: https://github.com/sophiamersmann/molecular-counting, the raw data used for molecular counting can be found here: . /zenodo. . all cellular processes are driven by coordinated networks of dynamically interacting molecular partners. to successfully function, these components typically need to be assembled into specific complexes or clusters. for example, receptor signalling generally requires the co-location of various sensory, regulatory and stimulatory partners; the precise makeup of these assemblies can tune the nature of the signal and resultant physiological output. molecular cell biology research has, thus far, largely focused on determining the identity of the components found in a given complex. however, it is becoming clear that the quantity of any given component is also vitally important. quantifying the number of molecules, or stoichiometry, within an assembly can be used to understand its ultrastructure and, ultimately, to create complete molecular models of entire cellular structures, as has been demonstrated for hiv capsids and the neurological synapse (briggs et al., ; wilhelm et al., ) . there are various approaches to investigate the number of molecules within a given complex; for example calibrated biochemical analysis or cryo-electron microscopy (cryo-em). however, such methods pose practical and/or technological barriers and, by their very nature, obscure heterogeneity within the sample. single molecule localisation microscopy (smlm) modalities, such as storm and palm, are potentially powerful techniques for counting (lee et al., ; coltharp et al., ; fricke et al., ; veatch et al., ; stein et al., ) . nonetheless, these approaches typically require detailed a priori knowledge of the experimental system (for instance, a thorough evaluation of the 'blinking' characteristics of the fluorophores (patel et al., ) ) and/or a well understood reference sample with which to calibrate the measurement (thevathasan et al., ) . these steps need to be performed independently for each different experimental model and microscope set up; this creates a high barrier to implementation and can make these methods vulnerable to experimental variation and artefacts. here we outline an alternative, and potentially complementary, approach that combines (non-smlm) fluorescent microscopy and statistical modelling to extract estimates of molecular numbers within a complex. the method requires differential binary labelling of a constituent (i.e. a protein of interest is labelled with fluorescent dye a or b); by appropriate mixing of the labelled components, any individual molecular complex can be simply scored as being positive or negative for a given label. the frequency of positive complexes is then analysed by statistical modelling to extract estimates of stoichiometry. this approach is simple and requires minimal calibration or a priori understanding of the experimental system. we demonstrate the feasibility and accuracy of this approach by studying the stoichiometry of virus-antibody complexes. adenovirus (adv) is a non-enveloped dna virus; its genome is enclosed within a proteinous shell, called a capsid (nemerow et al., ) . the major adv capsid protein is hexon; this assembles into trimeric subunits, that are hexagonal in shape, which in turn arrange to form an icosahedron with triangular faces (a molecular cartoon of the adv particle is provided in figure a ). the adv particle has vertices, each of which are occupied by a pentameric subunit (formed of the penton base protein) and a receptor binding 'spike' (formed of the fibre protein). antibodies (ab) that bind sites such as the spike can directly neutralise adv by blocking receptor interactions, therefore preventing the virus particle from entering the cell. however, antibodies targeting the hexon protein (which makes up the majority of the capsid) do not necessarily interfere with the mechanics of virus entry (fender et al., ; wohlfart et al., ) . nonetheless, anti-hexon antibodies can prevent virus infection by recruiting the intracellular antibody-sensor trim , which targets the virus for degradation and activates cell-intrinsic immune responses (keeble et al., ; mallery et al., ) . anti-hexon monoclonal antibody c inhibits adv infection via this mechanism and is used as a prototypical system to investigate trim . the stoichiometries of antibody and trim recruitment to incoming virus particles remain unclear and are likely to be a determinant of the nature of the resulting cellular response. previous studies, using alternative techniques, have provided estimates of the stoichiometry of adv- c complexes. each adv particle possesses identical hexon proteins, each of these represents a potential binding site for c . however, the hexon subunits are assembled as trimers, and are arranged in a specific geometry (as described above). moreover, antibodie are bivalent, therefore each c molecule has two hexon binding pockets. consequently, it is highly unlikely that each hexon molecule will be occupied by a single c molecule (i.e. antibodies per virus particle). analysis by cryo-em, immuno-gold em staining and calibrated fluorescence measurements suggest a true maximum stoichiometry of - antibodies per particle (mcewan et al., ; varghese et al., ) ; this maximum is likely determined by the limits to antibody binding and packing enforced by the geometry of the particle. we have applied our counting method to the adv- c complex and generated estimates that are in good agreement with these previous studies, therefore validating our approach. strategy. we used differential binary labelling and statistical modelling to extract estimates of stoichiometry, our strategy is outlined in figure ; note that this approach can be generalised to apply to many other multi-component systems (i.e. how many protein x are found in assembly y?). the hidden truth is the number of antibodies bound to a virus particle; the ab:virus stoichiometry is expected to increase with antibody concentration until it reaches a saturation point where a. the ground truth: the number of antibody molecules (k) per virus particle increases with antibody concentration up to a saturation point (k = nsat). b. extracting truth from data: adv particles (labelled with a green fluorescent dye) are incubated with a defined mixture of two batches of antibody; one batch has received fluorescent label a (magenta), whilst the second has received label b (blue). when viewed by microscopy, every virus particle has received at least one molecule of ab a , whereas only a minority have received any ab b and can be scored in a binary fashion. note, antibody molecules are not drawn to scale the maximum number of abs are bound ( figure a .). in our method ( figure b ), both components (virus and antibody) are fluorescently labelled, however, two separate batches of, the otherwise identical, antibody are given spectrally distinct dyes (resulting in ab a or ab b ). mixing of the differentially labelled antibody batches at appropriate proportions results in only a minority of virus particles receiving a particular fluorescent dye (b in the example cartoon). therefore, when imaged, we detect three distinct fluorescent signals: each virus particle can be identified by its reference dye (green in the cartoon example), every virus particle has also received antibodies with dye a (magenta), however, very few particles are positive for dye b (blue) and can be scored as positive or negative in a binary fashion. the frequency of virus particles that are positive for ab b is a function of the a:b mixing proportion and the stoichiometry of ab:virus interaction; this relationship between the data and the hidden truth can be modelled. consider a single virus to be capable of binding n sat antibodies at saturation. under the assumption that antibodies bind to the same virus independently from each other, k, the total number of (labelled and unlabelled) antibodies bound to a virus, can be modelled as a binomial random variable where p is the probability of an antibody binding to a particular binding site of the virus. if n sat and p are known, then the expected number of antibodies bound to a single virus is simply e[k] = n sat · p. however, in most cases n sat is not known and p cannot be expressed easily since it depends not only on the antibody concentration used but also the composition of the virus particle and the geometry of interaction, which can be difficult to obtain. to extrapolate an antibody count it is, therefore, necessary to estimate both, n sat and p. as described above, our experimental design utilizes antibody labelled with spectrally distinct dyes allowing binary scoring of individual virus particles as positive if they interact with at least one ab b molecule ( figure ). here, we describe this state as being a bernoulli random variable s that takes the value if the virus is in the positive state, and if it is in the negative state, i.e. where q is the probability a virus interacts with at least one labelled antibody. since q = p (s = ) = − p (s = ), we can derive a closed form for q by finding an expression for the probability of a virus not being complexed with any labelled antibody p (s = ). to this end, we simply sum over all possible virusantibody configurations under the constraint of all antibodies being unlabelled, i.e. a virus could bind zero, one, two, ... up to n sat unlabelled antibodies. the marginal probability of a virus being in a negative state is thus given by where p (s = |k = k) is the probability that given the virus binds to k antibodies, exactly zero of them are labelled. the conditional distribution of s given k = k is itself binomial, namely where f l is the proportion of antibodies that are fluorescently labelled. therefore and combining with the probability mass function of k, namely gives a closed form for q directly follows as note that here p (s = ) is expressed in terms of p and n sat , and will hereafter be referred to as p (s = ; θ), where θ = (p, n sat ). consider a single experiment (performed at a specific antibody concentration) to describe v viruses with states s = s , ..., s v where v + of these states are positive, i.e. v + viruses have been observed to interact with at least one labelled antibody. assuming independence among viruses, the likelihood of θ is then where q is as given in ( ). we are then interested in the posterior distribution of θ given by the central result of bayesian statistics, where π(θ) is a suitable prior for the unknown parameters θ. in most applications, more than one experiment is performed; consider m experiments to be conducted at varying antibody concentrations c , ..., c m , producing m sets of virus state observations s = {s , ..., s m }. parameter inference using the described single-experiment model would entail building m independent models, each estimating p j and n sat,j for experiment j. while estimating concentration-specific antibody binding probabilities is desired, inferring multiple n sat values is unintuitive since n sat is fixed for a particular virus-antibody pair, i.e. n sat should be common to all experiments regardless of the antibody concentration used. it is, therefore, preferable to develop a general model accounting for multiple experiments that estimates all p , ..., p m simultaneously while yielding only a single n sat estimate. such a general model contains a single likelihood l(θ; s), where θ = (p , ..., p m , n sat ). this can be expressed as where l j (θ j ; s j ) is the likelihood function for θ j = (p j , n sat ) as defined in ( ). hence, m + unknown parameters are estimated; a probability p j specific to each concentration c j for j = , ..., m, and crucially a single n sat shared over all experiments. to sample from the posterior distributions markov chain monte carlo (mcmc) is used, specifically the metropolis-hasting algorithm using pymc (patil et al., ) . no prior knowledge is incorporated by imposing a beta distribution beta( , ) on all p j , j = , ..., m, and a uniform distribution with a sufficiently large upper bound, uniform( , ), on n sat . the convergence of mcmc is checked by visual inspection of trace and autocorrelation plots for each parameter. model verification via simulations. the proposed method makes use of experimental parameters including the proportion of antibodies labelled (f l ) and the number of viruses sampled (v ). formally, these are not required to comply with specific bounds. however, certain configurations of an experimental set-up are not expected to yield data that lead to a sensible model. labelling almost all antibodies (i.e. f l close to ), for example, would result in a data set with low information content. to explore how different experimental design choices impact the model's ability to reliably estimate parameters, we analysed simulated data that assumed a range of possible experimental settings. we simulated a single experiment at a time and assumed the number of antibodies bound at saturation to be known. for this we used an upper limit estimate of adv- c stoichiometry that we previously derived from an alternative method, n sat = (mcewan et al., ) . in each simulation, the binding probability p of an antibody is thus the only parameter estimated. we considered a range of possible experimental settings by varying the total number of viruses sampled in an experiment (from to ) and the proportion of antibodies labelled (from . to . ). for an assumed true antibody binding probability p ∈ [ . , . ], data is simulated by drawing v virus states from s ∼ ber(q) with q as described in ( ). the antibody binding probability was then blindly estimated using mcmc and convergence checked using the gelman-rubin statistic (gelman and rubin, ) . a total of simulations were carried out that assess the model's ability to reliably estimate p in various experimental settings. as expected, high proportions of labelled antibodies produced data of low information content, reflected in the model's inability to accurately estimate p, even if the number of viruses used in an experiment was high ( fig. a) . by contrast, if f l is less than or equal to . , p was estimated with low bias and low variance ( fig. a) . simulations also suggested that for low f l , as a rule of thumb, at least viruses per experiment should be sampled (fig. b-c) . however, if the proportion of antibodies labelled is . (or higher), then the proposed model failed to produce a reasonable estimate of p for most underlying true values; higher number of viruses seemed to compensate this effect to some extent (fig. d ). in summary, these simulations put empirical bounds on experimental parameters and show that, if compliant, the method yields sensible estimates. experimental setup. successful implementation of our strategy requires sensitive and unambiguous measurements of individual virus-antibody complexes. we achieved this by immobilising adv particles onto coverslips for analysis by total internal reflection fluorescence microscopy (tirf-m; a detailed description of experimental methodology is provided in the supplementary information). prior to immobilisation, purified adv particles were directly labelled with alexa fluor ; when observed by tirf-m (figure bi ) they appear as monodisperse diffraction limited spots (the particle being ∼ nm in diameter; figure a ). to further verify that each object is a single virus particle we used srrf analysis (super-resolution by radial fluctuations (gustafsson et al., ) ); for each object we resolved a single maxima of fluorescence that was ∼ nm in diameter (figure bii and iii), consistent with the ultrastructure of adv particles ( figure a) . immobilised adv particles were incubated with mono- clonal antibody c conjugated to alexa fluor dye, and imaged by tirf-m. each adv particle was positive for antibody, indicating the assembly of virus-antibody complexes ( figure c ). moreover, individual adv- c complexes could be analysed in a quantitative manner over a > fold range in antibody concentration ( figure d ). we automated this process to allow measurements of whole populations of virus particles at varying concentrations of antibody ( figure e & f). c signal intensity was proportional to antibody concentration and reached a plateau at high values; this indicates increasing virus-antibody stoichiometries up to a saturation point at which maximum antibody binding is achieved (as outlined in figure a) . moreover, the populations of virus particles were quite homogenous in c signal; this suggests a relative uniformity of assembly. in conclusion, we were able to quantitatively analyse the assembly of individual virus-antibody complexes. to achieve differential labelling a second batch of c was directly conjugated with biotin; this served as the 'b' batch of antibody to be mixed with the c 'a' batch ( figure ) . it may be possible that conjugation with either biotin or alexa fluor has unexpected detrimental effects on antibody binding; therefore, to have confidence in our binary labelling system we needed to demonstrate fair competition between our differentially labelled antibody batches. to test this we incubated immobilised adv particles with a high concentration of antibody ( µg/ml) composed of different proportions of c or c biotin (e.g. . : . , . : . ). we then monitored the c fluorescence signal under each condition. as the proportion of c dropped we measured a stepwise reduction in fluorescence signal (figure a ). if both batches of antibody possess equivalent binding to adv we would expect a linear relationship between the proportion c and fluorescent signal. indeed, when normalised for units, we observed a near perfect linear relationship ( figure b , slope = . , r = . ); indicating a fair competition in binding between our a and b batches of antibody. as depicted in figure b , our approach requires detection of single antibody molecules (of b batch) within individual virus-antibody complexes. to explore this we incubated immobilised adv particles with µg/ml c spiked with % c biotin (i.e. f l . : . ). molecules of c biotin were detected using streptavidin (an ultra-high affinity biotin binding protein) conjugated to a quantum dot (qdot ). the photostability of quantum dots permits signal accumulation over prolonged exposure times (resch-genger et al., ; algar et al., ) , therefore increasing the sensitivity of detection. analysis by tirf-m revealed that whilst every particle was positive for c only a subset possessed c biotin -qdot signal ( figure c ); this suggests a population of adv particles receiving one, or very few, c biotin antibody molecules (as outline in figure b ). automated analysis revealed well-separated populations of adv particles that were positive or negative for c biotin ( figure d ). note that all particles were positive for c . these data are consistent with the assembly of virus-antibody complexes in which the vast majority of antibody molecules are from batch a ( c ), but a subset of complexes contain ∼ molecules of batch b antibody ( c biotin ); the proportion of c biotin positive particles will serve as the output data for statistical modelling. we have demonstrated quantitative analysis of c interaction with individual adv particles ( figure ) ; we have confirmed that differential labelling of antibody does not bias binding ( figure a & b) ; and that we could detect single molecules of c biotin allowing discrimination of positive and negative adv- c complexes ( figure c & d). therefore, we have satisfied the necessary technical requirements to implement the strategy outlined in figure . we proceeded with a series of experiments to generate data for statistical modelling and stoichiometric estimates. to achieve this we performed four independent overlapping titrations over a > fold range in antibody concentration ( . - µg/ml). guided by the simulations in figure , we explored a range of c biotin proportions, from . - . ( . - % ), and, where possible, collected > particles per sample (the average number of particles collected was > ). the proportion of positive particles was assessed for each sample, details of data analysis are provided in the supplementary information. supplementary figure provides representative data: scatter plots display adv , c and c biotin intensities for control samples (treated with unmixed c biotin or c ) and four representative test samples incubated with a range of antibody concentration, containing . c biotin . bar charts provide summary statistics for each channel: the particles have uniform adv reference signal, whereas the c fluorescence decreases with antibody concentration; likewise, the proportion of c biotin positive particles decreases with concentration. this data are consistent with the expected concentration-dependent stepwise reduction in virus-antibody stoichiometry. the four experiments generated sets of binary scores (supplementary figure and table ) which were integrated in to our statistical model, as outlined in the methods. this generated posterior distributions of p (probability of an antibody binding site being occupied) for each sample (supplementary figure a ) and an estimated n sat (the maximum number of antibodies that can bind per particle) of (maximum a posteriori (map) estimate; ci (credible interval) [ , ]; supplementary figure b ). absolute antibody numbers for each sample can be derived by multiplying the map estimate of each posterior distribution (for p) by n sat (supplementary figure c) . figure a displays the mean number of bound antibody at increasing c concentrations; adv- c stoichiometries range from to across the titration of antibody. for any given sample, alongside the proportion of particles that are positive for c biotin , the experimental setup provides fluorescent intensity values of c in each adv-ab complex (supplementary figure ) ; this provides an internal reference for adv- c interaction, therefore, the stoichiometric estimates should correlate with their experimentally-matched fluorescent intensity values. figure b demonstrates a near perfect linear correlation between stoichiometry and fluorescence intensities for an example experiment; this would suggest that our statistical modelling faithfully reports the relationship between antibody concentration and adv binding occupancy. the stoichiometric estimates generated by our method derive from an ensemble measurment and, therefore, represent the antibody interactions of the average virus particle; this obscures heterogeneity within the population. however, given the excellent agreement between the estimated antibody counts and c intensity values ( figure b ) we used the stoichiometric estimates to calibrate the c signals, therefore allowing us to infer population heterogeneity. to achieve this, for any given sample we matched the median c fluorescence intensity to its associated stoichiometic estimate (generated by the model); extrapolating from this we then converted the c fluorescence values to inferred antibody counts for individual virus particles. figure c provides histograms and frequency distributions of inferred antibody counts for a range of concentrations. being slightly skewed to the right the frequency data was best fitted using a log-normal distribution, this is particularly apparent at higher antibody concentrations. this would suggest that a significant proportion of virus particles are binding more antibodies than the average particle. however, no particles bound greater than antibody molecules. we provide an interpretation of these observations in the discussion. in summary we have used statistical modelling to derive stoichiometric estimates of adv- c complexes. this suggests that the most probable antibody binding maximum is molecules ( % ci - ). however, using stoichiometric estimates to calibrate fluorescent data revealed population heterogeneity with a small proportion of virus particles binding ∼ antibody molecules. notably, these values are in excellent agreement with previous estimates that we, and others, have derived using alternative methods (mcewan et al., ; varghese et al., ). various methods permit the investigation of molecular stoichiometries within biological assemblies but technical limitations often make it difficult to obtain reliable estimates. for example smlm, by its very nature, identifies single molecules and, if properly calibrated, can deliver accurate stoichiometries; however, successful counting by smlm requires a very detailed understanding of the photochemical behaviour of the chosen fluorescent dyes. here we outline a robust, and relatively facile, experimental framework for extracting accurate molecular counts using (non-smlm) fluorescent microscopy and statistical modelling labelling of a component of interest with spectrally distinct fluorescent dyes (label a or b), and mixing them at defined ratios such that b-labelled molecules are in the minority, allowed assembled complexes to be simply identified as being either positive or negative. the frequency of positive complexes was then related to the underlying stoichiometry of interaction through statistical modelling. by creating a scheme in which complexes need only be qualitatively scored for a particular label, our method negates the necessity for carefully calibrated measurements and an a priori understanding of the system. the only requirement is that the chosen label is clearly discerned from background; this is easily achievable with bright/stable fluorescent dyes and relatively inexpensive cameras; in this case we used quantum dots for sensitive detection, but many standard fluorescent dyes should also suffice. an obvious limitation is that our method relies on an ensemble measurement and obscures heterogeneity within the population of complexes. however, this information remains accessible via the a-label fluorescent signals measured from each complex. consequently, the ensemble-based stoichiometric estimates of the average complex can be used to calibrate these fluorescent signals and infer approximate molecular counts for individual complexes, therefore, restoring heterogeneity. we were able to make robust measurements of adv- c interactions by fluorescence microscopy and successfully implemented the differential labelling strategy. we performed multiple independent measurements at various antibody concentrations to derive molecular counts for adv- c complexes. our analysis estimated that the average adv particle interacts with a maximum of c antibody molecules. moreover, through examination of population heterogeneity we revealed that a small proportion of adv particles may bind up to antibody molecules. these data are can be reconciled with a molecular model of adv- c complexes. adv particles possess identical hexon subunits, each providing a potential binding site for c , however, previous estimates indicate an absolute maximum of antibody molecules per virion. this would suggest that particle geometry places packing constraints on the arrangement of antibody molecules. cryo-em analyses indicates a complex and heterogeneous interaction network in which particle geometry creates potential antibody clashes and, therefore, prevents binding to every site simultaneously (varghese et al., ) . whilst there are consistently five c molecules at each of the twelve vertices of the adv particle, additional antibody interactions occur through heterogeneous packing across the surface; the pattern of which is likely dictated by the random order in which binding sites become occupied on any given virus particle. as a consequence with optimal antibody packing there is likely to be an absolute maximum binding occupancy of ∼ molecules. our data indicate that the average particle binds fewer molecules ( ) than the threshold dictated by purely geometric limitations. this would suggest that the majority of particles do not achieve optimal antibody packing and saturate at lower occupancies. this model also offers explanation to our observation that no particles bind greater than ∼ molecules ( figure c ). this interpretation of our data exposes another potential flaw in our approach. our modelling strategy assumes that components bind independently, but in this test case the geometry of adv particles create clashes between adjacent c molecules such that complete saturation is not possible. consequently, antibody binding events can be influenced by prior antibody interactions and, therefore, are not independent. although this may have introduced inaccuracies in our estimates, the molecular counts derived from our approach mean stoichiometric estimates at increasing concentrations of c , error bars indicate standard error of the mean; data fitted with a binding curve, r = . (graphpad, prism). b. stoichiometric estimates were compared to c fluorescent values from an individual experiment; data fitted with a linear regression, r = . (graphpad, prism). c. stoichiometric estimates were used to calibrate fluorescent intensity values allowing inference of heterogeneity. histograms display the frequency of inferred antibody counts as a proportion of total particles. the frequency data were fitted with a log-normal distribution, r values were all > . (graphpad, prism). are in good agreement with previous values. moreover, we maintain that the assumption of independent binding is appropriate for a generalisable method that can be applied to other biological assemblies. for example, within virology, we intend on extending this method to investigate the molecular composition and antibody-mediated neutralisation of enveloped viruses such as human immunodeficiency virus, hepatitis c virus and sars-coronavirus- . beyond the confines of virology, our method could be applied to a variety of other biological assemblies of various scales, for example: bacteria; purified cellular organelles; cellular vesicles, such as exosomes; and supramolecular complexes such as ribosomes or inflammasomes. in conclusion, we have developed a novel and robust method for counting components within biomolecular complexes. this approach has provided accurate counts for a previously characterised system and could be applied in a variety of other contexts. moreover, we expect this system could be integrated with other complementary methods to enhance quantitative analysis; for example differential labelling and statistical modelling may provide a means of internally calibrating smlm-based counting schemes. semiconductor quantum dots in bioanalysis: crossing the valley of death the stoichiometry of gag protein in hiv- accurate construction of photoactivated localization microscopy (palm) images for quantitative measurements one, two or three? probing the stoichiometry of membrane proteins by single-molecule localization microscopy fast live-cell conventional fluorophore nanoscopy with imagej through super-resolution radial fluctuations counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (palm) antibodies mediate intracellular immunity through tripartite motif-containing (trim ) regulation of virus neutralization and the persistent fraction by trim structure of human adenovirus a hidden markov model approach to characterizing the photoswitching behavior of fluorophores pymc: bayesian stochastic modelling in python quantum dots versus organic dyes as fluorescent labels toward absolute molecular numbers in dna-paint nuclear pores as versatile reference standards for quantitative superresolution microscopy postentry neutralization of adenovirus type by an antihexon antibody correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting interaction between hela cells and adenovirus type virions neutralized by different antisera we would like to thank ricardo henriques for initiating some important conversations and providing technical assistance during image analysis. we would like to thanks niall adams for his help in supervising sfm during the development of the statistical methodology. molecular cartoon images were taken from the wellcome key: cord- -yc xtw s authors: lappin, michael r. title: microbiology and infectious disease date: - - journal: small animal clinical diagnosis by laboratory methods doi: . /b - - - - . - sha: doc_id: cord_uid: yc xtw s nan infectious agents may be identified directly by cytologic analysis, histopathologic evaluation, culture, viral isolation, antigen detection, or nucleic acid amplification techniques. polymerase chain reaction (pcr) assays are the most commonly used nucleic acid amplification techniques in small animal infectious diseases. detection of antibodies against infectious agents provides indirect evidence of prior exposure or current infection. this chapter describes methods for obtaining specimens, outlines currently used testing procedures for some of the more common infectious diseases, and discusses interpretation of results from the various procedures and tests.* infectious diseases should be on the differential list for most problems, especially those with fever or other signs of inflammation. history, physical examination findings, and routine clinical pathologic testing are seldom pathognomonic for an infectious cause, but they help the clinician rank differential diagnoses and develop a logical diagnostic plan. history can increase the degree of suspicion for infectious disease. exposure to other infected animals or contaminated fomites is important for agents with direct transmission, such as those inducing respiratory disease (e.g., feline herpesvirus , canine bordetellosis) or gastroenteritis (e.g., canine and feline giardiasis, canine and feline parvovirus infection). potential exposure to vectors (e.g., mosquitoes for dirofilariasis; ticks for lyme borreliosis [ixodes spp.], ehrlichiosis [rhipicephalus sanguineus], rickettsia rickettsii [rocky mountain spotted fever; dermacentor spp.], and babesia canis [r. sanguineus]) or appropriate travel history (e.g., coccidioidomycosis in the southwest; blastomycosis in the mississippi, missouri, and ohio river valleys) can also suggest infectious disease. vaccination history, deworming history, and determination of whether other animals or people in the environment are affected can aid in ranking infectious diseases on a differential diagnoses list. physical examination findings may suggest an infectious cause. infectious agents can induce fever. lymphadenomegaly as a result of reactive lymphoid hyperplasia can be infectious in origin. hepatosplenomegaly can be caused by immunologic stimulation induced by chronic intracellular infections (e.g., ehrlichiosis, brucellosis). endogenous uveitis commonly occurs after infections by feline immunodeficiency virus (fiv), feline infectious peritonitis (fip) virus, toxoplasmosis, and systemic mycoses. mucopurulent discharges can suggest primary or secondary bacterial infections. certain infectious diseases cause specific abnormalities such as dendritic ulcers (feline herpesvirus ), chorea myoclonus (canine distemper virus), or testicular swelling plus pain (canine brucellosis). finally, clinicopathologic abnormalities can suggest disease caused by infectious agents. neutrophilic leukocytosis, particularly if a left shift or degenerative neutrophils (see chapter ) are also present, is consistent with an infectious cause of disease. gram-negative sepsis is suggested by leukopenia with a degenerative left shift. monocytosis or lymphocytosis can be induced by persistent infection with a number of intracellular agents that result in persistent infection. examples include ehrlichiosis, toxoplasmosis, and bartonellosis. polyclonal (e.g., multiple infectious causes) or monoclonal (e.g., usually induced by neoplasia, rarely associated with canine ehrlichiosis) gammopathies may suggest chronic immune stimulation. neutrophils in aqueous humor, cerebrospinal fluid (csf), synovial fluid, or urine may indicate inflammation induced by infectious agents. morulae of ehrlichia spp. are rare in the cytoplasm of mononuclear cells (ehrlichia canis), neutrophils (ehrlichia ewingii; anaplasma phagocytophila [previously e. equi]), or platelets (ehrlichia platys). mycoplasma haemofelis (cats only), "candidatus m. haemominutum" (cats only), "candidatus m. turicensis" (cats only), m. haemocanis (dogs only), "candidatus m. haematoparvum" (dogs only), cytauxzoon felis (cats only), and babesia spp. sometimes will be identified cytologically on the surface or within canine or feline erythrocytes. for demonstration of cheyletiella spp., a piece of transparent adhesive tape is gently pressed against areas with crusts or dandruff and then placed on a microscope slide. next the hair is clipped, mineral oil is placed on the skin and on a microscope slide, and the skin is scraped using a blunt no. scalpel blade. for skin scrapings to look for demodex spp., the skin should be immobilized and mites expressed from follicles by pinching and scraping the extruded material. for scrapings to look for sarcoptes spp. or cheyletiella spp., the scraping is continued more superficially (inducing a mild capillary ooze) over a larger surface area. after transfer of the scraping, the microscope slide field is scanned at × for mites. for identification of dermatophytes, hairs are plucked from the periphery of a lesion, placed on a microscope slide, and covered with % to % potassium hydroxide to clear debris. the slide is then heated (not boiled) and examined under the × or × objective to search for hyphae, spores, conidia, budding yeasts, and fungusinduced damage (e.g., swollen or broken hair shafts). the × objective is used to identify arthrospores (dense aggregates of spherical structures that may cover the hair shaft [ figure - ]). failure to find arthrospores does not rule out dermatomycosis. culture is more sensitive for diagnosis of dermatophytosis (see fungal culture). romanowsky-type stains (e.g., wright) are used in preference to wet mount preparations and ink when looking for fungi other than dermatophytes (see chapter ) . romanowsky-type stains are also useful in advantages • cytology is inexpensive and readily available and may allow rapid confirmation and identification of an infectious agent. it assists in establishing normal flora and contaminants versus infection (e.g., interpretation of relative numbers of bacteria and yeasts in the ear canal). cytologic examination also permits visualization of relative numbers of organisms at the time of collection (culture results may be misleading in terms of fast-or slow-growing bacteria). disadvantages • infectious agents cannot always be found (e.g., ehrlichiosis, haemoplasmosis, infections with numbers of organism that are below sensitivity level of cytology). sometimes a presumptive cytologic diagnosis must be confirmed by other methods (e.g., histopathology, culture, pcr assay), and cytology is of limited value in detecting viral inclusions except in brief viremic stages of canine distemper. see chapter for discussion of cytologic techniques and cytologic conclusions. discharges from animals with suspected bacterial disease should be placed on a microscope slide, air dried, fixed, and stained with both gram and romanowsky-type stains (see chapter ) . the examination is started on low power ( × magnification), with oil immersion ( ×) used for inspection of bacterial morphologic features (i.e., rods, cocci) and gram stain characteristics (i.e., gram-positive [blue] or gram-negative [pink] ). the primary disadvantage of gram staining is that gramnegative bacteria may be difficult to find because background material stains pink. it is easier to find bacteria (dark-blue stain) and easier to study morphologic detail of other cells (i.e., inflammatory cells) using romanowsky-type stains. gram staining may be variable; organisms in body fluids may stain differently from those grown on a blood agar plate. gram stain demonstrates the gram-positive, branching filaments of actinomyces spp. and nocardia spp. (see . acid-fast stains can be used for mycobacterium spp. and to help differentiate nocardia spp. (acid-fast) from actinomyces spp. some bacteria have characteristic morphologic features. large rod-form bacteria containing spores found on fecal cytology of dogs or cats with diarrhea suggest clostridium perfringens (figure - ) . bipolar-staining, gramnegative coccobacilli found in aspirates of inflamed cervical lymph nodes from cats in the southwest or west suggest yersinia pestis. short spirochetes found on fecal cytology of animals with diarrhea suggest but do not prove campylobacteriosis. spirochetes found on cytology of gastric mucosa of vomiting animals suggest helicobacteriosis. to demonstrate inclusion bodies in acute feline chlamydial conjunctivitis, conjunctival scrapings are obtained with a flat spatula, smeared on a slide, stained with romanowsky-type stains, and examined for intracytoplasmic aggregations of chlamydophila felis (previously chlamydia). been resistant to medications. remember: skin and mucosal surfaces have a resident microflora (box - ); therefore care must be taken to avoid contamination. advantages • culture and antimicrobial susceptibility usually allows the most effective treatment to be administered. disadvantages • culture requires time for agents to grow; also, some organisms are fastidious or have special culture requirements. other disadvantages are the expense and the ease of contaminating or making inactivate cultures, rendering results worthless. body cavities • the site of skin puncture should be prepared as for blood culture (see discussion in cardiovascular system). if pyothorax or peritonitis seems likely but fluid cannot be aspirated, lavage (see chapter ) is indicated. because mixed infections are common and pure anaerobic infections may occur, aerobic and anaerobic cultures should be performed. cardiovascular system • blood cultures are indicated in suspected bacterial endocarditis or septicemia. a large vein prepared surgically with sequential iodine and alcohol scrubs is used for three blood culture specimens obtained during a febrile episode over a -hour period in dogs with suspected endocarditis. culture of fewer than three specimens significantly decreases the chance of positive results. at least ml of blood is placed directly into a transport medium that will support the growth of aerobic and anaerobic bacteria, and it is incubated at ° c for hours. clotted blood or blood containing ethylenediaminetetraacetic acid (edta) or citrate are unacceptable because this decreases isolation of organisms (bartonella spp. are exceptions; they can be cultured from edta tubes; see bartonellosis sections). if a patient is critically ill and sepsis is suspected, three cultures should be obtained over to hours before antimicrobial therapy is instituted. because the urinary system is a common portal of entry for bacteria into the body, urine identifying yeasts such as blastomyces dermatitidis, histoplasma capsulatum, sporothrix schenckii, coccidioides immitis, or cryptococcus spp. (see figure - ) in exudates, csf, lymph node aspiration cytology, or transtracheal aspiration cytology. canine distemper virus inclusions in lymphocytes, neutrophils, or erythrocytes ( figure common indications • culture and antimicrobial susceptibility are indicated in most suspected bacterial diseases (box - ), especially when clinical syndromes have eye • conjunctival culture should be performed before topical anesthesia or application of fluorescein stain by rolling a moistened sterile swab over the conjunctiva. ocular paracentesis is necessary for intraocular culture. gastrointestinal tract • primary bacterial gastroenteritis occasionally occurs. salmonella spp., campylobacter spp., c. perfringens, and escherichia coli are agents that can be involved. these organisms can also be isolated from normal animals, however. salmonella spp. and campylobacter spp. can cause small or mixed bowel diarrhea; c. perfringens is usually associated with large bowel diarrhea. approximately to g of fresh feces should be submitted to the laboratory for optimal results. if delayed transport of feces to the laboratory is expected, the clinician should is often cultured in patients when the source of septicemia or bacterial endocarditis is unknown. central nervous system • bacterial infection of the central nervous system (cns) is uncommon. even when infection occurs, low numbers of organisms make cytology and culture low-yield procedures. if increased numbers of neutrophils and increased protein are detected in csf (see chapter ) , however, aerobic and anaerobic bacterial culture and antimicrobial susceptibility testing are indicated. csf samples should be placed in transport media and delivered to the laboratory as soon as possible. aerobic and anaerobic bacterial culture should be performed when bacterial infection of the cns is suspected. culture of the third fraction of an ejaculate (preferred) or prostatic massage is recommended for prostatic culture. culture of the second fraction of an ejaculate is recommended for testicular culture. culture of prostatic or testicular material retrieved by aspiration or biopsy can also be performed. prostatic massage and closed prostatic aspiration or biopsy should be avoided in dogs with suspected prostatic abscesses. obtaining distal urethral specimens for quantitative culture before and after ejaculation may help avoid confusion caused by urethral contamination. anaerobic culture of urine or prostatic fluid is rarely useful. integument and ear • in superficial pyoderma, hair is clipped from the surrounding area, but disinfection is not attempted. a pustule is ruptured with a sterile fine-gauge needle, and a swab of pus is cultured. in deep pyoderma, hair surrounding the lesion is clipped and the area is disinfected with an antiseptic. the lesion is squeezed to express exudate, which is collected on a swab. gloves should be worn. for culture of ears, a sterile otoscope cone is inserted to the level of the horizontal canal and the ear is swabbed through the cone. when middle-ear infection is suspected, the animal is anesthetized and material for culture is retrieved by myringotomy by penetration of the tympanum with a sterile csf needle placed through a sterile otoscope cone. musculoskeletal system • no normal flora exists in musculoskeletal tissues. dogs with radiographic evidence of discospondylitis should be evaluated for brucella canis and bartonella spp. infections (see diagnostic tests for select bacterial infections). intervertebral joints can be cultured after fluoroscopically guided aspiration or when decompressive spinal surgery is required. most cases of discospondylitis develop after hematogenous spread of bacteria from an extravertebral source. blood and urine are commonly cultured from patients with discospondylitis; staphylococcus spp. are commonly involved. dogs or cats with suppurative arthritis (with or without cytologic visualization of bacteria) should have the synovial fluid cultured for aerobes and mycoplasma spp. (see chapter ) . likelihood of positive culture results increases if the synovial fluid contains degenerative neutrophils. l-form bacteria usually cannot be grown from joint fluid via routine culture techniques. synovial biopsy for culture plus histopathologic evaluation for l-form bacteria is more sensitive than only culture of fluid. borrelia burgdorferi is almost never isolated by routine culture from joints of dogs with lyme disease. use of pcr assays on synovial fluid to amplify dna of mycoplasma spp., b. burgdorferi, a. phagocytophilum, and ehrlichia spp. may prove to be an effective way of documenting the presence of the organisms in the joints of affected animals, but objective data are lacking. in osteomyelitis, culture of fistulous tracts is less sensitive than culture of affected bone. culture for infectious myositis is seldom performed unless suspicion for an anaerobic infection (e.g., clostridium spp.) is based on foul odor, subcutaneous (sc) emphysema, or empyema. the clinician can better evaluate for other infectious myopathies (e.g., toxoplasmosis, leptospirosis) using serologic testing or pcr assays. respiratory system • lower airway specimens are best obtained by transtracheal aspiration or bronchoalveolar lavage during bronchoscopy. fine-needle pulmonary aspiration biopsy can be used but carries more risk (see chapter ) . bacteria can be isolated from the trachea in some clinically healthy dogs. these bacteria are probably transient; common isolates are listed in box - . because many organisms isolated from normal dogs have also been associated with lower respiratory tract inflammation, all transtracheal aspiration samples should be evaluated by culture, antimicrobial susceptibility, and cytology. with cytology, the clinician should look for squamous cells coated with bacteria (which indicate oropharyngeal contamination) (see figure - ). bacteria should not be considered significant unless accompanied by neutrophilic inflammation. mycoplasma spp. have been isolated in pure culture from lower airways of patients with clinical signs of respiratory disease. , culture for mycoplasma spp. should be performed on all transtracheal aspiration samples; these samples need to be transported to the laboratory in amies medium or modified stuart bacterial transport medium. mycoplasma spp. culture should be specifically requested. amplification of mycoplasma spp. dna from airway secretions by pcr assay can also be used to document infection. nasal specimens are best obtained from nasal lavage or core biopsy, or by passing a swab through a sterile otoscope cone (see chapter ) . the clinician can best obtain pharyngeal specimens using a guarded swab taken during pharyngoscopy. nasal and pharyngeal cultures can be difficult to interpret because of extensive normal flora in the nasal cavity and nasopharynx (see . for aerobic culture, no special transport medium is required if the swab remains moist and can be inoculated onto the culture medium within hours. swabs containing liquid or gel transport media are frequently used, a disk containing a fixed amount of antibiotic. the procedure is qualitative and allocates organisms to the sensitive (susceptible), intermediate (indeterminate), or resistant category. advantages • advantages of the disk diffusion test are its simplicity and suitability for most routine cultures, that it can be performed in the office, and its applicability for rapidly growing organisms (e.g., enterobacteriaceae, staphylococcus aureus). disadvantages • this test is not suitable for slowgrowing organisms and anaerobes. in addition, there is inaccuracy in predicting susceptibility of poorly diffusing antibiotics (e.g., polymyxins), and factors that influence the test (e.g., ph and thickness of the medium, concentration of organisms, incubation time) must be standardized. it is imperative that proper procedures be followed to avoid errors in diagnosis. artifacts • artifacts result from improper sample collection (i.e., wrong sample, contamination), improper sample transport, failure to notify the laboratory of suspected pathogens (e.g., salmonella spp., anaerobic bacteria, campylobacter spp., mycoplasma spp.), recent antibiotic treatment, and culture for a secondary rather than a slowgrowing primary pathogen (i.e., insufficient duration of culture). failure to grow fastidious anaerobes may be caused by short, seemingly insignificant exposure to oxygen or failure to use prereduced culture media. interpretation • recognizing normal flora (see box - ) is necessary for correct interpretation. preliminary identification is expected in to hours, and antibiotic sensitivity is reported in to hours. most aerobic and facultative organisms are identified within days; identification of anaerobic organisms or mycoplasma spp. may require an additional to days. bacterial pathogens commonly isolated from various body systems are listed in box - . the overlap between resident and pathogenic organisms should be noted. staphylococcus pseudintermedius is the major pathogen isolated from the skin of dogs with pyoderma. gramnegative organisms are likely to be contaminants in superficial pyoderma and secondary to s. pseudintermedius in deep pyoderma. primary bacterial rhinitis is rare in dogs and cats but can result from infection with bordetella bronchiseptica, mycoplasma spp., and chlamydophila felis (cats). primary bacterial pneumonia can result from b. bronchiseptica or mycoplasma spp., whereas other organisms are usually secondary to viral infections or aspiration. bacterial growth from urine obtained by cystocentesis is significant because the bladder is normally sterile. urine cultures, however, are best interpreted in conjunction with a urinalysis. if growth occurs despite absence of significant pyuria (see chapter ), sample contamination, improper sample transport, or diseases causing immune suppression (e.g., hyperadrenocorticism, diabetes mellitus, fiv infection) must be considered. in quantitative culture of urine obtained by catheterization or midstream voiding, greater than or equal to , colonies/ml is significant. lower concentrations may however. routine cultures can be safely stored in transport media at room temperature for up to hours. after this time, overgrowth is a potential problem because of various growth rates of different organisms. refrigerated, routine specimens can be stored in transport media for at least days. tissue samples can be refrigerated for up to days. fluids (e.g., urine) can be safely stored at room temperature for to hours, refrigerated for hours, and refrigerated in transport media for hours. quantitative culture is not accurate for fluids stored in transport media because of artifactual dilution. for anaerobic culture, fluid should be aspirated into a syringe, the needle capped with a rubber stopper, and the sample inoculated onto culture medium within minutes of collection. transport media that support the growth of anaerobic bacteria are available but are not ideal for all fastidious organisms. with these limitations, samples can be refrigerated for days in an appropriate transport medium. analysis • blood agar plates grow most routine bacterial pathogens. a biplate containing blood agar and macconkey agar is frequently used. the common anaerobic culture medium is thioglycolate. the decision to perform in-office testing instead of using a commercial laboratory is based on case load, available equipment, and expertise. sensitivity testing • sensitivity testing gives an in vitro estimation of suitability of a given concentration of an antimicrobial agent. two techniques are used: ( ) the dilution test and ( ) the disk diffusion test. dilution test • this test is quantitative and determines the least amount of antimicrobial agent needed to prevent growth of a microorganism (minimum inhibitory concentration [mic]). quantitative susceptibility testing is indicated when antimicrobial dosing schedules need to be monitored closely (e.g., gentamicin) or when disk test results are inapplicable, equivocal, or unreliable (e.g., slow-growing organisms, confirmation of susceptibility to polymyxins, confirmation of susceptibility or resistance to given doses of aminoglycosides). other indications include anaerobes and testing for synergism or antagonism between antimicrobials. advantages • the dilution test may be effective even though disk diffusion techniques suggest otherwise (e.g., antibiotics concentrated in urine). disadvantages • disadvantages of the dilution test include expense, inability to perform the test in the office, and need to determine if required concentrations of a certain antibiotic are feasible. ideally, blood concentrations of drugs should be more than times the mic and urine concentrations to times the mic. mic sensitivity for topically administered antimicrobials is seldom determined because these methods are based on blood or urine concentrations. disk diffusion test • this is the most widely used method in clinical practice (i.e., kirby-bauer technique). a zone of inhibition of bacterial growth is noted around these assays prove previous exposure to a bartonella spp., but as serologic cross-reaction occurs between b. henselae, b. clarridgeaie, and b. koehlerae, assays based on b. henselae antigens cannot differentiate between the agents. current bartonella spp. infection is proven by culture or pcr assay results. results of some pcr assays or genetic sequencing can be used to identify the species of bartonella involved with the infection. cats can be infected with more than one bartonella spp. concurrently. detection of local antibody production by the eye and documentation of bartonella spp. dna in aqueous humor has been used to document uveitis as a result of bartonellosis. (see appendix i for laboratories for infectious diseases.) up to % of healthy cats exposed to c. felis can be seropositive, and so antibody tests results alone cannot be used to prove clinical bartonellosis. between % and % of seronegative cats are bacteremic, and so antibody test results cannot be used to exclude bartonella spp. infection from the differential list. positive blood culture or pcr results prove current infection but do not document clinical illness. repeated bacteremia has been detected in experimentally inoculated and naturally infected cats; therefore a single negative blood culture or pcr result does not exclude infection. because of these findings, it is currently recommended to combine serologic test results with those of blood culture or pcr assay results when evaluating clinically ill cats for bartonellosis. (see appendix i for laboratories for infectious diseases.) if test results are positive and there is no better explanation for the cause of illness, treatment may be indicated. to date, there has been no proven clinical benefit to following bartonella spp. assay results after positive response to therapy. because serologic test results do not accurately correlate with presence of bacteremia and individual culture or pcr assay results can be falsely negative, there is no indication for testing healthy cats for bartonella spp. infection. , the centers for disease control and prevention recommends maintaining flea control and avoiding bites and scratches to avoid bartonellosis. healthy cats used for blood donors should be seronegative and culture or pcr negative and should be maintained on flea control products. occasional indications • dogs from endemic areas or with an appropriate travel history with unexplained myocarditis, endocarditis, granulomatous lymphadenitis, cutaneous vascular disease, hemolytic anemia, polyarthritis, idiopathic effusions, granulomatous meningoencephalitis, granulomatous rhinitis, or thrombocytopenia should be considered for bartonella vinsonii testing. , the full spectrum of b. henselae-associated illnesses in dogs has not been determined but appears to be similar to that for b. vinsonii. , based on seroprevalence studies, rural dogs with fleas or ticks are most likely to be exposed to bartonella spp. analysis, artifacts, and interpretation • circulating antibodies against b. vinsonii and b. henselae are most commonly detected by ifa. infection can be documented be significant in chronic infections or in females. in samples of prostatic fluid obtained by ejaculation, infection is diagnosed if the specimen contains greater than or equal to times more bacteria than the urethral sample. culture of prostatic aspirates may be more accurate. blood cultures can be difficult to interpret. falsepositive results are caused by contamination with normal cutaneous microflora, including corynebacterium spp., bacillus spp., coagulase-negative staphylococci, anaerobic diphtheroids, streptococci, and clostridium spp. isolation of the same organism from two or more cultures strongly suggests that it is pathogenic, whereas growth in only one culture is less certain unless it is a pathogenic bacterium unlikely to be a contaminant. csf and synovial fluid are normally sterile; any growth in an aseptically obtained sample is significant. for dermatophyte culture, hair is clipped from the lesion periphery; hair shafts are plucked with forceps and cultured on dermatophyte test medium (dtm) or derm duet. sc and deep fungal infections are best diagnosed by cytologic or histopathologic evaluation, with or without serology. if organisms cannot be identified, cutaneous lesions can be cultured, but these are rarely useful owing to overgrowth by resident bacteria and fungi. the lesion is prepared as for dermatophytes, and a swab is cultured onto sabouraud and mycose medium. systemic and sc fungi may require weeks' cultivation on sabouraud medium for growth to occur. bartonellosis, feline (bartonella henselae, b. clarridgeaie, b. koehlerae) occasional indications • to date, most cats with clinical bartonellosis have been infected with bartonella henselae. however, b. clarridgeaie, b. koehlerae, and b. quintana have been identified in some clinically ill cats. , bartonella henselae, b. clarridgeaie, and b. koehlerae are transmitted among cats by ctenocephalides felis and so cats with a history of flea infestation are more likely to be infected. most cats exposed to bartonella spp. maintain subclinical infections; however, fever, uveitis, lymphadenopathy, endocarditis, myocarditis, hematuria, and hyperglobulinemia have been documented convincingly in experimentally infected or naturally exposed cats. results have been equivocal for a link between bartonella spp. infection and gingivitis or stomatitis. bartonella testing may be indicated in cats with one or more of these clinical syndromes for which another explanation is not readily apparent. analysis, artifacts, and interpretation • circulating antibodies are detected by immunofluorescent antibody assay (ifa), enzyme-linked immunosorbent assay (elisa), and western blot immunoassay. , results of laboratories for infectious disease); however, clinical utility of this assay has not been documented. definitive diagnosis requires demonstration of the organism by culture, histopathologic evaluation of tissue, or pcr assay. presumptive diagnoses of clinical lyme disease in dogs can be based on appropriate clinical, historic, and laboratory evidence of disease combined with positive serologic testing and response to therapy. occasional indications • dogs with reproductive tract abnormalities, lymphadenomegaly, hyperglobulinemia, discospondylitis, or uveitis should be suspected of having brucellosis and be screened for antibodies against brucella canis. analysis, artifacts, and interpretation • circulating antibodies are most commonly detected in serum by rapid slide agglutination test (rsat), tube agglutination test (tat), agar gel immunodiffusion (agid), and elisa. , the rsat and tat are screening procedures; an rsat for point-of-care use is commercially available. both assays should be performed with -mercaptoethanol ( -me) to eliminate heterologous igm agglutinins responsible for most false-positive reactions. false-positive reactions in the -me tat may be the result of autoagglutination in hemolyzed samples. agid can be performed using cell wall antigens or cytoplasmic antigens. agid performed with cytoplasmic antigens is the most specific antibody assay; agid performed with cell wall antigens is the most sensitive. because of nonspecific precipitin reactions, positive results in agid with cell wall antigens are difficult to interpret. minimal time between infection and a positive test result varies with the test, but most infected dogs are seropositive in the -me tat and agid by week to after infection. -me tat titers from different laboratories cannot be meaningfully compared; however, a titer of : to : is generally suspicious, whereas a titer greater than or equal to : usually correlates with isolation of b. canis from blood culture. after cessation of bacteremia, -me tat titers rapidly decrease to less than : within a few weeks and remain low ( : to : ) for months or longer. in agid, antibodies to external antigens persist for a few weeks, whereas antibodies to internal (i.e., cytoplasmic) antigens persist up to months after cessation of bacteremia. although these animals are abacteremic, b. canis can be isolated from selected organs (e.g., epididymis, prostate). when the -me rsat or tat is used as a screening test and results are positive, a tentative diagnosis of brucellosis is made; positive blood culture or agid should be used to confirm results. if blood culture or agid is negative, brucellosis is unlikely. if -me rsat or tat results are negative in a dog strongly suspected of having brucellosis, the test should be repeated in weeks to preclude the possibility of early infection. definitive diagnosis requires isolation of b. canis, although this is not always achieved. although blood culture is ideal, it is inconvenient and expensive. culture of urine or an ejaculate may also be performed in males. occasional indications • dogs from areas endemic for ixodes ticks or with an appropriate travel history and fever, lameness, glomerulonephritis, or nonseptic, suppurative polyarthritis should be suspected of having lyme disease (borreliosis) and screened for antibodies against b. burgdorferi. , , analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa, elisa, and western immunoblot. the organism can be documented in tissues by culture, histologic techniques, or pcr assay result. (see appendix i: select laboratories for infectious disease.) serum antibodies are generally used to screen for exposure to b. burgdorferi in dogs. immunoglobulin m (igm) and immunoglobulin g (igg) antibodies against b. burgdorferi can be detected in canine serum. titers considered significant vary by laboratory and assay. both antibody classes can persist in serum for months after exposure. depending on the assay used, cross-reactivity with b. burgdorferi antigens used in ifa and elisa can occur with other spirochetes; thus a positive titer does not always document exposure to b. burgdorferi. b. burgdorferi vaccines induce antibodies that are detected by some ifa and elisa. western immunoblot can be used to differentiate vaccine-induced antibodies from antibodies resulting from natural infection. antibodies against the c peptide of b. burgdorferi are rarely induced by vaccination; point-of-care assays using this peptide are commercially available. because b. burgdorferi migrates through the tissues, most dogs with borreliosis are positive for antibodies by the time illness is detected. healthy dogs develop the same antibody responses as clinically ill dogs, however. because of these factors, interpretation of positive serum antibody titers is difficult. serum antibodies against b. burgdorferi only document exposure to b. burgdorferi (or a similar antigen), not clinical disease. an assay to quantify antibodies against the c peptide is commercially available (see appendix i: select or oral ulceration, particularly if tick exposure, rabbit ingestion, or potential for human infection is confirmed. tularemia is a direct zoonosis from clinically ill cats to people. analysis, artifacts, and interpretation • clinicians measure antibodies in serum using a microscopic agglutination (ma) assay. , (see appendix i: select laboratories for infectious disease.) time between acquisition of infection and a positive titer is not known. in dogs, titers of : to : are commonly detected in acute infections. in cats with tularemia, ma titers are generally greater than : . development of a positive titer or a fourfold increase in titer between acute and convalescent sera ( weeks later) is presumptive evidence of infection. definitive diagnosis is obtained by isolation of the bacterium in a culture of a blood specimen or by identification in tissue by immunofluorescence. occasional indications • dogs and cats with nasal or pulmonary disease can be serologically screened for antibodies against aspergillus fumigatus; cats are affected less frequently than dogs. results are generally interpreted in conjunction with cytology, radiology, histopathology, and culture. analysis, artifacts, and interpretation • agid, counterimmunoelectrophoresis (ciep), and elisa have been used to detect circulating antibodies against a. fumigatus in serum. presence of serum antibodies can represent either exposure or infection, and some dogs with nasal aspergillosis are falsely negative for serum antibodies. in one recent study of dogs with and without nasal aspergillosis, the sensitivity, specificity, and positive and negative predictive values for serum anti-aspergillus antibody results were %, %, %, and %, respectively. owing to persistence of titers in some treated dogs (i.e., months), monitoring titers to assess therapeutic response is not recommended. dogs or cats infected with penicillium spp. will be seronegative if assessed only in assays using a. fumigatus antigens. radiographic demonstration of nasal turbinate destruction suggests aspergillosis or nasal neoplasia. cytologic analysis (see figure - ) and culture of canine nasal exudate alone are not diagnostic because fungal elements may be nondetectable in affected dogs whereas they may be found in noninfected dogs (including dogs with nasal tumors). the organism is sometimes difficult to culture from an aspergilloma (fungal ball). nasal lavage is a low-yield procedure for demonstration of the organism; nasal biopsy is suggested (see chapter ) . definitive diagnosis should be based on three factors: ( ) histopathologic evidence of tissue invasion, ( ) an aspergilloma combined with serologic and culture evidence of infection, or ( ) serologic and radiographic evidence of infection (i.e., bone lysis). in rare cases with growth usually occurs within days, but cultures should be held for to weeks before being discarded. at least three cultures from specimens obtained several days apart are recommended. occasional indications • serologic testing for antibodies against leptospira spp. should be considered in dogs with undiagnosed fever, ecchymoses, vomiting, diarrhea, muscle pain, uveitis, coughing, dyspnea, renal pain, thrombocytopenia, renal failure (particularly acute), or increased activities of hepatic enzymes. , the most common pathogenic serovars in dogs include leptospira canicola, l. icterohaemorrhagiae, l. grippotyphosa, l. bratislava, l. autumnalis, and l. pomona. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by the microscopic agglutination test (mat), elisa (igm, igg), and microscopic microcapsular agglutination test (mcat). most diagnostic laboratories use mat. the primary disadvantage of serologic testing is that it is difficult to determine whether positive titers are caused by active infection, previous infection, or vaccination. in addition, when results from different laboratories are compared, results commonly vary. a laboratory that assesses for antibodies against multiple serovars and participates in the international leptospirosis society proficiency program should be used. antibodies are detected by mat days to weeks after infection. acutely infected dogs are often mat negative; dogs with suggestive clinical signs of disease but negative mat results should be retested in to days; development of a positive titer confirms recent infection. a fourfold increase in antibody titer also can confirm recent infection. vaccination can induce positive mat titers. because of the presence of cross-reactive antibodies, one cannot assume that the serovar inducing the highest titer during acute infection is the serovar causing infection. the combination of increasing antibody titers with appropriate clinical pathologic abnormalities and clinical findings suggests clinical leptospirosis. definitive diagnosis requires demonstration of the organism by urine dark-field microscopy, phase-contrast microscopy, culture, or pcr assay. examination of urine for leptospires is a low-yield procedure. demonstration of spirochetes by histopathologic evaluation of renal tissue leads to a presumptive diagnosis, which may be confirmed by tissue culture. culture or pcr assay can be of most benefit early in the course of infection when mat results are negative. the organism is in high levels in blood for the first days of infection and then is highest in urine. repeated culture may be needed because of intermittent shedding. administration of antimicrobial therapy can result in false-negative results. rare indications • testing for tularemia (i.e., rabbit fever) should be considered in animals from endemic areas developing fever, lymphadenomegaly, weight loss, organism is not demonstrated by cytology (figure - ) , histopathology, or culture. feline disease is rare but has been associated with nodular or ulcerative skin lesions, pulmonary interstitial disease, osteomyelitis, uveitis, and cns disease. an antigen test has been evaluated for use with samples from humans but has not been validated for use with samples from dogs or cats. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by complement fixation (cf), agid, elisa, latex agglutination (la), and tube precipitin (tp) tests. tp detects igm antibodies; cf and agid detect igg antibodies. false-negative results in tp occur in early infections (< weeks), chronic infection, rapidly progressive acute infection, and primary cutaneous coccidioidomycosis. false-positive results in the cf test are caused by anticomplementary serum, which may be caused by bacterial contaminants or immune complexes. finally, cross-reactions in patients with histoplasmosis and blastomycosis may occur with all tests. after resolution of disease, cf titers decrease over weeks but remain positive at a low titer (e.g., : ) for months. definitive diagnosis requires demonstration of the organism on smears, aspirates, histopathologic evaluation, or culture. the organism is often difficult to demonstrate. wet mount examination of unstained or stained (periodic acid-schiff) smears or aspirates is more suitable than dry mounts, which may distort the spherules. common thoracic radiographic findings are mixed interstitial, bronchial, and alveolar pulmonary patterns and hilar lymphadenomegaly. positive serologic test results and characteristic radiographic changes allow tentative diagnosis. disseminated disease, cytologic evaluation of aspirates of affected tissue may be useful. if the organism cannot be demonstrated by biopsy samples obtained through the nares, positive serologic test results may support exploratory surgery. occasional indications • dogs from endemic areas with fever, weight loss, pulmonary interstitial disease, lymphadenomegaly, uveitis and blindness, ulcerative or draining skin lesions, undiagnosed prostatic or testicular disease, intracranial disease, osteomyelitis, or (rarely) renal disease can be serologically screened for antibodies against blastomyces dermatitidis and b. dermatitidis antigens if the organism is not demonstrated by cytology (see figure - ), histopathology, or culture. , in endemic areas, screening for antibodies against b. dermatitidis should be considered in cats with pulmonary interstitial disease, intracranial disease, lymphadenomegaly, ulcerative or draining skin lesions, or uveitis and blindness. analysis, artifacts, and interpretation • circulating antibodies are most commonly detected in serum by agid. blastomyces antigens in urine and blood of dogs have been measured using the mvista blastomyces antigen eia (miravistalabs.com). because subclinical canine infections are unusual, positive serum antibody test results are considered significant. false-negative results occur in animals with peracute infection or with advanced cases overwhelming the immune system. in dogs, the sensitivities of antigen testing of urine, antigen testing of serum, and agid serum antibody testing were . %, . %, and . %, respectively. thus dogs with a high index of suspicion for blastomycosis that are negative for serum antibodies should be screened for urine or serum antigens. many cats with blastomycosis are serum antibody negative. whether serum or urine antigen testing will aid in the diagnosis of blastomycosis in cats remains to be determined. definitive diagnosis requires identification of the yeast by cytology, histopathology, or fungal culture. impression smears from skin lesions and aspirates from enlarged lymph nodes frequently reveal organisms; recovery of organisms from transtracheal aspiration, pulmonary aspiration biopsy samples, or urine is less consistent. culture requires to days and is of lower yield than cytology or biopsy. diffuse nodular interstitial pulmonary disease and hilar lymphadenomegaly are common radiographic findings. positive serologic results combined with appropriate clinical signs and radiographic abnormalities allow presumptive diagnosis. occasional indications • dogs from endemic areas with pulmonary interstitial disease, fever of undetermined origin, hilar lymphadenomegaly, osteomyelitis, uveitis, pericarditis, and nodular or ulcerative skin lesions can be screened for antibodies against c. immitis if the occasional indications • cats and rarely dogs with undiagnosed respiratory (especially nasal), cns, eye (especially uveal tract), and skin (especially nodular or ulcerative lesions) infections can be screened for cryptococcus neoformans and c. gattii antigens if the organism is not demonstrated by cytology, histopathology, or culture. , analysis, artifacts, and interpretation • measurement of antibodies against c. neoformans or c. gattii is not clinically useful. cryptococcal antigen is detected in serum, aqueous humor, or csf using latex agglutin ation. negative serum la titers may occur in early disease or uncommonly in chronic low-grade infections, in chemotherapy-induced remission, or in nondisseminated disease. specificity of the serum la is high. a titer of greater than : in serum or csf is positive; very high titers are commonly detected. in some animals, decreases in serum titer parallel response to therapy. positive titers occur in some animals after apparently successful clinical responses, suggesting persistent low-grade infection or false-positive results. , cryptococcal encephalitis may cause a positive csf la titer despite a negative serum la. definitive diagnosis is based on cytologic, histopathologic, or culture demonstration of the organism or a positive la test result. cytology is commonly positive (see figure - ) because there are usually numerous yeasts found in affected tissues (i.e., nasal and cutaneous lesions, aqueous and vitreous humor). the organism can occasionally be recovered from nasal washings of normal animals. csf may contain the yeast, but concentration techniques (i.e., cytocentrifugation) should be used. routine cytology stains (e.g., wright) are adequate to demonstrate the organism. large numbers of organisms are usually visible despite little or no inflammation. culture is seldom necessary. serologic testing is used if the yeast cannot be demonstrated cytologically or to monitor response to treatment. a pcr assay has been used to amplify the organism dna from tissue but has not been assessed extensively to date. rare indications • animals with weight loss, pulmonary interstitial disease, uveal disease, diarrhea, or lymphadenomegaly can be serologically screened for antibodies against histoplasma capsulatum if the organism is not demonstrated by cytology, histopathology, or culture. analysis, artifacts, and interpretation • primarily, agid is used to detect circulating antibodies in serum. presence of serum antibodies confirms exposure but not clinical illness because of infection. agid has questionable clinical usefulness because titers persist longer than year after resolution of disease in some animals, and both false-positive and false-negative results occur. antibody testing is even less rewarding in cats. definitive diagnosis requires demonstration of the organism by cytology (see figure - a), biopsy, culture, or pcr assay. an enzyme immunoassay test used to detect histoplasma antigen in the urine of people has not been validated for use with samples from dogs and cats. the organism is more difficult to demonstrate than b. dermatitidis; however, cytologic examination of rectal scrapings in dogs with colonic histoplasmosis is often diagnostic. fine-needle aspiration of other organs may demonstrate the organism. in most cats with systemic histoplasmosis, the organism is identified on bone marrow cytology. thoracic radiographs are indicated if pulmonary histoplasmosis is suspected; a nodular interstitial pattern is expected. culture of h. capsulatum is of lower yield than biopsy. rare indications • babesia canis vogeli and b. gibsoni infect dogs in the united states, and diagnostic tests can be performed in dogs from endemic areas or in those with an appropriate travel history that have fever, anemia, icterus, splenomegaly (i.e., acute babesiosis), or intermittent fever and weight loss (i.e., chronic babesiosis). although babesiosis can cause anemia in cats, species infecting cats are not found in the united states. exposure to r. sanguineus ticks (b. canis) or pit bull terriers (b. gibsoni) are risk factors for exposure to the two agents in dogs. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa. (see appendix i: select laboratories for infectious disease.) in most laboratories, titers greater than : are considered positive. experimentally infected dogs develop detectable igg titers approximately weeks after infection. false-negative results can occur in immature dogs, in peracute cases, or in dogs with concurrent immunosuppression. antibodies against b. gibsoni and b. canis may or may not cross-react, depending on the antigen source used by a particular laboratory, and so specific ifa should be used for both organisms. dna of b. canis and b. gibsoni can be amplified by pcr assay, and positive results indicate current infection. , however, both antibodies and dna can be detected in dogs that are healthy and those that are clinically ill. antibody titer magnitude does not correlate to the presence or absence of disease. a titer of greater than : was suggested for b. gibsoni, but not all infected dogs achieve a titer of this magnitude. it is important to determine which species are involved in a case because response to treatment varies. duration of positive titers after resolution of disease is unknown. in untreated experimentally infected dogs, titers remained high for at least months. untreated, seropositive dogs should be considered carriers of the infection. treatment is indicated only for seropositive, clinically ill dogs. definitive diagnosis requires demonstration of the organism in blood smears stained with romanowskytype preparations (e.g., wright and giemsa) (figure - ) or pcr assay. organisms are best found in blood (particularly in acute disease) from a microcapillary system (e.g., ventral surface of ear or toenail). note: shape of the organism may be distorted in old blood. infection, not clinical disease. however, if antibodies are detected in an animal with appropriate clinical signs of disease, treatment may be indicated in an attempt to slow progression. definitive diagnosis is based on demonstration of the organism in tissues. the organism can be differentiated from t. gondii structurally, by immunohistochemistry, and by pcr assay. n. caninum oocysts are found in the feces of some dogs. occasional indications • healthy cats • t. gondiispecific antibodies form in serum, aqueous humor, and csf of healthy and diseased cats or dogs. antibodies do not directly correlate with clinical toxoplasmosis. no serologic test is currently available that accurately predicts when a seropositive cat previously shed oocysts. a seropositive cat is less likely than a seronegative cat to shed the organism if re-exposed. elisa, ifa, and western blot immunoassay can be adapted to detect various antibody classes; igm and igg are those usually assessed. t. gondii-specific igm is detectable in serum by elisa in approximately % of subclinically ill cats to weeks after experimental induction of toxoplasmosis; these titers generally are negative less than weeks after infection. detectable igm titers were present in the serum of . % of cats in a study of clinical toxoplasmosis; igg titers were detected in %. igm titers persist in some clinically ill cats for greater than weeks; these cats are frequently co-infected with fiv or have ocular toxoplasmosis. after repeat inoculation with t. gondii, primary inoculation with the petaluma isolate of fiv, and administration of glucocorticoids, some cats with chronic toxoplasmosis experience short-term recurrence of detectable igm titers. healthy and clinically ill dogs occasionally develop detectable igm titers. kinetics of postinfection igm titers in dogs is unknown. after experimental induction of infection in subclinically ill cats, t. gondii-specific igg can be detected by elisa in serum from most cats by weeks. positive igg antibody titers generally persist for years after infection. single high igg titers have been suggested to indicate recent or active infection. the author, however, has demonstrated igg antibody titers greater than : , in subclinically ill cats years after experimental induction in chronic disease or asymptomatic carriers, demonstration of organisms is unreliable, and a tentative diagnosis is based on clinical signs and a positive titer. dogs with babesiosis are often coombs positive (see chapter ). rare indications • serology for neospora caninum can be performed in dogs with clinical evidence of polyradiculomyositis, including progressive ascending rigid paralysis, dysphagia, muscle atrophy, and (rarely) myocardial dysfunction or pneumonia. analysis, artifacts, and interpretation • circulating antibodies are detected in serum by ifa. (see appendix i: select laboratories for infectious disease.) a presumptive diagnosis of neosporosis can be made by combining appropriate clinical signs of disease and positive serology or presence of antibodies in csf with the exclusion of other causes inducing similar clinical syndromes, in particular, toxoplasma gondii. igg antibody titers greater than or equal to : have been detected in most dogs with clinical neosporosis; minimal serologic cross-reactivity exists with t. gondii at titers greater than or equal to : . because the organism is a tissue protozoan, seropositivity may correlate with permanent infection. circulating antibodies against neospora caninum only document base an antemortem diagnosis of clinical toxoplasmosis on these tests alone. antemortem diagnosis of clinical toxoplasmosis can be tentatively based on the combination of the following: • demonstration of serologic evidence of infection • clinical signs of disease referable to toxoplasmosis • exclusion of other common causes • positive response to appropriate treatment t. gondii was detected by pcr in aqueous humor of . % of cats with uveitis. the organism also can be detected transiently in aqueous humor and blood of healthy, experimentally inoculated cats, however, making the positive predictive value of the pcr for clinical disease less than %. rare indications • serologic testing for antibodies against trypanosoma cruzi should be considered in dogs from endemic areas and those with generalized lymphadenomegaly, neurologic signs, or myocardial dysfunction (especially second-or third-degree heart block or ventricular tachycardia). analysis, artifacts, and interpretation • ifa, direct hemagglutination, and cf usually detect circulating antibodies in canine sera. dogs are generally seropositive weeks after infection. a positive titer documents exposure to the organism, not clinical disease. positive titers vary by assay. definitive diagnosis requires demonstration of the organism on blood smear, lymph node impression, or buffy coat and plasma interface smear. t. cruzi is occasionally found in peripheral blood without demonstrable organisms in tissue. a standard workup for myocardial disease, including chest radiographs, electrocardiogram, electrolytes, and echocardiography (if available), is indicated. alternatively, t. cruzi amastigotes can be demonstrated in tissues. pcr can be used to amplify organism dna. indications • dogs living in ixodes spp. endemic areas with acute fever or polyarthritis should be screened for antibodies against anaplasma phagocytophilum (previously ehrlichia equi) or for a. phagocytophilum dna in blood by pcr assay. , , the role this organism plays in chronic disease syndromes in dogs is unknown. analysis, artifacts, and interpretation • antibodies against a. phagocytophilum in serum can be measured by ifa or commercially available elisa (snap dx, table - , idexx corporation, westbrook, me). the antibodies have only variable cross-reactivity with other anaplasma spp. ehrlichia spp., or neorickettsia spp. and so positive test results likely indicate exposure to a. phagocytophilum. infected dogs can be seronegative when clinical signs of of toxoplasmosis. a positive igg antibody titer in a single serum sample only documents exposure, not recent or active disease. demonstration of an increasing igg titer can document recent or active disease. unfortunately, the time span from the first detectable positive igg titer to the maximal igg titer is approximately to weeks, leaving a very narrow window for documenting an increasing titer. many cats with clinical toxoplasmosis have chronic low-grade signs, and they are not tested until their igg antibody titers have reached maximal values. in humans with reactivation of chronic toxoplasmosis, igg titers only rarely increase; cats appear to be the same. several agglutination tests have been evaluated using cat serum. an la and an indirect hemagglutination assay (iha) are commercially available. these assays are not species specific and potentially detect all classes of serum immunoglobulins directed against t. gondii. unfortunately, la and iha rarely detect antibody in feline sera when positive for only igm by elisa. modified agglutination using formalin-fixed tachyzoites is the most sensitive procedure for detection of t. gondii antibodies in cat sera, but it is generally unavailable commercially. local production of t. gondii-specific igg in csf and aqueous humor occurs in experimentally inoculated, subclinically ill cats and in cats and dogs with clinical disease because of toxoplasmosis. local igm production has only been detected in csf and aqueous humor of animals with clinical disease. most cats with uveitis and production of t. gondii-specific antibodies in aqueous humor have responded to administration of anti-toxoplasma drugs, suggesting that aqueous humor antibody testing aids in diagnosis of clinical ocular feline toxoplasmosis. (see appendix i: select laboratories for infectious disease.) fecal examination • fecal oocysts can be demonstrated using flotation techniques with various solutions with specific gravities from . to . . sugar solution centrifugation is probably the optimal technique. oocysts of t. gondii are to µm in diameter, approximately oneeighth the size of toxocara cati eggs. focusing on only one plane of the microscope slide or coverslip can result in oocysts being overlooked. the oocysts cannot be distinguished grossly from hammondia hammondi or besnoitia darlingi (nonpathogenic coccidians infecting cats). sporulated oocysts isolated from feces can be inoculated into mice or tissue cultures for definitive identification. because oocyst shedding has rarely been documented in cats with subfatal, clinical toxoplasmosis, the diagnostic usefulness of fecal examination is limited. cats with clinical signs referable to t. gondii should undergo fecal evaluation, however, because of potential zoonotic risk. interpretation • exposure to t. gondii is suggested by finding antibodies in serum, aqueous humor, or csf. recent or active toxoplasmosis is suggested by finding an igm titer greater than : or a fourfold or greater increase in igg titer, or documenting local antibody production in aqueous humor or csf. because t. gondii-specific antibodies can also be detected in the serum, csf, and aqueous humor of healthy, infected animals, one cannot assay results in clinically ill dogs, e. canis appears to be the organism in this group that is most commonly associated with clinical illness. serologic testing or pcr assay for e. canis is indicated for dogs from endemic areas or with an appropriate travel history and thrombocytopenia, anemia, leukopenia, hyperglobulinemia, proteinuria, polyarthritis, fever, uveitis, lymphadenomegaly, hepatosplenomegaly, or inflammatory cns disease, particularly if the animal has a history of exposure to rhipicephalus ticks. analysis, artifacts, and interpretation • circulating antibodies against e. canis are detected in serum by ifa or elisa; they do not cross-react with rickettsia rickettsii or anaplasma platys (see canine thrombocytotropic anaplasmosis later). serologic cross-reactivity between e. canis antibodies and those against a. phagocytophilum (previously ehrlichia equi; see canine granulocytotropic anaplasmosis earlier), e. chaffeensis, e. ewingii (see canine granulocytotropic ehrlichiosis earlier), and neorickettsia risticii (previously e. risticii) is variable. multiple serologic tests are needed to exclude all of the ehrlichia spp., anaplasma spp., and neorickettsia spp. from the differential list. thus pcr assays are often combined with serologic tests. in addition, pcr assay results may be positive before seroconversion in some dogs. in experimentally infected dogs, antibodies against e. canis can be detected as early as days and are almost always present by days after inoculation. antibody titers continue to increase for weeks to months after inoculation in untreated, experimentally infected dogs. e. canis titers of less than : are suspect and should be rechecked in approximately to days; a titer of : or higher is diagnostic. initial positive results in a recently marketed point-of-care test occur at approximately : . positive titers revert to negative to months after resolution of infection; persistence of titers for greater than or equal to months suggests a carrier state. however, positive antibody titers have been detected for months after apparently successful therapy in some naturally infected dogs. clinically ill, seropositive dogs should be treated a minimum of days and until clinical and laboratory abnormalities have resolved. whether to treat healthy, seropositive dogs is controversial; the issues involved in this decision were recently reviewed. , the clinician can make a definitive diagnosis of e. canis infection by demonstrating morulae (i.e., clusters of the organism) in mononuclear cells, culture, or pcr assay. morulae are rarely found on routine blood smear or bone marrow aspiration cytology unless the dog has been immunosuppressed. cytology and pcr assay results can be falsely negative in dogs that have been treated. ehrlichia spp. can be isolated by tissue culture of heparinized infected canine blood or bone marrow aspiration samples, but culture is of limited availability, expensive, and of low yield. ehrlichia spp. can be detected in whole blood by pcr, which has potential benefit for use in monitoring treatment. (see appendix i: select laboratories for infectious disease.) the consensus statement on ehrlichial disease of small animals from the infectious disease study group of the american college of veterinary internal medicine (acvim) states the following: disease first occur and can be immediately assessed by pcr assay or have repeat serology performed in approximately weeks to evaluate for seroconversion. alternately, both assays can be performed at the same time. antibiotic therapy can lead to falsely negative pcr assay results. a. phagocytophilum dna and antibodies can be detected in both healthy and clinically ill dogs, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. indications • cats living in ixodes spp. endemic areas with fever, mild thrombocytopenia or clinical evidence of polyarthritis should be evaluated for a. phagocytophilum dna in blood by pcr assay. , antibodies can be detected in serum by ifa, but a standardized test is not available. (see appendix i: select laboratories for infectious disease.) analysis, artifacts, and interpretation • dna of a. phagocytophilum has been amplified from several cats with clinical signs of anaplasmosis. some of the cats were seronegative when first assayed but had seroconverted when assayed at a later date. whether the point-of-care assay used to detect a. phagocytophilum antibodies in canine serum is valid for use with cat serum is unknown. antibiotic therapy can lead to falsely negative pcr assay results. a. phagocytophilum dna and antibodies can be detected in both healthy and clinically ill cats, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. untreated healthy cats can be pcr positive for weeks after tick exposure. indications • dogs living in the midwest united states with evidence of fever or clinical evidence of polyarthritis should be evaluated for ehrlichia ewingii dna in blood by pcr assay. , specific antibodies can be detected in serum, but a standardized test is not currently available. analysis, artifacts, and interpretation • there is variable cross-reactivity between e. canis antigens and e. ewingii antigens, and so serologic tests for e. canis will not always be positive when the infecting agent is e. ewingii. pcr assays that amplify the dna of e. ewingii are available and should be performed on blood from dogs with suspected acute infection. antibiotic therapy can lead to falsely negative pcr assay results. healthy dogs can be pcr positive for e. ewingii dna in blood. common indications • ehrlichia canis, e. chaffeensis, and neorickettsia risticii all infect monocytes of dogs and can be associated with clinical illness. based on pcr occasional indications • serologic testing for rickettsia rickettsii antibodies (rocky mountain spotted fever [rmsf]) is indicated for dogs from endemic areas or with an appropriate travel history and acute onset of fever, lymphadenomegaly, petechiae, neurologic signs, stiff gait, peripheral edema, dyspnea, or scleral congestion. history of tick exposure is inconsistent. exposed dogs either develop acute disease with approximately a -day clinical course or are subclinically infected. the primary tick vectors are active from spring to fall in most of the united states; therefore rmsf should only be considered a principal differential diagnosis for clinically ill dogs during this time span. the majority of cases are diagnosed in southeastern states. analysis, artifacts, and interpretation • antibodies against r. rickettsii in canine serum can be measured by ifa, elisa, and la. elisa or ifa can detect igm and igg antibodies against rmsf. la is not antibody class-specific. cutoffs for positive antibody titers, as well as specificity and sensitivity, vary by assay. antibodies against the nonpathogenic spotted fever group rickettsia (r. belli, r. montana, r. rhipicephali) cross-react with r. rickettsii antigens. in dogs with clinical illness because of rmsf, igm antibody titers are generally positive. because igm has short duration in serum, false-negative results may occur with igm testing. false-positive results are most common in the igm elisa. positive igg titers are detectable to days after infection. serum samples with igg titers greater than or equal to : are generally considered positive. if igg or igm antibodies are not detected in a patient with clinical and laboratory evidence of rmsf, a convalescent igg titer to weeks later is recommended. timing of the second titer is not critical because igg antibody titers do not decrease for at least to months after infection. documentation of seroconversion or a fourfold increase in igg titer is consistent with recent infection. a presumptive diagnosis of canine rmsf can be based on the combination of appropriate clinical, historic, and clinicopathologic evidence of disease; serologic test results; exclusion of other causes of the clinical abnormalities; and response to anti-rickettsial drugs. documentation of seroconversion or an increasing titer to weeks after initial serologic testing suggests recent infection. diagnostic criteria used in one recent study included a fourfold rise in antibody titer or a single titer of greater than or equal to : if the initial titer was submitted week or more after initial onset of clinical abnormalities. positive serum antibody test results alone do not prove rmsf because subclinical infection is common. in addition, positive serum antibody tests do not document infection by r. rickettsii because infection with nonpathogenic spotted fever group agents induce cross-reacting antibodies. demonstration of r. rickettsii by inoculating affected tissues or blood into susceptible laboratory animals or by documenting the organism in endothelial cells using direct fluorescent antibody staining leads to a definitive diagnosis of rmsf, but these techniques are not clinically practical. pcr can be used to document the if pcr is used to monitor treatment, the pcr assay should be repeated after antimicrobial therapy has been discontinued for weeks. if pcr results are positive, an additional weeks of treatment should be given with the pcr assay repeated after antimicrobial therapy has been discontinued for weeks. if pcr results are positive after treatment cycles, use of an alternate antiehrlichial drug should be considered. if pcr results are negative the test should be rechecked in months; if still negative therapeutic elimination is likely. however, the organism may be sequestered in other tissues like the spleen. rare indications • cats with thrombocytopenia, anemia, leukopenia, hyperglobulinemia, proteinuria, polyarthritis, fever, or lymphadenomegaly should be evaluated for ehrlichia spp. dna in blood by pcr assay if no other obvious cause exists. , , to date, e. canis is the monocytotropic strain amplified from naturally exposed cats. antibodies can be detected in serum by ifa, but a standardized test is not available. (see appendix i: select laboratories for infectious disease; protatek reference laboratories.) analysis, artifacts, and interpretation • while serum antibodies against e. canis have been detected in the serum of some cats, a number of cats with e. canis dna amplified from blood were seronegative. , thus while ifa testing is available for e. canis antibodies in feline serum, it should be combined with pcr assay. antibodies against e. canis can be detected in serum from healthy cats and therefore cannot be used alone to make a definitive diagnosis of ehrlichiosis. a tentative diagnosis of feline ehrlichiosis is based on the combination of clinical signs, positive serologic or pcr assay results, exclusion of other known causes, and response to tetracyclines. occasional indications • testing for anaplasma platys infection is indicated for dogs from endemic areas or with appropriate travel history and thrombocytopenia or endogenous uveitis. , analysis, artifacts, and interpretation • circulating igg antibodies against a. platys are detected in serum by ifa. antibodies against a. platys react with a. phagocytophilum antigen used in a commercially available kit. experimentally infected dogs become antibody positive to days after infection. infected dogs can be seronegative when clinical signs of disease first occur and can be immediately assessed by pcr assay or have repeat serology performed in approximately weeks to evaluate for seroconversion. alternately, both assays can be performed at the same time. antibiotic therapy can lead to falsely negative pcr assay results. a. platys dna and antibodies can be detected in both healthy and clinically ill dogs, and so positive test results do not document clinical illness. morulae are only rarely documented cytologically in clinical specimens. analysis, artifacts, and interpretation • determining serum antibodies to feline or canine parvoviruses or coronaviruses is rarely performed clinically because positive results do not correlate with clinical disease. a point-ofcare assay for detection of canine parvovirus antibodies is available. vaccinated dogs seropositive in this assay probably do not need to be boosted. detecting fecal shedding of canine parvovirus viral antigen by electron microscopy, virus isolation, fecal hemagglutination, fecal la, or elisa can be used to confirm current infection. in-office elisa for canine parvovirus in feces seems to accurately detect fecal shedding of parvovirus in acute cases (see chapter ) . the specificity of the assays is good, but they cannot differentiate vaccine strains of parvovirus and virulent strains. falsenegative reactions can occur. these assays also detect feline parvovirus. virus isolation, electron microscopy, or molecular assays can be used to document coronaviruses in feces, but results do not correlate with the presence of illness. rare indications • fip is an appropriate differential diagnosis in cats with fever; uveitis; retinal hemorrhage; nonseptic abdominal or pleural exudates or modified transudates; anemia; hyperglobulinemia; and renal, hepatic, or neurologic abnormalities. results of currently available serum or blood tests cannot be used alone to definitively diagnose fip. analysis, artifacts, and interpretation • circulating antibodies against coronaviruses can be detected by ifa and elisa in feline serum. antibody to coronavirus indicates prior exposure to either enteric coronaviruses or fip-inducing coronaviruses. a positive titer does not diagnose fip or protect against disease. feline vaccines containing bovine serum occasionally cause false-positive results. cats with fip can rarely have negative results because of rapidly progressive disease with a delayed rise in titer, disappearance of antibody in terminal stages of the disease, or immune complex formation. a positive coronavirus antibody titer does not predict whether a cat will ever develop fip. titer magnitude cannot distinguish between exposure to enteric coronaviruses or fip-inducing strains. rarely, positive titers can be induced by vaccination for coronavirus. kittens can be seropositive because of colostrumderived antibodies until weeks of age. if adult cats in the environment infect kittens, antibodies can be detected again to weeks later. current coronavirus infections can be detected by fecal virus isolation, electron microscopy of feces, or rt-pcr of feces. however, positive test results do not indicate fip because antibody-positive, healthy cats can pass coronaviruses. definitive diagnosis of fip requires histopathologic evaluation of tissues. lesions visible by light microscopy are generally pathognomonic, but immunohistochemistry can be used to confirm coronavirus particles. rt-pcr can amplify coronavirus rna from effusions, tissues, and blood and usually correlates with the presence of fip. amplification of coronavirus by pcr in effusions and tissues predicts fip, but detection in presence of rickettsial agents in blood, other fluids, or tissues and will likely be clinically useful in the future. rare indications • dogs with appropriate signs of cns disease can have antibodies in csf and serum against canine distemper virus. analysis, artifacts, and interpretation • the clinician can measure csf and serum igg antibodies against canine distemper virus by serum virus neutralization, ifa, or elisa. elisa can be used to measure serum igm antibodies. csf antibodies to distemper virus are increased in some dogs subsequently diagnosed histopathologically as having distemper encephalitis. false-positive results can occur in csf samples contaminated with blood. concurrent measurement of serum antibody concentrations can be helpful; if csf concentrations are greater than serum concentrations, the antibody in csf had to be produced locally and suggests cns distemper. detection of serum igg antibodies is of minimal diagnostic value because a positive titer could develop secondary to vaccination or previous exposure. a fourfold increase in serum igg titer over a -to -week period suggests recent infection. detection of circulating igm antibodies is consistent with recent infection but not clinical disease. a point-of-care assay for detection of canine distemper antibodies is available. vaccinated dogs that are seropositive in this assay probably do not need to be boosted. a presumptive diagnosis of distemper encephalitis can be made with increased csf protein and leukocytes (lymphocytes predominating) plus a positive csf antibody titer in a sample not contaminated with peripheral blood. definitive diagnosis of canine distemper infection requires demonstration of viral inclusions by cytologic examination (see figure - a and b), direct fluorescent antibody staining of cytologic or histopathologic specimens, histopathologic evaluation, or reverse transcriptase-pcr (rt-pcr) documentation of distemper viral rna in peripheral blood, csf, or conjunctival scrapings. (table - ; see appendix i: select laboratories for infectious disease.) positive rt-pcr test results can be induced by modified live vaccination. viral inclusions can rarely be found in erythrocytes, leukocytes, and leukocyte precursors of infected dogs. inclusions are generally present for only to days after infection and therefore often are not present when clinical signs occur. inclusions may be easier to find in smears made from buffy coats or bone marrow aspirates than those made from peripheral blood. viral particles can be detected by immunofluorescence in cells from the tonsils, respiratory tree, urinary tract, conjunctival scrapings, and csf for to days after infection. indications • viral enteritis induced by parvoviruses, coronaviruses, and other viruses should be suspected in young animals with fever and diarrhea, particularly if neutropenia is present (i.e., parvoviruses). neoplastic, reproductive, immunologic, or hematologic disease, as well as in clinically normal cats exposed to felv-positive cats. analysis, artifacts, and interpretation • viral antigen (p ) is detected by ifa in neutrophils and platelets from blood or bone marrow, or in blood, plasma, serum, saliva, or tears by elisa. nucleic amplification assays can also be used to assess the stage of infection. , when evaluating for antigen, testing of serum or blood gives the best results; tears and saliva should not be tested. several point-of-care elisa tests are available in the united states. other assays are also available in other countries. antibody titers to felv envelope antigens (neutralizing antibody) and against virustransformed tumor cells (feline oncogenic cell membrane antigen, or focma, antibody) are available in some laboratories, but the prognostic significance of the results is currently unknown; therefore the tests are not used clinically. felv infection has four major outcomes. cats with inappropriate immune responses develop progressive infection and usually develop felv-associated diseases. cats with regressive infection can be transiently positive for p antigen in blood or serum but ultimately become negative. abortive exposure occurs in cats with good immune responses and infection never occurs. rarely, focal infection of tissues such as the spleen, lymph nodes, small intestine, or mammary glands can occur. cats with regressive infection, abortive exposure, or focal infection rarely become ill. elisa test results generally become positive within days of exposure to felv and can revert to negative in cats that develop regressive infection. cats suspected to have regressive infection can be isolated from other cats and retested by elisa in several weeks or be tested by ifa or pcr assay. positive ifa test results prove the bone marrow has been infected and has % correlation with virus isolation. these cats generally develop progressive infection. false-negative ifa reactions may occur when leukopenia or thrombocytopenia prevents evaluation of an adequate number of cells. false-positive reactions rarely occur from nonspecific staining of eosinophils. a positive ifa indicates that the cat is viremic and contagious. virtually all ifa-positive cats are elisa-positive. finding an ifa-positive but elisa-negative cat suggests technique-related artifact. a negative elisa result is approximately % correlated with negative ifa and an inability to isolate felv. cats that are elisa-positive but ifa-negative are called discordant. discordant results are usually caused by false-positive elisa results, falsenegative ifa results, or early stages of regressive infections. use of pcr assays to detect viral rna or cell-associated dna (provirus) can be performed on blood, bone marrow, or tissues and be used to evaluate cases with discordant elisa and ifa results. some cats with focal infection localized to bone marrow have positive bone marrow ifa results. the most reliable means of identifying focal felv infections is virus isolation or pcr assay. a cat with focal infection may become viremic after extreme stress or administration of glucocorticoids. blood does not. , hyperproteinemia and polyclonal gammopathy (detected by electrophoresis; see chapter ) can occur, particularly in the noneffusive form. monoclonal gammopathy rarely occurs. classic nonseptic pyogranulomatous exudate or modified transudate with high protein and relatively low cell count (see chapter ) is commonly used for presumptive diagnosis. electrophoresis can also be performed on body fluids. a gamma globulin fraction greater than or equal to % is highly suggestive of fip, whereas an albumin:globulin ratio in body fluid greater than . probably rules out fip. in another study, an albumin:globulin ratio of . had a positive predictive value of % and an albumin:globulin ratio of . had a negative predictive value of %. common indications • cats with chronic weight loss, fever, rhinitis, conjunctivitis, gingivitis, dermatitis, diarrhea, uveitis, recurrent abscessation, clinical toxoplasmosis, any chronic infectious disease, chronic renal failure, or lymphadenomegaly should be evaluated for fiv infection. analysis, artifacts, and interpretation • igg antibodies are detected in serum by elisa, ifa, and western blot immunoassay. , there are many different in-clinic kits available depending on the country. western blot immunoassay is performed in some commercial laboratories. in the united states, one in-office elisa is available to detect fiv antibodies and feline leukemia virus (felv) antigen combined. this assay is available with and without dirofilaria immitis antigen assay. seroconversion occurs to weeks after inoculation in experimentally infected cats. seropositive cats are probably infected with fiv for life. false-positive reactions can occur in the elisa but are thought to be rare. positive elisa results should be confirmed via western blot immunoassay or ifa, particularly in healthy cats unlikely to have been exposed to fiv. detection of circulating antibodies only confirms infection, not clinical illness. kittens can have detectable colostrum-derived antibodies until to weeks. because many clinical syndromes associated with fiv infection are caused by opportunistic infections, further diagnostic procedures may determine treatable causes. for example, many fiv-seropositive cats with endogenous uveitis are co-infected by t. gondii. virus isolation and rt-pcr are available in some laboratories and can be used to confirm infection. a recently marketed fiv vaccine induces serum antibodies that are indistinguishable from antibodies induced by natural exposure, at least by use of currently available antibody tests. the ability of virus isolation or rt-pcr to accurately differentiate naturally exposed and vaccinated cats is currently unknown and varies between laboratories. common indications • because of diverse manifestations of felv infection, testing is indicated in all clinically ill cats, especially those with evidence of infectious, in dogs or cats with clinical signs, laboratory abnormalities, or thoracic radiographic changes consistent with dirofilariasis. the test can also be used to assess efficacy of adulticide treatment. analysis, artifacts, and interpretation • elisa can detect circulating heartworm antigen in serum; several kits are commercially available. there is greater sensitivity when compared with microfilaria detection. in dogs, d. immitis antigen tests may be positive as early as to months and are usually positive to months after infection. false-negative results usually occur in early stages of infection and may occur in single-sex infections (male only) or in dogs or cats with low worm burdens (< to worms). retesting in to months should be performed to detect dogs in which results were negative in early stages of infection. after successful adulticide treatment, test results become negative in approximately weeks. in experimental infections, cats testing positive did so about months after infection. however, single-sex or low worm burden infections can lead to false-negative results. therefore a positive antigen test result is specific for infection, but a negative result does not rule out dirofilariasis. in cats, the combination of serum antigen test results with serum antibody test results is more sensitive than performing either test alone (see heartworm antibody titer, next). heartworm antibody titer (feline) rare indications • a heartworm antibody titer should be obtained in cats with coughing, unexplained vomiting, syncope, or radiographic evidence of heartworm disease. analysis, artifacts, and interpretation • several elisas detect antibodies to d. immitis in feline sera. the assays are more sensitive than microfilaria demonstration techniques. the assays are very specific; no cross-reactivity exists with d. reconditum. the positive predictive value for heartworm disease is less than %, however, because circulating antibodies can be present in cats that have cleared the infection naturally. false-negative antibody test results also occur; therefore serum antibody and antigen tests should be performed in concert in cats with suspected dirofilariasis. common indications • cytologic evaluation for microfilaria is indicated in dogs with signs consistent with heartworm disease (right-sided heart disease, coughing, dyspnea, eosinophilia, polyclonal hyperglobulinemia, protein-losing nephropathy [pln] ), in dogs about to begin prophylactic therapy (with diethylcarbamazine, ivermectin, or milbemycin), and rarely in cats with signs consistent with heartworm disease (i.e., dyspnea, cardiomegaly, unexplained vomiting). analysis, artifacts, and interpretation • cytologic testing for dirofilaria immitis is very specific (microfilaria morphology differentiates d. immitis microfilaria from those of dipetalonema reconditum), quick, and inexpensive; all concentration techniques (knott's and filter tests) are much more sensitive than examination of fresh blood smears and are reasonably sensitive in dogs that have not been treated with filaricidal drugs. up to % of dogs have spontaneous occult dirofilariasis and must be diagnosed by serologic testing and radiographic examination. all cytology tests have poor sensitivity in cats. thus, microfilaria techniques should no longer be used as stand-alone diagnostic tests and should only be used conconcurrently with antigen tests.* , † a positive test result diagnoses heartworm disease, except in juveniles less than months of age that could have received the microfilaria by transplacental transfer. common indications • a heartworm antigen titer should be included in annual screening of dogs to evaluate for exposure to d. immitis, and should be performed continued geographic distribution of babesiosis among dogs in the united states and association with dog bites: cases babesia gibsoni infections in dogs from north carolina feline granulocytic ehrlichiosis-a report of a new clinical entity and characterization of the new infectious agent clinical ehrlichiosis in a cat prevalence and geographic distribution of dirofilaria immitis, borrelia burgdorferi, ehrlichia canis, and anaplasma phagocytophilum in dogs in the united states: results of a national clinic-based serologic survey a study of naturally occurring feline coronavirus infections in kittens interpretation and misinterpretation of serological test results american trypanosomiasis post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline clinicopathological findings in dogs seroreactive to bartonella henselae antigens infectious diseases of the dog and cat infectious diseases of the dog and cat comparison of latex agglutination, indirect immunofluorescent antibody, and enzyme immunoassay methods for serodiagnosis of rocky mountain spotted fever in dogs infectious diseases of the dog and cat coccidioidomycosis in cats: a retrospective study ( - ) experimental canine leptospirosis caused by leptospira interrogans serovars pomona and bratislava detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis comparison of different tests to diagnose feline infectious peritonitis quality of different in-clinic test systems for feline immunodeficiency virus and feline leukaemia virus infection clinical relevance of annual screening using a commercial enzymelinked immunosorbent assay (snap dx) for canine ehrlichiosis how molecular methods change our views of felv infection and vaccination cryptococcal infection in cats: factors influencing treatment outcome, and results of sequential serum antigen titers in cats rapid identification and differentiation of bartonella species using a single-step pcr assay laboratory diagnosis of bacterial infections pcr detection of the cryptococcus neoformans caps gene from a biopsy specimen from a case of feline cryptococcosis guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents relapsing bacteremia following blood transmission of bartonella henselae in cats feline toxoplasmosis: interpretation of diagnostic test results prevalence of bartonella species dna in the blood of cats with and without fever molecular and serological evidence of anaplasma phagocytophilum molecular evidence supporting ehrlichia canis-like infection in cats bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings association of feline practitioners panel report on diagnosis, treatment, and prevention of bartonella spp. infections the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr canine brucellosis: a diagnostician's dilemma babesia canis canis, babesia canis vogeli, babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal rna genes mycoplasmal respiratory infections in small animals: cases ( - ) performance of a commercially available in-clinic elisa for the detection of antibodies against anaplasma phagocytophilum, ehrlichia canis, and borrelia burgdorferi and dirofilaria immitis antigen in dogs experimental transmission of bartonella henselae by the cat flea accuracy of polymerase chain reaction assays for diagnosis of feline immunodeficiency virus infection in cats morphologic, immunohistochemical, and ultrastructural characterization of a distinctive renal lesion in dogs putatively associated with borrelia burgdorferi infection: cases ( - ) association of bartonella species, feline calicivirus, and feline herpesvirus infection with gingivostomatitis in cats toxoplasmosis and neosporosis a combined approach for the enhanced detection and isolation of bartonella species in dog blood samples: pre-enrichment liquid culture followed by pcr and subculture onto agar plates clinical characteristics and predictors of mortality for cryptococcus gattii infection in dogs and cats of southwestern british columbia clinical and serologic findings in cats with cryptococcosis serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test canine rocky mountain spotted fever: a retrospective study of cases seroprevalences of antibodies against bartonella henselae and toxoplasma gondii and fecal shedding of cryptosporidium spp, giardia spp, and toxocara cati in feral and pet domestic cats evaluation of peptide-and recombinant protein-based assays for detection of anti-ehrlichia ewingii antibodies in experimentally and naturally infected dogs suspected ehrlichial infection in five cats from a household comparison of serologic evaluation via agar gel immunodiffusion and fungal culture of tissue for diagnosis of nasal aspergillosis in dogs prevalence of mycoplasmal and ureaplasmal recovery from tracheobronchial lavages and prevalence of mycoplasmal recovery from pharyngeal swab specimens in dogs with or without pulmonary diseases comparative strain analysis of anaplasma phagocytophilum infection and clinical outcomes in a canine model of granulocytic anaplasmosis protein electrophoresis on effusions from cats as a diagnostic test for feline infectious peritonitis performance of serologic tests used to detect heartworm infection in cats antigen and antibody testing for the diagnosis of blastomycosis in dogs feline ehrlichiosis: literature review and serologic survey acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention re-examination of feline leukemia virus: host relationships using real-time pcr wanke mm: canine brucellosis prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) canine and feline blood donor screening for infectious disease bartonella spp. antibodies and dna in aqueous humor of cats infectious diseases of the dog and cat american association of feline practitioners' feline retrovirus management guidelines effect of vaccination against feline immunodeficiency virus on results of serologic testing in cats utility of an in-office c elisa test kit for determination of infection status of dogs naturally exposed to borrelia burgdorferi quantitative measurement of c antibody following antibiotic treatment of borrelia burgdorferi antibody-positive nonclinical dogs ehrlichiosis and anaplasmosis in dogs and cats acvim small animal consensus statement on lyme disease in dogs: diagnosis, treatment, and prevention novel chemically modified liquid medium that will support the growth of seven bartonella species detection of antibodies to francisella tularensis in cats infectious diseases of the dog and cat canine nasal aspergillosis/ penicilliosis clinical presentation of anaplasma phagocytophilum-seropositive dogs residing in an endemic area dogs are definitive hosts of neospora caninum pcr detection of acute ehrlichia canis infection in dogs consensus statement on ehrlichial disease of small animals from the infectious disease study group of the acvim key: cord- -i jm p s authors: hu, jing; gao, mingyan; wang, zuobin; chen, yujuan; song, zhengxun; xu, hongmei title: direct imaging of antigen–antibody binding by atomic force microscopy date: - - journal: appl nanosci doi: . /s - - -w sha: doc_id: cord_uid: i jm p s direct observation of antigen–antibody binding at the nanoscale has always been a considerable challenging problem, and researchers have made tremendous efforts on it. in this study, the morphology of biotinylated antibody-specific immunoglobulin e (ige) immune complexes has been successfully imaged by atomic force microscopy (afm) in the tapping-mode. the afm images indicated that the individual immune complex was composed of an ige and a biotinylated antibody. excitingly, it is the first time that we have actually seen the ige binding to biotinylated antibody. alternatively, information on the length of ige, biotinylated antibodies and biotinylated antibody-specific ige immune complexes were also obtained, respectively. these results indicate the versatility of afm technology in the identification of antigen–antibody binding. this work not only lays the basis for the direct imaging of the biotinylated antibody-ige by afm, but also offers valuable information for studying the targeted therapy and vaccine development in the future. electronic supplementary material: the online version of this article ( . /s - - -w) contains supplementary material, which is available to authorized users. antigen-antibody binding research has particular significance in medical diagnosis, vaccine exploration, immunological study and disease mechanism analysis (nergiz et al. ; bruno et al. ) . in recent years, many immunoassay methods have been used to detect antigen-antibody binding, which include many additional biochemical reagents (meyer et al. ; stentzel et al. ; zhuang et al. ) . the extensive use of biochemical agents not only requires tedious process, but also causes serious environmental pollution. in addition, although the immunological techniques were able to monitor and quantify the signal of antigen-antibody, they require a large number of protein samples and there is no way to detect a single molecule. furthermore, the fatal drawback of these immunological approaches is that they cannot provide a visual image of antigen-antibody binding. accordingly, it is urgent to find an uncomplicated method to directly observe and determine the specific interactions between antigen and antibody at the molecular level. traditionally, electron microscopy, ultrasound imaging and optical microscopy have been widely known as biomolecular imaging methods (gopal et al. ; negishi et al. ; maresca et al. ). however, their actual use has been suffered from some inherent disadvantages, such as complex sample preparation and demanding operating environment (jung et al. ) . in , the greatest invention of the atomic force microscopy (afm) resoundingly opened the door to nano-imaging and nano-manipulating (krieg et al. ) . atomic force microscope is a multi-functional electronic supplementary material the online version of this article (https ://doi.org/ . /s - - -w) contains supplementary material, which is available to authorized users. biomolecular research instrument with particularly high spatial resolution, exceptionally low invasiveness, and incomparably elegant simplicity, which has attracted increasing attention in the past decades (alsteens et al. ) . actually, many biomolecules, such as proteins and dnas, have long been satisfactorily imaged by afm (ikai et al. ) . for instance, kumar et al. ( ) observed the morphologies of rc-lh -pufx complexes and intact atp-synthase by gentle tapping-mode afm. in addition, valero et al. ( ) performed afm to image the interlocked double-stranded dna nanostructures, including rotaxanes, catenanes and daisychain rotaxanes. additionally, the interaction between proteins and dna via afm imaging has also been extensively studied by taking advantage of their morphological differences. nevertheless, most studies on antigen-antibody interactions have focused on the afm-based single-molecule force spectroscopy, and only a few are based on afm imaging (tanja et al. ) . even afm imaging is studied at its height, and there is very little research on their appearance (wang et al. ; ouerghi et al. ) . to the best of our knowledge, the biotinylated antibody-specific ige immune complexes imaged by afm have never been reported. while it is conceptually straightforward to use images to analyze the ige interactions with biotinylated antibodies, the data analysis is complicated by the many conformations of the protein. in this work, we characterized the interaction between the ige and biotinylated antibodies by measuring changes in the morphology and length of each sample by tappingmode afm. it is exciting that afm image can be used to accurately describe ige binding to biotinylated antibodies. moreover, the length data of different samples can enhance the accuracy of our results. ige and biotinylated antibodies were purchased from human ige enzyme-linked immunosorbent assay (elisa) kit (solarbio science & technology ltd., beijing, china). magnesium chloride hexahydrate (mgcl · h o) was obtained from beijing chemical works ltd. (beijing, china). the mm × mm fluorphlogopite mica was supplied by taiyuan fluorphlogopite mica ltd. (changchun, china). all the chemicals were used as received without purification during the study. ultrapure water with resistivity no less than . mΩ·cm was employed in the experiments. experimentally, ige was determined by an elisa kit in accordance with the manufacturer's specifications. to image the antibody-specific ige immune complexes, the sample was prepared as displayed in fig. . in a typical experiment, μl of aqueous mgcl · h o ( mm) solution was first incubated on the surface of newly cleaved mica for min, and then μl of ige ( ng ml − ) solution was dripped. after min, the surface covering the ige was exposed to a biotinylated antibody solution ( μl, ng ml − ) for min. finally, the mica surface was thoroughly rinsed with water for three times and dried with nitrogen. similarly, the mg + -modified mica surfaces covered with the ige and biotinylated antibodies were also obtained, respectively. for comparison, the sample without the protein solution was prepared according to the above method. the surface morphology measurement and data acquisition of all samples were carried out by the afm system (nano wizard iii, germany) with the tapping-mode in air. the tap ai-g cantilevers (resonance frequency: khz, spring constants: n m − , curvature radius: nm) were used for the experiment, and the × pixels images were collected at the line rate of hz. after unremitting efforts, a japanese couple teruko and kimishige ishikaka discovered the skin-sensitizing ige in (ribatti ) . the discovery of ige was a major breakthrough in the area of immunology and allergy (hirohisa et al. ). since ige plays an absolutely essential role in allergic responses and can be used for the diagnosis and treatment of allergic diseases (han et al. ) , it is crucial to understand the interaction between the specific antigens and ige antibodies. herein, the binding of ige to biotinylated antibodies was first examined by the classic elisa method, and the corresponding results were obtained, as shown in fig. . it can be seen that the absorbance tends to grow in proportion to the ige concentration, which is in fair agreement with the manufacturer's instructions, indicating a good affinity between the ige and biotinylated antibodies. nevertheless, the minimum detection limit of elisa method was . ng ml − , which could not meet our requirement to study the interaction between antigen and antibody at the molecular level. to gain an insight into the details of a single ige binding to a single biotinylated antibody, the afm was further employed to visualize the morphology of ige before and after incubation with biotinylated antibodies, and the relevant data were also obtained. there was no significant difference in the afm image morphology of mica surface before and after the modification of mg + (fig. s ). therefore, the modification of mg + had no effect on the observation of mica surface proteins at the current resolution scale. additionally, the mica surface modified by mg + was very smooth, while some randomly distributed nanoparticles were found on the mica surface after incubation with ige and biotinylated antibodies (fig. ) , indicating that these proteins had been successfully adsorbed on the mg + -modified mica surface, respectively. as shown in fig. a and b, the ige molecules in various orientations individually dispersed on the surface of mica, which was consistent with our previous results (hu et al. ). an analogous phenomenon was observed in fig. c and d, where the biotinylated antibodies with different conformations were found to be evenly adsorbed on the mica surface. interestingly, there was a lot of little tail-like structures appeared in the view. it must be kept in mind that the biotinylated antibodies used in this study are biotinylated anti-human ige antibodies, which are immunoglobulin g (igg) antibodies with biotins. furthermore, igg shares the similar molecular architecture as ige, consisting of two antigen binding fragments (fab) and one crystallizable fragment (fc), and appears as a single globular morphology in most cases (funari et al ; gould and sutton ) . hence, it would be reasonable to believe that the biotin attached to igg molecule possesses the little taillike structure. however, it can be seen from the figures that not every igg is connected to a biotin. this phenomenon can be attributed to the following two principal reasons. first, the biotinylated antibodies were randomly distributed on the mica surface. second, their structures were imaged from the top view, and various morphologies could be obtained. besides that, the length of ige and biotinylated antibodies were also calculated from more than molecules, respectively. as shown in table , the length of ige was mainly between and nm ( %), which is close to the length of the biotinylated antibodies ( nm and nm, %). it is worth mentioning that the statistical length of the biotinylated antibodies does not include the biotin length. it should be noted that after incubation with biotinylated antibodies, the ige morphology changed significantly, resulting in a new characteristic image that was completely different from either ige or biotinylated antibody alone (fig. ) . furthermore, compared with the single ige and biotinylated antibody molecules, some larger particles (fig. ) could be detected in the field of view. it is worth noting that the corresponding results are derived from calculations and statistics of more than particles. these results strongly demonstrate that the ige has reacted with biotinylated antibodies, and we have successfully probed the biotinylated antibody-specific ige immune complexes. although it is easy to track and image individual biotinylated antibody-specific ige immune complexes that are well isolated from particles, not all of these complexes can be tracked, even on well-dispersed images. the untracked complexes included those that clustered with other particles, as well as those that were not completely within the image boundaries. in addition to the immune complexes, a number of nanoparticles were also observed with the structures corresponded to the ige, biotinylated antibodies and their detached fragments (the circles in fig. are typical representations of the structures). as observed, only some of the complexes were detected in the fig. , and some of the ige and biotinylated antibodies were not involved in the formation of the antigen-antibody complex. the main reason of this phenomenon is that the binding of ige to the biotinylated antibodies depends on the ability of the antibodies to capture biotinylated antibodies, and this specific ability is largely dependent on the orientation and conformation of the antibody. in the case of physical adsorption, when the ige orientation is random, only a small fraction of the adsorbed antibodies can capture biotinylated antibodies and retain them. in fact, the biotinylated antibodyspecific ige immune complexes have a wide variety of morphologies and sizes on the mica surface due to their threedimensional structures. it was extremely difficult to find all the orientations of the biotinylated antibody-specific ige immune complexes. hence, we randomly selected several immune complex molecules with high resolution to reveal interesting details about them, and their corresponding afm phase, three-dimensional and possible spatial images were shown in fig. a -d, e-h and i-l, respectively. specifically, fig. a , e and i would correspond to the biotinylated antibody-specific ige immune complex deposited on the surface by the fab and fc fragments of ige or biotinylated antibody. furthermore, when the immune complex is attached to the support via the ige fab fragment and the three fragments of biotinylated antibody, it would be shown in fig. b , f and j. additionally, the immune complex molecule could also flat on the mica surface by its six fragments. the immune complex with this conformation would produce the images as shown in fig. c , g and k. meanwhile, fig. d , h and l would represent the biotinylated antibody-specific ige immune complex adsorbed on the mica surface by the ige fragment and the two fab fragments of biotinylated antibody. it is also possible that the immune complex is located on the support by the ige fab and fc fragments and the two fab fragments of biotinylated antibody. overall, the quite good match of the d complex revealed the molecular mechanism by which a single biotinylated antibody interacts with one of the two fab fragments immobilized on the mica surface with ige. from fig. a -d, we can find that the biotin is sometimes linear and sometimes blocky, and the linear structure is easier to be discriminated than the blocky structure. there is no doubt that the flexible three-dimensional structure results in different forms of biotin on the mica surface. moreover, the tip moves over the tight biotin making the biotin image blurry. in modern medicine, antibody-targeted immunotherapy is a powerful tool for the treatment of cancer and autoimmune diseases. the growing prevalence of antibody-targeted treatment requires a deeper understanding of the functions of antibodies, especially their interaction with antigens (lu and sun ) . affinity is not only a vital parameter to determine the interaction between molecules, but also an important indicator to understand the molecular recognition, biological process, drug discovery and screening (oh et al. ; linke et al. ) . the results from the elisa depicted that the ige had a high affinity with biotinylated antibodies, which laid a foundation for further observation of biotinylated antibody-specific ige immune complexes. the length distribution histogram of the biotinylated antibodyspecific ige immune complexes as expected, the results from afm indicate that the different orientations of the antibody-specific ige immune complexes are formed on the mica surface, from which the ige and biotinylated antibodies can be readily identified because the biotinylated antibodies possess a little tail-like structure. admittedly, there are some immune complexes that confuse ige and biotinylated antibodies due to the inevitable factors of the tightness of the immune complexes themselves and the randomness of their distribution on the mica surface. in addition, due to the highly variable in the length of the antibody-specific ige immune complexes, we made a statistical analysis of the relevant data. although the length of these antibody-specific fig. afm phase (a, b, c and d) , three-dimensional (e, f, g and h) and spatial (i, j, k and l) images of the individual biotinylated antibodyspecific ige immune complexes ige immune complexes vary slightly in the range of - nm, it is consistent with the expected length of single immune complex. moreover, since the length of the immune complex is larger than that of ige and biotinylated antibody, we can also determine whether the ige is bound to the biotinylated antibodies by the particle length. thus, in our case, it is possible to distinguish between the antigen, antibody and antigen-antibody immune complex by the length and topography. obtaining knowledge about the topological structure of the proteins and their interactions is the key to understanding the fundamental causes of disease and developing drugs. in this respect, afm imaging technology provides a unique possibility for the study of proteins. in this paper, the morphology and length of ige, biotinylated antibodies and biotinylated antibody-specific ige immune complexes were analyzed by afm, respectively. the results of afm imaging demonstrated that the immune complexes exhibited various morphologies, and we were able to identify the ige and biotinylated antibody based on the protein morphology in most cases. moreover, the individual immune complex shows the larger size in length compared with the ige and biotinylated antibody. by combining the above information, we can quickly determine whether ige is bound to biotinylated antibodies. therefore, the morphology and length analysis can be extremely helpful to investigate antigen-antibody interactions at the molecular level and provide a new way for the studies of vaccines and targeted therapies. we are confident that our successful visualization of the biotinylated antibody-specific ige immune complexes at the single molecular scale will also enable the direct imaging of other immune complexes. atomic force microscopy-based characterization and design of biointerfaces antibody modified gold nanoparticles for fast colorimetric screening of rheumatoid arthritis single molecule characterization of uv-activated antibodies on gold by atomic force microscopy ige in allergy and asthma today immunogold fib-sem: combining volumetric ultrastructure visualization with d biomolecular analysis to dissect cell-environment interactions specifically immobilizing his-tagged allergens to magnetic nanoparticles for fast and quantitative detection of allergen-specific ige in serum samples landmark papers in our journal: articles i to iii of the series describing the discovery of ige by the ishizakas imaging the substructures of individual ige antibodies with atomic force microscopy atomic force microscope as a nano-and micrometer scale biological manipulator: a short review molecular imaging of membrane proteins and microfilaments using atomic force microscopy atomic force microscopy-based mechanobiology direct imaging of protein organization in an intact bacterial organelle using highresolution atomic force microscopy potent affinity material for tracing acetone and related analytes based on molecular recognition by halogen bonds structural mechanism of high affinity fcγri recognition of immunoglobulin g nonlinear x-wave ultrasound imaging of acoustic biomolecules technical report: xmapr-high-dynamic-range (hdr) quantification of antigenspecific antibody binding ag -modified bubble liposomes for targeted ultrasound imaging of tumor neovasculature an epoxysilane modified indium tin oxide electrode for the determination of pak : application in human serum samples ultra-sensitive and label-free probing of binding affinity using recognition imaging investigating antibody-antigen binding with atomic force microscopy the discovery of immunoglobulin e specific serum igg at diagnosis of staphylococcus aureus bloodstream invasion is correlated with disease progression single molecule force spectroscopy reveals two-domain binding mode of pilus- tip protein rrga of streptococcus pneumoniae to fibronectin design, assembly, characterization, and operation of double-stranded interlocked dna nanostructures characterizing antibody specificity to different protein morphologies by afm immunoglobulin g-encapsulated gold nanoclusters as fluorescent tags for dot-blot immunoassays conflict of interest the authors declare no conflict of competing interest. key: cord- -ent vu z authors: tan, joshua; sack, brandon k; oyen, david; zenklusen, isabelle; piccoli, luca; barbieri, sonia; foglierini, mathilde; fregni, chiara silacci; marcandalli, jessica; jongo, said; abdulla, salim; perez, laurent; corradin, giampietro; varani, luca; sallusto, federica; sim, b kim lee; hoffman, stephen l; kappe, stefan h i; daubenberger, claudia; wilson, ian a; lanzavecchia, antonio title: a public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein date: - - journal: nat med doi: . /nm. sha: doc_id: cord_uid: ent vu z immunization with attenuated plasmodium falciparum sporozoites (pfspz) has been shown to be protective, but the features of the antibody response induced by this treatment remain unclear. to investigate this response at high resolution, we isolated igm and igg monoclonal antibodies from tanzanian volunteers who were immunized by repeated injection of irradiated pfspz and who were found to be protected from controlled human malaria infection (chmi) with infectious homologous pfspz. all igg monoclonals isolated bound to p. falciparum circumsporozoite protein (pfcsp) and recognized distinct epitopes in the n-terminus, nanp repeat region, and c-terminus. strikingly, the most effective antibodies, as assessed in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the pfcsp n-terminal junction that is not in the rts,s vaccine. these dual-specific antibodies were isolated from different donors and used vh - or vh - alleles carrying tryptophan or arginine at position . using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies. investigate this response at high resolution, we isolated igm and igg monoclonal antibodies from tanzanian volunteers who were immunized by repeated injection of irradiated pfspz and who were found to be protected from controlled human malaria infection (chmi) with infectious homologous pfspz. all igg monoclonals isolated bound to p. falciparum circumsporozoite protein (pfcsp) and recognized distinct epitopes in the n-terminus, nanp repeat region, and cterminus. strikingly, the most effective antibodies, as assessed in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the pfcsp n-terminal junction that is not in the rts,s vaccine. these dual-specific antibodies were isolated from different donors and used vh - or vh - alleles carrying tryptophan or arginine at position . using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies. malaria is a serious global health threat, causing , deaths and million clinical cases in . much of the effort to develop a vaccine against the disease has focused on plasmodium falciparum sporozoites (pfspz), the asymptomatic parasite stage that is injected by mosquitoes into the host skin to initiate a malaria infection. after entering the skin, pfspz migrate to the liver, multiply in hepatocytes and then emerge in the blood, where the parasites cause malaria symptoms and differentiate into sexual stages for transmission. while natural infection by pfspz elicits little or no protective immunity to this stage of the life cycle , , subunit or whole organism vaccines based on pfspz can induce robust immune responses [ ] [ ] [ ] . the most advanced malaria vaccine candidate, rts,s, incorporates part of the p. falciparum circumsporozoite protein (pfcsp), which coats the pfspz surface and plays a key role in parasite migration out of the skin, entry into the liver parenchyma and invasion of hepatocytes [ ] [ ] [ ] [ ] [ ] [ ] . multi-site clinical trials in sub-saharan africa have shown that rts,s confers significant but modest and short-lived protection against clinical illness , , . an alternative approach that has shown promise is the use of whole attenuated pfspz as immunogens. this line of research is based on key early discoveries that immunization with irradiated p. berghei sporozoites protected mice against subsequent challenge and that immunization of humans with > irradiated mosquitoes carrying pfspz conferred sterilizing protection against controlled human malaria infection (chmi) [ ] [ ] [ ] . these studies have led to efforts to develop whole attenuated pfspz as a vaccine , and recent trials have shown that immunization with attenuated pfspz was highly protective in malaria-naïve volunteers and gave significant protection in malian adults , [ ] [ ] [ ] . while these results are promising, the specific mediators of this protective immune response have yet to be fully elucidated. studies of the antibody response have mainly investigated polyclonal serum responses to pfspz and pfcsp , [ ] [ ] [ ] , and highresolution analysis of the monoclonal antibodies generated by vaccination and their target antigens on the pfspz surface remains to be performed. these experiments could provide useful information for the improvement of whole sporozoite-based vaccines and for the identification of new antigens as subunit vaccine candidates, as well as to generate tools for prophylaxis of p. falciparum infection. we characterized the antibody response of tanzanian volunteers living in malaria-endemic regions who were immunized by repeated intravenous injection of irradiated pfspz (pfspz vaccine) and then underwent chmi with live homologous parasites (fig. a) . serum igm and igg antibodies, as measured by flow cytometry on live pfspz, increased following immunization, but did not show a clear association with protection from chmi (fig. b,c and supplementary fig. a,b) . memory b cells from five protected individuals were immortalized and screened by staining of intact pfspz to isolate human monoclonal antibodies against any surface antigen on pfspz ( supplementary fig. c) . most of the igg monoclonal antibodies bound to pfspz with high affinity (fig. d,e) . interestingly, in the two donors from whom both pfspz-specific igm and igg monoclonal antibodies were isolated, the igm antibodies were recovered at much higher numbers (fig. f ). the igm antibodies had fewer, but still substantial, mutations compared to the igg antibodies, consistent with an origin from igm memory b cells (fig. g ). in particular, the finding of an antibody lineage containing both igm and igg members suggests an incomplete switch in this response despite repeated immunization ( supplementary fig. d ). these results demonstrate that immunizations with pfspz vaccine induce a robust antibody response that retains a significant igm component. to identify the features of the most effective neutralizing monoclonal antibodies produced by the protected individuals, we tested a panel of igg antibodies in vitro for their capacity to inhibit pfspz traversal and invasion of a human hepatocyte cell line (fig. a) . the invasioninhibitory activity varied among antibodies and was significantly correlated with binding affinity to pfspz (fig. b) . a subset of antibodies was further tested in an in vivo mouse humanized liver model for their capacity to protect against natural, mosquito bitetransmitted infection by pfspz. some antibodies, such as mgg , mgg , mgh , mgu and mgu , were very potent in reducing liver burden by up to . %, while others, such as mgg , mgh , mgh and mgu , were less effective (fig. c) . these findings suggest that in vivo neutralizing activity may be related to the fine specificity of the antibodies. next, we set out to identify the target antigens of the monoclonal antibodies. strikingly, although we had used an antigen-agnostic approach to identify antibodies that bound to the pfspz surface regardless of specificity, we found that all of the antibodies bound to recombinant pfcsp (fig. d,e) , confirming that this is the most immunogenic protein on the pfspz surface [ ] [ ] [ ] . to understand the basis for effective neutralization, we mapped the specificity of the monoclonal antibodies using synthetic peptides that cover the n-terminus, the nanp repeat region, the n-terminal junction (connecting region between the n-terminal domain and nanp repeats), and the c-terminus of pfcsp. binding to the classical nanp repeats (nanp peptide), the pfcsp c-terminus, recombinant pfcsp or pfspz did not correlate with efficacy ( supplementary fig. a-d) . interestingly, however, binding to a minimal -mer peptide (npdp ) that covers the junction between the n-terminal domain and the nanp repeats was a shared characteristic of the most potent in vivo neutralizing antibodies (fig. f) . these potent antibodies also recognized the nanp peptide, suggesting that the capacity to bind both to the nanp repeats and to the n-terminal junction of pfcsp is the main feature of efficient neutralization. another distinctive characteristic of the most potent antibodies was the common usage of vh - family alleles carrying tryptophan or arginine at position (vh - f , here defined as including vh - , vh - - , vh - - and vh - alleles sharing > % identity) (fig. e , supplementary fig. , , supplementary table ). strikingly, vh - f was the most common vh gene used by igm antibodies isolated from donors g and u, with almost % of such antibodies carrying w or r ( supplementary fig. e ). collectively, these data indicate that the most potent antibodies have a dual specificity and share common vh gene usage. importantly, such antibodies were isolated from four out of five donors, suggesting that these antibodies belong to a public lineage and therefore have the potential to be readily induced by vaccination. to investigate the influence of somatic mutations on binding of vh - f antibodies to pfcsp, we focused on two clonally related and highly mutated antibodies, mgu and mgu (fig. a) . the unmutated common ancestor (uca) of these antibodies, which carries tryptophan at position , was able to bind to pfcsp and pfspz with low affinity, while substitution to serine , which is commonly found in vh - alleles, resulted in loss of binding (fig. b,c) . these findings, in conjunction with the high frequency of w in the igm sequences and the identification of putative vh - f alleles carrying w in the germline sequences obtained from non-b cells of donors g, u and w ( supplementary fig. , ) , identify vh - f alleles carrying w as a preferred feature for the initiation of this lineage-specific antibody response to pfcsp. the branch point of this clone achieved, through several mutations, high affinity binding to pfspz, pfcsp and nanp , while further mutations in mgu , but not mgu , increased breadth by conferring the unique ability to bind to npdp (fig. b-e, supplementary fig. ). despite their similarities in binding to pfspz, pfcsp and nanp , mgu was substantially more potent than mgu in the in vivo assay (fig. c) , suggesting that acquisition of binding to npdp is the key factor for potent neutralization. interestingly, mutagenesis studies of mgu suggest that w remains a critical residue for binding to npdp , but becomes dispensable for high affinity binding to nanp and full-length pfcsp in the fully mutated antibody (fig. f ). in contrast, in a second clonal family consisting of mgu and mgu , full binding to pfcsp, nanp , npdp and pfspz was already achieved by the branch point while the remaining mutations appeared redundant ( supplementary fig. a -e). to investigate the original specificities of germline vh - f antibodies, we analysed binding of the ucas of various vh - f antibodies to pfcsp peptides using a more sensitive beadbased assay. most ucas bound to nanp but not to npdp , suggesting that the antibodies generally started as nanp-specific and gained affinity for npdp through somatic mutations ( supplementary fig. f-m) . these data delineate a pathway of antibody development that is dependent on specific vh alleles and leads to antibodies with dual specificity. to identify the minimal residues recognized by antibodies at the pfcsp nterminal junction, we performed a mutational analysis on npdp , a -mer version of npdp that was used to provide a longer scaffold for binding (fig. g) . the loss of binding to certain peptides identified a specific motif (dpnanp) that was recognized by most antibodies regardless of vh gene usage. these findings, combined with data from peptide array experiments ( supplementary fig. ) , identify the n-terminal junction binding site of the most potent neutralizing antibodies as including the first unit of the nanp repeat region and flanking non-repeat sequences, providing a molecular basis for the dual specificity of these antibodies. to gain structural insights into the recognition of the n-terminal junction, we attempted to crystallize several vh - f antibodies and successfully crystallized mgg in complex with the ac- kqpadgnpdpnanp -nh peptide ( fig. a and supplementary table ) . only the c-terminal half (npdpnan, residues - ) of the peptide was visible, with most of the contacts being made by heavy-chain residues as shown by the fab buried surface area (fig. b) . specifically, the heavy-chain cdr loops form a groove in which the peptide resides ( fig. a and supplementary fig. a ). in addition, three interfacial waters are involved in an extensive hydrogen-bonding network, connecting the side chain of n to the base of cdr h and cdr h ( supplementary fig. b ). the ch/π interaction between p and w ( fig. a and supplementary fig. b ) confirms that the latter residue is critical for binding as indicated by the mutagenesis experiments. while most peptide residues, except for n , display a relatively large buried surface area (fig. c) , weak electron density for the residues visible at the peptide's termini (n , p , a and n ) ( fig. e and supplementary fig. c ) indicates structural flexibility and highlights the sequence dpn as the principal binding motif. interestingly, the dpn sequence displays a pseudo turn, which is stabilized by hydrogen bonding of the aspartate side chain to the asparagine backbone amide. such a conformation is very similar to turns observed for unbound nanp peptides in solution and in crystal structures of free and antibody-bound peptides (fig. d) [ ] [ ] [ ] [ ] [ ] . an isolated dpn motif is also present in the - c-terminal peptide, which may explain its binding to mgg (fig. e) , notwithstanding the significant differences between the dpn flanking residues in the - c-terminal and the n-terminal junctional peptides (ndpnr versus pdpna, respectively). binding to both npdp and nanp repeats suggests that the aspartate residue in the dpn motif is interchangeable with an asparagine. binding studies of mgg to npdp peptide mutants validate the specificity of mgg toward dpn and npn motifs (fig. g) . only when the central dpn and npn motifs within the npdp peptide are mutated to dpa and aan is mgg binding completely abrogated. in contrast, a third dpn motif present at the c-terminus of the npdp peptide does not contribute to binding, possibly indicating the importance of flanking residues for optimal binding. overall, these data imply that potential peptide-binding promiscuity allows mgg to bind to diverse epitopes on pfcsp. since other vh - f antibodies cannot bind to the - peptide, we expect that they will be less promiscuous and bind slightly larger or more specific sequences. the finding that the most potent monoclonal antibodies recognize a defined n-terminal junctional peptide suggests that this region could be a component of an effective subunit vaccine. in an initial attempt to investigate whether the npdp peptide might be sufficient to induce a protective response, we immunized balb/c mice with npdp conjugated to klh. all mice produced igg antibodies that were specific for the npdp peptide, but were at best weakly reactive for the nanp peptide ( supplementary fig. a,b) . strikingly, in spite of their ability to bind to pfspz, the mouse sera were unable to inhibit pfspz invasion of a hepatocyte cell line in vitro, suggesting that dual specificity for nanp and the n-terminal junction may be required for potent neutralizing function ( supplementary fig. c,d) . the reliance of the dual-specific antibodies that we isolated on specific human vh - f alleles suggests that mice, which do not have the counterparts of human vh - f genes, may not be the most suitable model organism to test a novel csp-based vaccine. rather, an organism such as the aotus monkey, which has a more similar vh gene repertoire to humans and contains vh - f -like genes carrying the equivalent of w , may be a more suitable choice. this study shows that the antibodies produced by vaccinated and protected african individuals contain a highly mutated igg component, as well as an important igm component with fewer but substantial mutations, as also seen in the response to blood-stage plasmodium antigens . the large igm component would be consistent with stimulation of marginal zone b cells in the spleen following intravenous immunization with a particulate antigen . strikingly, all of the igg antibodies that we identified recognized pfcsp, consistent with previous studies describing the immunodominance of this protein and its abundance on the pfspz surface [ ] [ ] [ ] , . our findings are in agreement with previous work that separately describes the importance of the pfcsp nanp repeat region and of the n-terminus , - , but, importantly, highlight the fact that antibodies that target epitopes in both regions simultaneously are more potent than antibodies that exclusively recognize each individual site. interestingly, the structural analysis and peptide mutational data indicate that these antibodies, which are originally specific for nanp motifs, do not acquire a completely unrelated specificity, but rather gain promiscuity for sequences centred on a dpn motif. this dual specificity is to a large extent encoded by vh - f alleles, but also requires extensive somatic mutations. the importance of dual specificity is highlighted by the fact that immunization with a single npdp peptide is not sufficient to confer protection despite eliciting pfspz-binding antibodies. the increased potency of the dual-specific antibodies could be due to the proximity of the n-terminal junction to region i (klkqp) of pfcsp, which is involved in the cleavage of the n-terminus to allow pfspz invasion of hepatocytes , . this n-terminal junction region is not included in the most advanced malaria vaccine candidate rts,s, which may explain its limited efficacy in malaria-endemic regions . these findings support continued work to develop whole pfspz vaccines, which contain the entire pfcsp, and provide a rationale for further attempts to develop refined vaccination approaches to elicit dual-specific antibodies using prime-boost strategies with improved carriers , . the finding that the most potent antibodies share common vh gene usage in multiple donors is consistent with a public antibody response that can be readily induced by vaccination. these results are reminiscent of previous work on the use of particular vh genes and their allelic forms in the response to the stem of influenza haemagglutinin , and justify further efforts to investigate the role of vh gene polymorphisms in protective antibody responses. nevertheless, whether these antibodies are sufficient to protect humans and why some individuals were not protected remain to be established. possible reasons for the latter include the lack of a vh - f allele, the incomplete maturation of the vh - f antibodies, or insufficient production of the potent antibodies. the antibodies described could be used to obtain proof of concept that antibodies alone can be protective in vivo in humans, as previously shown in mice and non-human primates [ ] [ ] [ ] , and pave the way for the use of antibodies in the prophylaxis of p. falciparum infection and for the development of improved antibody-based subunit malaria vaccines. aseptic, purified cryopreserved pfspz of the nf strain provided by sanaria ® were used in serum and monoclonal antibody binding experiments. pfspz produced by the center for infectious disease research, seattle was used in all in vitro and in vivo functional assays. following informed consent, blood samples used in this study were collected from malaria pre-exposed volunteers during a clinical phase clinical trial of the safety, immunogenicity and protective efficacy of the sanaria ® pfspz vaccine in bagamoyo, tanzania for serum preparation, whole blood was collected in vacutainer tubes (bd) containing clot activators and kept at room temperature until a clot was formed. the tube was centrifuged at , × g for min at °c and the serum fraction was stored at − °c. peripheral blood mononuclear cells (pbmcs) were isolated from whole blood by ficoll density gradient centrifugation and resuspended in freezing medium for long-term storage in liquid nitrogen. cryopreserved pfspz (sanaria ® ) were thawed and stained with different concentrations of tanzanian sera or monoclonal antibodies in . × sybr green i (thermofisher scientific) for min at °c. the pfspz were washed twice by centrifugation at × g for min. human serum antibody binding was detected using . μg ml − alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, - - ) or alexa fluor conjugated goat anti-human igm (jackson immunoresearch, - - ). mouse serum antibody binding was detected using μg ml − pe-cy -conjugated goat anti-mouse igg (biolegend, ) or pe-cy -conjugated rat anti-mouse igm (bd biosciences, ). facs diva (version . ) was used for acquisition of samples and flow-jo (version . ) was used for facs analysis. the pfspz were gated based on high fluorescence in the fitc channel. median fluorescence intensity (mfi) of the pfspz in the alexa fluor or pe-cy channel was calculated to quantify igg or igm binding. the concentration of antibody needed to achieve mfi , (conc ) was calculated by interpolation of binding curves fitted to a sigmoidal curve model (graphpad prism ) as a measure of affinity. the gating strategy can be found in supplementary figure . igm or igg memory b cells were isolated from frozen peripheral blood mononuclear cells (pbmcs) by magnetic cell sorting with . μg ml − anti-cd -pecy antibodies (bd, ) and mouse anti-pe microbeads (miltenyi biotec, - - ), followed by facs sorting using . μg ml − alexa fluor -conjugated goat anti-human igg (jackson immunoresearch, - - ), μg ml − alexa fluor -conjugated goat anti-human igm (invitrogen, a ) and / pe-labeled anti-human igd (bd, ). as previously described , sorted b cells were immortalized with epstein-barr virus (ebv) and plated in single cell cultures in the presence of cpg-dna ( . μg ml − ) and irradiated pbmc-feeder cells. two weeks post-immortalization, the culture supernatants were tested (at a / dilution) for binding to pfspz by flow cytometry using a no-wash protocol. briefly, cryopreserved pfspz were thawed, stained with the supernatants in . × sybr green i for min at room temperature, and incubated with . μg ml − alexa fluor -conjugated goat anti-human igg or anti-human igm for hour at °c. only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies. the ability of monoclonal antibodies and serum to prevent pfspz invasion and traversal in vitro was tested as previously described , . briefly, monoclonal antibodies at μg ml − were mixed with freshly dissected pfgfp_luc sporozoites in dmem media containing fitc-dextran, % fbs, pen/strep, fungizone and l-glutamine and incubated at °c for min. these pfspz were then added to hc hepatoma cells plated one day prior at , cells/well in a -well plate for a final moi of . ( , pfspz: , hc ). plates were then spun at × g for min and the pfspz were left to infect for min at °c. cells were then fixed, stained with μg ml − of the monoclonal antibody a conjugated to alexafluor- and analyzed by flow cytometry for invaded cells ( a /alexafluor- positive) or traversed cells (fitc-dextran positive). frg huhep mice were purchased from yecuris, inc. and infected by bite of pfgfp_luc mosquitos - hours following intraperitoneal injection of μg/mouse of each monoclonal antibody or human igg control as described previously . parasite liver burden was determined by bioluminescent imaging using an ivis imager at day at the peak of liver burden. reductions in liver burden were calculated by normalization to the mean of control mice injected with an equivalent dose of human igg within each bite experiment. all animal procedures were conducted in accordance with and approved by the center for infectious disease research institutional animal care and use committee (iacuc) under protocol sk- . the seattle biomed iacuc adheres to the nih office of laboratory animal welfare standards (olaw welfare assurance # a - ). cdna was synthesized from selected b-cell cultures and both heavy chain and light chain variable regions (vh and vl) were sequenced as previously described . the usage of vh and vl genes and the number of somatic mutations were determined by analyzing the homology of vh and vl sequences of monoclonal antibodies to known human v, d and j genes in the imgt database (version . . ) . antibody-encoding sequences were amplified and sequenced with primers specific for the v and j regions of the given antibody. sequences were aligned with clustal omega (version . . ) . unmutated common ancestor (uca) sequences of the vh and vl were inferred with antigen receptor probabilistic parser (arpp) ua inference software, as previously described , or constructed using imgt/v-quest . phylogenetic trees were generated with the dna maximum likelihood program (dnaml) of the phylip package, version . , . antibody heavy and light chains were cloned into human igg , igκ and igλ expression vectors and expressed by transient transfection of expi f cells (thermofisher scientific) using polyethylenimine. cell lines were routinely tested for mycoplasma contamination. the antibodies were affinity purified by protein a chromatography (ge healthcare). the kqpadgnpdpnanp peptide was ordered from innopep inc. with a purity of > % and containing chlorine counter ions. the peptides have n-terminal acetylation and cterminal amidation to eliminate charges at the peptide termini. the mgg -peptide complex was crystallized from a solution containing mgg at . mg ml − in tbs buffer ( mm tris-hcl, mm nacl, . mm kcl, ph . ) with a : molar ratio of ac-kqpadgnpdpnanp-nh peptide to fab. crystals were grown using sitting drop vapor diffusion with a well solution containing . m kh po , % glycerol, % peg at k and typically appeared within days. crystals were cryo-cooled without additional cryoprotection. x-ray diffraction data were collected at the advanced light source (als) . . . data collection and processing statistics are outlined in supplementary table . data sets were indexed, integrated, and scaled using the hkl- package (version ) . the structures were solved by molecular replacement using phaser (version . . ) with a homology model (swiss-model - and pigspro ) for mgg as a search model. after refinement of the fab using phenix.refine (version . - ) combined with additional manual building cycles in coot (version . . ) , positive fo-fc density was observed in the fab combining site for the peptide. the peptide was manually built into the difference density fo-fc map, followed by additional rounds of refinement of the complex in phenix.refine and manual building cycles in coot . buried surface areas (bsa) were calculated with the program ms (version . ) , using a . -Å probe radius and standard van der waals radii . total iggs were quantified using half-area, high-binding -well plates (corning) with μg ml − goat anti-human igg (southernbiotech, - ) using certified reference material (erms-da , sigma-aldrich) as a standard. to test specific antibody binding, elisa plates were either directly coated with μg ml − of recombinant pfcsp (sanaria ® , sequence previously shown ), μg ml − of peptide - or μg ml − of peptide - , or first with μg ml − of avidin (sigma-aldrich), followed by μg ml − of nanp (nanpnanpnanpnanpna), npdp (kqpadgnpdpnanpnvdpn), npdp (kqpadgnpdpnanpn) or various npdp peptide mutants. non-specific binding to plates coated with an irrelevant control peptide was tested to exclude polyreactivity of the antibodies. all peptides and mutants were synthesized with biotin attached to the c-terminus (a&a labs). plates were blocked with % bovine serum albumin (bsa) and incubated with titrated antibodies, followed by / ap-conjugated goat anti-human igg (southern biotech, - ). plates were then washed, substrate (p-npp, sigma) was added and plates were read at nm. streptavidin beads with different levels of fitc labelling (svfb- - k, spherotech) were coated with μg ml − of biotinylated nanp , npdp , npdp or a negative control peptide for min at room temperature. the beads were washed and incubated with titrations of monoclonal antibodies for min at room temperature. antibody binding was detected with . μg ml − alexa fluor -conjugated goat anti-human igg or anti-human igm. the ucas were compared for binding to nanp and npdp at a concentration of μg ml − (supplementary fig. f ). biotinylated npdp and nanp peptides were diluted ( nm) in hepes buffered saline (hbs) ( mm hepes, ph . , mm nacl, mm edta, . % surfactant . hbs was also used as running buffer. an irrelevant biotinylated -mer peptide was used as a control for non-specific interactions. a neutravidin-immobilized nlc proteon sensor chip (biorad) was pre-conditioned with an nacl solution ( m) and the biotinylated peptides were injected onto the chip. the monoclonal antibodies were diluted and titrated in hbs ( - . - . - . - . nm) and injected onto chip; one channel of the chip was injected with hbs and used as reference for the analysis. all injections were made at a flow rate of μl/min. injection time and dissociation time were s and s, respectively. each binding interaction of the monoclonal antibodies with the biotinylated peptides was assessed using a proteon xpr instrument (biorad) and data were processed with proteon manager software (version . . . ). k a , k d and k d were calculated by applying the langmuir fit model. peptides of -amino acid lengths spanning the entire pfcsp (with a shift of a single amino acid between peptides) were synthesized and coated onto a microarray chip (peppermap® linear epitope mapping, pepperprint gmbh). the peptides were incubated with μg ml − of monoclonal antibodies for h at °c, followed by incubation with dylight conjugated goat anti-human igg to detect antibody binding. female balb/c mice ( - weeks of age) were obtained from envigo laboratories. all procedures were performed in accordance with guidelines by the swiss federal veterinary office and after obtaining ethical approval from the ufficio veterinario cantonale, bellinzona, switzerland (approval number: ). keyhole limpet haemocyanin (klh)conjugated npdp (genscript) was reconstituted in water and formulated with % mf (addavax, invivogen) according to the manufacturer's instructions. mice were immunized subcutaneously with μg of peptide on day and . mice were bled on day . recovered sera were used for staining of pfspz by flow cytometry and for binding to pfcsp and pfcsp peptides by elisa. the number of mutations in the heavy chains of igg (n= antibodies) and igm (n= antibodies) isolated from the tanzanian volunteers were compared by a two-sided t-test. results are shown as mean ± s.d.. in this test, n refers to the number of antibodies, p = . , t = . , df = . a two-tailed spearman's correlation was performed to correlate invasion with binding affinity to pfspz (from n= representative experiment out of ), p = . . in the in vivo test of the monoclonal antibodies, error bars show s.d. and were calculated from n = or mice for each antibody. a one-sided anova with kruskal-wallis post test was used to compare the percentages of liver burden with that of control mice injected with irrelevant human igg; for the anova, p = . , f = . , df = , df = . for the kruskal-wallis post test, the results for each individual antibody are presented as *p≤ . , **p≤ . , ****p< . . a two-tailed spearman's correlation was performed to correlate affinity for npdp , pfcsp, nanp , - or pfspz with in vivo antibody efficacy (from n= representative experiment out of ). p = . , . , . , . and . , respectively. the confidence intervals were not determined by prism as n< for each correlation. in all other cases, n refers to the number of independent experiments. sequence data of the monoclonal antibodies isolated in this study will be deposited in genbank (https://www.ncbi.nlm.nih.gov/genbank/). the x-ray structure factors and coordinates have been deposited in the protein data bank (pdb id bqb). refer to web version on pubmed central for supplementary material. a, binding interface of mgg in complex with the kqpadgnpdpnanp peptide; only residues to of the peptide have interpretable electron density (indicated in bold). the peptide is shown in the cartoon representation with sidechains as sticks, while the heavy and light chains of mgg are shown as dark and light grey surfaces respectively. the cdr loops are in the cartoon representation: cdrh (green), cdrh (blue), cdrh (magenta), cdrl (light green), cdrl (light blue) and cdrl (pink). the w sidechain is shown as blue sticks and the interfacial waters are highlighted as red spheres. b, buried surface area (bsa) for the heavy chain (hc) and light chain (lc) with the peptide. c, bsa for individual peptide residues with the fab. d, pseudo turn for the dpn motif of the bound peptide (yellow carbons) and type i β-turn for the previously published crystal structure of the unbound anpna peptide (green carbons) . stabilizing hydrogen bonds between the sidechain of d /n and the amide backbone of n /n in the two structures are highlighted by the dashed line. e, fo-fc electron density map for the n-terminal peptide contoured at . σ (dark blue) and . σ (light blue). naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications an intensive longitudinal cohort study of malian children and adults reveals no evidence of acquired immunity to plasmodium falciparum infection the rts,s malaria vaccine antibody and b cell responses to plasmodium sporozoites protection against malaria at year and immune correlates following pfspz vaccination the basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of plasmodium falciparum sporozoites malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes the plasmodium circumsporozoite protein is proteolytically processed during cell invasion efficacy and safety of rts s/as malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase individually randomised controlled trial heparan sulfate proteoglycans provide a signal to plasmodium sporozoites to stop migrating and productively invade host cells the malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host four-year efficacy of rts,s/as e and its interaction with malaria exposure seven-year efficacy of rts,s/as malaria vaccine among young african children protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei immunization of man against sporozoiteinduced falciparum malaria sporozoite induced immunity in man against an ethiopian strain of plasmodium falciparum protection of humans against malaria by immunization with radiationattenuated plasmodium falciparum sporozoites development of a metabolically active, non-replicating sporozoite vaccine to prevent plasmodium falciparum malaria protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine sterile protection against human malaria by chemoattenuated pfspz vaccine safety and efficacy of pfspz vaccine against plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in mali: a randomised, double-blind phase trial rationale for development of a synthetic vaccine against plasmodium falciparum malaria the circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites natural parasite exposure induces protective human anti-malarial antibodies conformational preferences of synthetic peptides derived from the immunodominant site of the circumsporozoite protein of plasmodium falciparum by h nmr crystal structure of an npna-repeat motif from the circumsporozoite protein of the malaria parasite plasmodium falciparum structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein t-dependent b cell responses to plasmodium induce antibodies that form a highavidity multivalent complex with the circumsporozoite protein identification of five different ighv gene families in owl monkeys (aotus nancymaae) somatically hypermutated plasmodium-specific igm + memory b cells are rapid, plastic, early responders upon malaria rechallenge human marginal zone b cells total and putative surface proteomics of malaria parasite salivary gland sporozoites interrogating the plasmodium sporozoite surface: identification of surfaceexposed proteins and demonstration of glycosylation on csp and trap by mass spectrometrybased proteomics an immunologically cryptic epitope of plasmodium falciparum circumsporozoite protein facilitates liver cell recognition and induces protective antibodies that block liver cell invasion the n-terminal domain of plasmodium falciparum circumsporozoite protein represents a target of protective immunity proteolytic cleavage of the plasmodium falciparum circumsporozoite protein is a target of protective antibodies versatile virus-like particle carrier for epitope based vaccines design of a hyperstable -subunit protein icosahedron rapid development of broadly influenza neutralizing antibodies through redundant mutations vaccine-induced antibodies that neutralize group and group influenza a viruses monoclonal, but not polyclonal, antibodies protect against plasmodium yoelii sporozoites inability of malaria vaccine to induce antibodies to a protective epitope within its sequence humoral protection against mosquito bite-transmitted plasmodium falciparum infection in humanized mice synthesis and immunological characterization of -mer and -mer peptides corresponding to the n-and c-terminal regions of the plasmodium falciparum cs protein molprobity: all-atom structure validation for macromolecular crystallography an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus development of a quantitative flow cytometrybased assay to assess infection by plasmodium falciparum sporozoites efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning imgt, the international immunogenetics information system a new bioinformatics analysis tools framework at embl-ebi reconstructing a b-cell clonal lineage. i. statistical inference of unobserved ancestors co-evolution of a broadly neutralizing hiv- antibody and founder virus processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software swiss-model: modelling protein tertiary and quaternary structure using evolutionary information protein structure homology modeling using swiss-model workspace the swiss-model workspace: a web-based environment for protein structure homology modelling pigspro: prediction of immunoglobulin structures v phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot the molecular surface package side-chain torsional potentials: effect of dipeptide, protein, and solvent environment we would like to thank m. nussenzweig (rockefeller university) and h. wardemann (german cancer research center) for providing reagents for antibody cloning and expression. this work was supported in part by the swiss the authors would like to thank first and foremost the study volunteers for their participation in the study. we also thank the entire study team at the bagamoyo branch of the ifakara health institute and the manufacturing, quality control, regulatory and clinical teams at sanaria, inc. for their contributions to the conduct of the trial. we would like to thank prof. marcel tanner (former director of the swiss tropical and public health institute, basel) for his vision and support of the development of the clinical trial platform enabling whole sporozoite-based malaria vaccine trials in bagamoyo, tanzania. key: cord- -sh w mye authors: lu, shuai; li, yuguang; wang, fei; nan, xiaofei; zhang, shoutao title: leveraging sequential and spatial neighbors information by using cnns linked with gcns for paratope prediction date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: sh w mye antibodies consisting of variable and constant regions, are a special type of proteins playing a vital role in immune system of the vertebrate. they have the remarkable ability to bind a large range of diverse antigens with extraordinary affinity and specificity. this malleability of binding makes antibodies an important class of biological drugs and biomarkers. in this article, we propose a method to identify which amino acid residues of an antibody directly interact with its associated antigen based on the features from sequence and structure. our algorithm uses convolution neural networks (cnns) linked with graph convolution networks (gcns) to make use of information from both sequential and spatial neighbors to understand more about the local environment of the target amino acid residue. furthermore, we process the antigen partner of an antibody by employing an attention layer. our method improves on the state-of-the-art methodology. a ntibody, also known as immunoglobulin, is a yshaped protein consisting of two light chains and two heavy chains [ ] , and can bind to a specific surface of the antigen, named epitope. amino acid residues of an antibody directly involved in binding epitope is called paratope [ ] . the accurate recognition of paratope on a given antibody would greatly improve antibody affinity maturation [ ] - [ ] and de novo design [ ] - [ ] . we can get high resolution structure of antibodyantigen complex by experimental methods, such as xray [ ] , nrm [ ] and cryo-em [ ] . however, it remains time consuming and empirical [ ] . as more and more protein structures including antibody-antigen complexes were analyzed, the machine learning-based methods can be used for predicting paratope by learning the paratope-epitope interaction patterns from known antibody-antigen complex structures. according to the type of selecting neighbors of target residue for representing and predicting, the machine learning-based methods can be divided into two categories, leveraging sequential neighbors or spatial neighbors. as for methods leveraging sequential neighbors, a part of the antibody sequence was used consisting of the target residue and additional forward and backward sequential neighbors. sequential neighbors were selected from the whole sequence of antibody like the methods in [ ] - [ ] , and others only took advantage of the sequence of cdr region [ ] , [ ] . although the sequence was always available at the stages of an antibody discovery campaign earlier than the structure, machine learning methods using spatial neighbors can provide more precise definition of the paratope. in [ ] , the antibody surface patch which was a set of amino acid residues adjacent to each other on the antibody surface, were represented by d zernike descriptors. and the stateof-art method [ ] represented an antibody as a graph where each amino acid residue was a node and k nearest spatial neighbors were used in the convolution operator. in this work, we utilize the sequential and spatial neighbors of the target antibody residue by using convolutional neural networks (cnns) linked with graph neural networks (gcns) for paratope prediction. first, we construct an antibody residues feature matrix form sequence-based and structure-based information. next, we employ cnns which take the residues feature matrix as input with a fixed window size for considering the influence of sequential neighbors. then, the output of cnns are directly fed to gcns for learning the local environment of spatial neighbors. at last, our program predicts the binding probability of each antibody residue. we also compare results with those from other paratope predictors, and our framework achieves the best performances. moreover, we add an attention layer to our model performs best attempting gain more information from antigen partner. we use the dataset the same as [ ] . all the complexes in training set are collected by [ ] from the training set used to train paratome [ ] , antibody i-patch [ ] and parapred [ ] predictors. the complexes in test set are fetched from abdb database [ ] . the antibody-antigen complexes present in abdb are split into two categories depending on whether their antigen is a protein or not. in both training and test sets, the complexes whose resolution better than Å or the antibody sequence which has more than % sequence identity are removed. the training set is further split into two disjoint sets: a reduced training set and a validation set,and the validation set is used to tune the hyper parameters in the predictive model. structures with nonprotein-binding antibodies are removed in the state-of-art method [ ] resulting in complexes for training, for validation and for testing. specifically, the complexes with pdb id ap and kve, only has one chain in antibody which are still retained. to construct the input matrix, we encode the d antibody sequence as a d numerical matrix with the dimension of (l, n ), where l is the length of the antibody sequence and n is the residue features vector dimension ( here). as shown in fig. , the feature representation for amino acid residue a is donated by x a . different components of the feature representation are denoted by the superscripts . each box indicates the program used to extract a given set of features. all those features can be classified into two classes according to the source: sequence-based and structure-based. one-hot encoding: the type of amnio acid residue (only possible natural types are considered) is encoded to a dimensional vector, where where each element is either or and indicates the existence of a corresponding amino acid residue. seven physicochemical parameters: those parameters are about physicochemical properties of residues summarized by [ ] . profile features: we run psi-blast [ ] against the nonredundant (nr) [ ] database for every antibody sequence. then we get the pssm and psfm matrix, both with dimension (l, ) , as well as a d vector related with column entropy with dimension l, where l is the length of the antibody sequence. relative accessible surface area, secondary structure, phi and psi torsion angles for each residue: those features are computed using dssp [ ] . the secondary structure totally has eight classes and is represented by one-hot encoding. half sphere amino acid composition: hsaac captures the amino acid residue composition in the direction of the side chain of a residue, defined as the number of times a particular amino acid occurs in that direction within a minimum atomic distance threshold of . Å from the residue of interest. residue depth: we calculate the average distance of the atoms of a residue from the solvent accessible surface by msms [ ] . protrusion index: the protrusion index of a nonhydrogen atom is calculated using psaia [ ] which is defined as the proportion of the volume of a sphere with a radius of . Å centered at that atom that is not filled with atoms [ ] . each element of this vector is normalized to have the range from to as in [ ] . b-factor: the b-factor (or temperature factor) is an indicator of thermal motion about an atom. we use the maximum b-factor of any atom for each residue. we represent an antibody as a graph [ ] , where each residue is a node whose features represent the properties of the residue. we define the spatial neighbors of a residue as a set of k ( , in our work) closest residues determined by the mean distance between their heavy atoms [ ] . fig. shows sequential and spital neighbors of a target residue. from the analyzed d structure of an antibody-antigen complex, a residue on antibody is judged to belong to the paratope if at least one of its atoms is located within . Å from any antigen atoms like previous methods [ ] , [ ] . the sequence of the input antibody with length l is considered as a set of sequential nodes s and each node is represented as a d vector s i : all the nodes of the antibody sequence compose a d features matrix as said in sectioon . . our cnns uses one filter function, where the input is s i−w:i+w = {s i } l i= = s ( ) and the output is a hidden vector s where f is a non-linear activation function (e.g. relu), w c is the wight matrix, and the b c is the bias vector. here we use residual connections which act as a shortcut connection between inputs and outputs of some part of a network by adding inputs to outputs shown as as a result, we apply the function to obtain s set of hidden vector of every position of the residue sequence: we use the graph convolution [ ] which enables orderindependent aggregation of properties over spatial neighbors of target residue and together contributes to the formation of a binding interface. for a node s i , the receptive field consisting of k spatial neighbors g i = {g i } k j= from the input graph. after processing by cnns, result of a node is s (t) and its spatial neighbors are g the parameters of this operation include the aggregation weight matrix w t for the target node, the aggregation weight matrix w g for the neighboring nodes, and the bias vector b g . thus, multiple layers can be stacked to produce high-level representations for each node s finally, two fully connected layers perform classification for each antibody residue z (t) i after processing by cnns and gcns. an inverse logit function transforms each residue's output y i to indicate the probability of belonging paratope shown as we implement our model using pytorch [ ] v . . validation sets are used to find the optimal set of network training parameters for final evaluation. the training details of these neural networks are as follows: optimization: momentum optimizer with nesterov accelerated gradients; learning rate: . ; batch size: ; dropout: . ; sequential neighbors size: (fixed); spatial neighbors in the graph: (fixed); number of layers in gnns: , or ; number of layers in cnns: , or . training times of each epoch vary from roughly - minutes depending on network depth, using a single nvidia rtx gpu. for each combination, networks are trained until the performance on the validation set stops improving or for a maximum of epochs. gcns have the following number of filters for , and layers, respectively: ( ), ( , ), ( , , ). all weight matrices are initialized as in [ ] and biases are set to zero. training is carried out by minimizing the weighted cross-entropy loss function [ ] . in this secction, we compute precision and recall by predicting residues as paratope with probability above . [ ] . as the area under the receiver operating characteristics curve (auc roc) is threshold-independent and increases in direct proportion to the overall prediction performance, we take it to assess the overall predictive abilities. beside, we consider the area under the precision recall curve. to provide robust evaluation of performance, we have trained and tested all networks five times, and computed the mean and standard error. results comparing the auc roc and auc pr of various layers combination of cnns and gcns are shown in table and table . our first observation is that the all the cnns linked with gcns methods, with auc roc around . and auc-pr around . , outperform the individual cnns or gcns methods which have distinct lower auc prs, showing that the incorporation of combined information from a residue's sequential and spatial neighbors improves the accuracy of interface prediction. this matches the biological intuition that the region around a residue should impact its binding affinity [ ] . we also observe that the effect of the combination number of cnns and gcns layers is not linear, i.e. more layers will not achieve better performance. indeed, in protein interface prediction, networks with more than four layers performed worse in [ ] . in addition, one layer gcn achieves better performance than two layer gcns about paratope prediction in task-specific learning in [ ] . we agree with these findings and draw the same conclusions. as said in secttion . , residue features are classified into two classes: sequence-based and structure-based according to the source. furthermore, sequence-based features can be divided into three parts: residue type one-hot encoding(a), profile features(b) and the seven physicochemical parameters(c) as their different properties. all the structurebased feature are considered as a individual part(d). because the residue type is the most basic features, all kinds of combination must include it's one-hot encoding, e.g. a+b, a+c, a+d, a+b+c, a+b+d, a+c+d, a+b+c+d. we obtain the best performance form the model with layers cnns linked with layers gcns as shown in table and table . hence, we train this model again using the other kinds of residue features combination. each combination was evaluated by averaging all the auc roc and auc pr of all the antibodies in testing set. both mean value and standard deviation are reported in fig. and fig. . from fig. , we can see that three residue features combinations(a+b: . ± . , a+b+c: . ± . , a+b+d: . ± . ) almost achieve the optimal performance. all of them contain the profile features(b). as for the auc pr in fig. , we can see that performance vary from all kinds combination. the model using all the features still works best. as shown in fig. . and fig. . , we compare our method to other existing methods specifically for paratope prediction, i.e. antibody i-path which pays attention to energetic importance(auc roc: . , auc pr: . ) [ ] , parapred which consists of cnn and rnn-based networks(auc roc: . , auc pr: . ) [ ] , models using d zernike descriptors(auc roc: . , auc pr: . ) [ ] and graph convolution and attention mechanism(auc roc: . , auc pr: . ) [ ] . note that these methods only considering sequential or spatial neighbors of target antibody residue. our model achieves competitive or greater performance compared to these methods on both auc roc( . ± . ) and auc pr( . ± . ). an attention layer was used to explore the specific interaction between antibody and antigen pairs on paratope and epitope prediction [ ] . the contributions of attention layer only were accessed about epitope predictor. in this study, we add an attnetion layer to our best model resulting in lower performance(auc roc: . ± . , auc pr: . ± . ) for paratope prediction. fig. shows the heatmap of attention score between every pairs of residues from the complex on which our model performed best(pdb id k ). but we can not see outstanding performance as in epitope predictor, which could be caused by the different environment components of epitope and paratope [ ] , [ ] . in this study, we design and implement a new structurebased paratope predictor leveraging sequential and spatial neighbors of target antibody residue. our model is trained on the antibody-antigen complex structures collected from datasets of some paratope predictors which includes the most structures. moreover, we utilize more residue features consisting of sequence-based and structure-based. experimental results with a training dataset and an independent validation dataset demonstrate the efficiency of our method. the superior performances of our method are due to several reasons, including a rich dataset, more sufficient features selection, and careful construction of the prediction model considering sequential and spatial neighbors at same time. we note that our program has two potential disadvantages. first, the predictor needs antibody structure as it takes structure-based residue features as input. second, at the stage of extracting residue features, it consumes long computer time as psi-blast [ ] needs to be performed. in our future work, we will take adjacent information from antibody sequence so that the predictor can make use of gcns without structure. we will attempt to accelerate the computation speed by using several servers to concurrently perform psi-blast [ ] . biomolecule binding motifs mining is a long-term challenge for understanding their function. the forming incorrect interaction between some critical molecules has been revealed as one of the import causes for diseases like covid- [ ] . the method proposed in this study is specifically for identifying the antibody-antigen binding residues. in the future work, we will future investigate the applicability of our model to other types of molecules binding residues prediction problem, e.g., drug-target interaction prediction [ ] . structure, function and properties of antibody binding sites antibody and antigen contact residues define epitope and paratope size and structure effective optimization of antibody affinity by phage display integrated with high-throughput dna synthesis and sequencing technologies insights into the structural basis of antibody affinity maturation from next-generation sequencing the effects of framework mutations at the variable domain interface on antibody affinity maturation in an hiv- broadly neutralizing antibody lineage in silico methods for design of biological therapeutics computational design of epitope-specific functional antibodies epitope-directed antibody selection by site-specific photocrosslinking watching a protein as it functions with -ps time-resolved x-ray crystallography methodological advances in protein nmr towards atomic resolution structural determination by single-particle cryo-electron microscopy computer-aided antibody design paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure prediction of site-specific interactions in antibody-antigen complexes: the proabc method and server antibody i-patch prediction of the antibody binding site improves rigid local antibody-antigen docking parapred: antibody paratope prediction using convolutional and recurrent neural networks attentive cross-modal paratope prediction antibody interface prediction with d zernike descriptors and svm learning context-aware structural representations to predict antigen and antibody binding interfaces abdb: antibody structure database-a database of pdb-derived antibody structures generation and evaluation of dimension-reduced amino acid parameter representations by artificial neural networks gapped blast and psi-blast: a new generation of protein database search programs blast: at the core of a powerful and diverse set of sequence analysis tools dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features reduced surface: an efficient way to compute molecular surfaces psaia -protein structure and interaction analyzer cx, an algorithm that identifies protruding atoms in proteins pairpred: partner-specific prediction of interacting residues from sequence and structure protein interface prediction using graph convolutional networks pytorch: an imperative style, high-performance deep learning library progress and challenges in predicting protein interfaces structural basis for the recognition of sars-cov- by full-length human ace drug-target interaction prediction from chemical, genomic and pharmacological data in an integrated framework origins of specificity and affinity in antibody-protein interactions this work was supported by the 'created major new drugs' of major national science and technology (no. zx - ), and leading talents fund in science and technology innovation in henan province( ). xiaofei nan and shoutao zhang are the corresponding authors for this paper. key: cord- -ghc pf authors: sun, zehua; yan, lixin; tang, jiansong; qian, qian; lenberg, jerica; zhu, dandan; liu, wan; wu, kao; wang, yilin; lu, shiqiang title: brief introduction of current technologies in isolation of broadly neutralizing hiv- antibodies date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: ghc pf hiv/aids has become a worldwide pandemic. before an effective hiv- vaccine eliciting broadly neutralizing monoclonal antibodies (bnmabs) is fully developed, passive immunization for prevention and treatment of hiv- infection may alleviate the burden caused by the pandemic. among hiv- infected individuals, about % of them generated cross-reactive neutralizing antibodies two to four years after infection, the details of which could provide knowledge for effective vaccine design. recent progress in techniques for isolation of human broadly neutralizing antibodies has facilitated the study of passive immunization. the isolation and characterization of large panels of potent human broadly neutralizing antibodies has revealed new insights into the principles of antibody-mediated neutralization of hiv. in this paper, we review the current effective techniques in broadly neutralizing antibody isolation. antibody responses to neutralize human immunodeficiency virus- (hiv- ) are mediated by direct binding to viral spikes, which are trimers composed of glycoproteins gp and gp (pincus et al., a; pincus et al., b; blair et al., ; morris et al., ; micoli et al., ; pegu et al., ; haynes and mascola, ; liao et al., ; brodine et al., ; ward and wilson, ; debnath et al., ; moore et al., ) . gp with the attachment of virus particles to the target cells, and the gp engages in virus-cell fusion (durham and chen, ; desai et al., ; dale et al., ; bieniasz et al., ; rausch et al., ) . while successful in-vitro, recombinant gp has failed to generate cross neutralizing antibodies in vivo possibly due to the natural conformation of gp (prevost, ; wang et al., ; doores et al., ; doores et al., ; zhou et al., ; wu et al., ; watkins et al., ; robert-guroff et al., ) . monoclonal antibodies were first generated in mice in using a hybridoma technique (patke et al., ; holzlohner and hanack, ; bhatia et al., ; pogson et al., ; abusneina and gauthier, ; milstein, ) , and were subsequently accepted globally by manufacturers. now, it is well understood that rodent and murine derived antibodies are immunogenic in humans; when binding to antigens within the human body, the complexes can be recognized as immunogenic agents. to solve this problem, human equivalent forms were generated from rodent or murine antibodies, known as humanized antibodies (pardi et al., ; wiehe et al., ; rothenberg, ; bhowmick et al., ; hamanoue et al., ) . in recent years, new techniques have evolved to isolate human monoclonal antibodies against hiv- directly which offer an advantage of reduced immunogenicity. a panel of hiv- specific potent broadly neutralizing antibodies has been isolated in recent decades. the first generation antibodies, f , g and e , were used in combination for passive immunization (morris et al., ; mehandru et al., ; armbruster et al., ) . a second generation antibody, igg b , was observed to neutralize numerous virus isolates with ic in nm level ( nm is equal to about . ug/ml) for the first time (li et al., ; bunnik et al., ; ashish et al., ; zwick et al., ; sun et al., ) . the new generation of potent broadly neutralizing antibodies, including vrc -class antibodies, nih - , pg / , e , bg , ioma, o , acs , can neutralize virus isolates ranging from % to % with ic less than μg/ml, and can neutralize %- % of virus isolates with ic less than μg/ml (morales et al., ; huang et al., ; braibant et al., ; thenin et al., ; ringe et al., ; euler et al., ; davenport et al., ; pancera et al., ; wu et al., ; scheid, ; scheid et al., ) . worth noting, broadly neutralizing antibody e neutralizes % of tested viruses with geometric mean ic of . μg/ml. e binds to the cell-surface envelope as opposed to phospholipids. the binding of e and mper reveals a highly conserved gp -hydrophobic residue and a critical arginine or lysine just before the transmembrane region. the breadth and potency of e demonstrates a conserved site for hiv neutralization and potential target for hiv vaccine design (huang et al., ) . the highly potent vrc -class broadly neutralizing antibodies (bnabs) individually neutralize up to % of hiv- isolates. vrc class antibodies are considered a sub group of bnabs and have already been isolated from several hiv- infected patients. they each neutralize different virus isolates through the interaction with hiv gp cd binding site. vrc -class bnabs are derived from human vh - gene family, which occupies % of the human antibody repertoire. structural studies revealed that these vrc -class abs interact with gp cd binding site by mimicking the cd molecule; specifically the secondary structure formed by heavy chain structure and cdr in light chain. additionally, an important deletion in cdrl present in many vrc class abs can facilitate the interaction with the cd binding site and avoid conflict with glycan at asn (n ) on loop d (zhou et al., ; georgiev et al., ; wu et al., ; zhou et al., ) . nextgeneration sequencing (ngs)-derived sequencing reveals that vrc lineage is comprised of at least six distinct heavy chain clades and five light chain clades. this observation suggests that extraordinary variation in antibody immunity may only occur within a few antibody lineages − or even a single lineage (wu et al., ) . generation of an unmutated common ancestor of vrc -class antibodies could be a potential mechanism for opposing hiv- . after stimulation by virus and co-evolution with the virus, the unmutant common ancestor has ability to mature to bnabs. human antibodies can be elicited by b cells to eliminate "foreign immunogens" directly or indirectly. some antibodies can be produced to neutralize foreign antigens by various mechanisms, while some nonneutralizing antibodies can perform effector functions such as mediating nk cells via antibody-dependent cellular cytotoxicity (adcc) (liao et al., ; lin et al., ; thomann et al., ; ito et al., ; mandi et al., ) . serum immunoglobulins produced naturally by human immunity have been applied in passive immunotherapy against many infectious diseases since . but this approach suffers a number of problems. not only have these polyclonal immunoglobulins displayed mixed efficacies, but they also pose the potential threat of bloodborne pathogen transmission. in addition, the resource of serum is limited and expensive. these disadvantages limit the application of polyclonal immunotherapy (berry, ; stevens et al., ; wang et al., ; sili et al., ; lang et al., ) . recent technology developments have made it possible to isolate human antibodies against hiv- from both in vivo and in vitro sources. these improved techniques include display methods, epstein-barr virus (ebv) immortalization, classical hybridoma procedures, and single-cell sorting followed by molecular cloning. . techniques to make human monoclonal antibodies in the mid- s, hybridoma technology was invented for production of mouse monoclonal antibodies with defined antigen specificities and neutralization activity for application in clinical therapies (kelso et al., ; hugwil, ; tomita and tsumoto, ; martin-lopez et al., ; hencsey et al., ; honda et al., ; köhler and milstein, ) . hybridomas can be generated by effective fusion of b cells and partner cells followed by screening of individual antibody producing cells. production of monoclonal antibody in individual hybridoma cells can be easily quantified by surface plasmon resonance imaging (stojanovic et al., ) . b cell hybridomas can be an important source for screening of monoclonal antibodies. highthroughput screening is used to characterize mouse igg antibodies; including sub-isotypes, binding activity, and neutralizing activities (liu et al., ; szafran et al., ) . fluorescent antigen can be used to sort antigen binding hybridoma cells from a mixture as opposed to the traditional way of screening using multi-micro well plate screening and limiting dilution. as an extension of this technology, t cell hybridoma was also generated (krishnan et al., ) . however, mouse monoclonal antibodies were shown to be problematic in humans due to their immunogenic properties and low effector function. high immunogenicity prevented their application in humans where prolonged dosing was required. differences in sequence and glycosylation pattern of the fc region make rodent mabs poorly effective in mediating effector functions in human (li et al., ) . human b cells proved to be difficult to immortalize using hybridoma technique (li et al., ; cafri et al., ; park et al., ) . the primary issue was that mabs produced by human b cell hybridoma cells could react against selfantigens. in addition, the lack of sufficient maturation outside of the germinal centers and low binding affinities of mabs derived by hybridoma, meant that only . % of screened clones typically reacted to the target antigen. due to these problems, antibody humanization was developed as an alternative to generate human format antibodies from rodent derived antibodies (martin and rees, ; choi et al., ; olimpieri et al., ; safdari et al., ; tsurushita et al., ; ohtomo et al., ) . humanized antibodies are produced by methods of genetic engineering and can potentially reduce the immune response against non-human derived antibodies. this is done by combining the variable (v) region binding domain of a rodent antibody with human antibody constant (c) regions. this kind of chimeric antibody retains the binding specificity with less immunogenicity to humans. in some cases, the chimeric antibodies retain the effects in mediating human complement-dependent cytotoxicity (cdc) or adcc. it is also possible to produce a humanized antibody without creating a chimeric intermediate first. once the precise sequences of the desired cdrs are known, these dna sequences can be directly cloned into antibody expression vectors with human antibody "scaffold". a wide variety of human antibody expressing vectors has been developed using this method. for example, pdr vector is a human igg antibody expressing vector which was originally used for expression of human hiv- potent neutralizing antibody igg b . antibody vrc was cloned into a different cloning vector described previously by tiller et al. (tiller et al., ) . another method to immortalize human memory b cells is by epstein-barr virus (ebv) mediated transformation (lee et al., ; klein, ; straub and zubler, ; ohashi et al., ; mclaughlin et al., ; traggiai et al., ) . the c b, ebv-binding receptor positive b cells can be immortalized to generate antibodies for isolation using single cell sorting, or can be cultured with feeder cells for further screening or cloning. the optimized methodology is to activate b cells using toll-like receptor (tlr ) agonist cpg dna before or during ebv infection, which has been used to isolate human monoclonal antibodies against various pathogenic viruses (katsumura et al., ; bass and darke, ; sun et al., ) . it has also been reported that human b cells can be immortalized by transforming a retrovirus encoding anti-apoptotic factor b cell lymphoma (bcl ) and bcl-xl in the presence of interleukin (il- ) and cd ligands (schrader et al., ; rydstrom et al., ; kusam et al., ). this process can trigger memory b cells to differentiate into antibody secreting cells. the immortalization of human b cells provides a substantial resource of human b cells for the subsequent screening. after culturing for - weeks, antibodies can be collected and isolated from the supernatant to perform neutralization against different viruses. immortalized b cells can also be sorted by antigen using flow-based technology, and the heavy and light chain genes of antibodies secreted by immortalized b cells can then be cloned. the famous g , f and e antibodies are generated by this method (buchacher et al., ) . however, due to the low efficiency of ebv-induced b cell transformation, this method has limitation in application. though optimization methods were studied based on ebv-induced b cell transformation, the efficiency still needs a dramatic improve (sun et al., ; lu et al., ; kwakkenbos et al., ) . some reports show that memory b cells can be cultured short-term, and the supernatant can be used directly for screening of neutralization activity in a high throughput neutralization assay (micro-neutralization). a number of famous neutralizing antibodies have been isolated based on these methods including pg , pg , pgt - and e (huang et al., ; ringe et al., ; walker et al., ; walker et al., ; falkowska et al., ) . hiv- potent neutralizing antibodies pg and pg are classic antibodies isolated by microneutralization. these were originally isolated from , activated memory b cells from one clade a infected donor screened from approximately characterized hiv infected donors. the memory b cells were cultured at clonal density for the purpose of cloning antibody heavy and light chain pair from each culture well. pg neutralized out of viral isolates and pg neutralized out of viruses with a median potency (ic ) less than . μg/ml. e is an anti-gp antibody isolated from , memory b cells, sorted and cultured with il- , il- , and cd -ligand expressing feeder cells. e neutralized % of the tested pseudoviruses with an ic below μg/ml, and % of the tested viruses with an ic values below ug/ml. b cell culture and microneutralization can isolate potent broadly neutralizing hiv- antibodies. however, the large scale screening of b cells makes this method labor intensive and costly, in addition to low yield; typically ten antigen-specific clones can be isolated out of ten thousands of b cells ( fig. a and b) . in current reports, ebv transformation was optimized with efficiency from . %- % to . %, which enabled the generation of immortalized b cell libraries. however the efficiency still needs to be improved to enable greater b cell survival (sun et al., ) . phage display is a library method which permits screening of antibodies from a large recombinant library (falkowska et al., ; boots et al., ; chan et al., ; aghebati-maleki et al., ; rahbarnia et al., ; finlay et al., ) . antibodies can be displayed in the form of either single chain variable fragments (scfv) or antigen-binding fragments (fab). recent reports show that single domain antibody can also be displayed in phage display system (duarte et al., ; kazemi-lomedasht et al., ; li et al., ; rotman et al., ; tang et al., ) . many antibodies against different viruses including rabies virus, severe acute respiratory syndrome (sars) virus, ebola virus, yellow fever virus, hepatitis c virus, dengue virus, hepatitis a virus, influenza virus, and hiv, have been successfully isolated using this technology. the principle for phage display technique in isolating antibodies is to construct a phage display recombinant antibody library first. recombinant antibody library can increase the diversity of antibodies in b cell repertoires, which promotes the screening of antibodies with novel properties (graus et al., ) . antibody libraries are in general constructed by randomly assembling antibody heavy and light chain variable region, and through gene shuffling of the heavy and light chain to further increase the diversity. in summary, phage display creates greater diversity of antibodies and provides rapid and high throughput way of screening. m and m are hiv- cross-reactive human monoclonal antibodies isolated from a recombinant phage display library by competitive antigen panning (zhang et al., ; zhang et al., ; zhang et al., ; zhang et al., a; zhang et al., b) . m is a gp specific cross-reactive antibody, and m can recognize both gp and gp . b is another potent neutralizing antibody isolated by phage display from recombinant antibody libraries, and it is considered one of the most potent neutralizing antibodies isolated by phage display, as it can neutralize about % of known hiv- isolates (li et al., ; mantis et al., ; haussner et al., ; wu et al., ). m , m and b can be considered standard antibodies isolated by phage display; however the potencies are much lower than antibodies isolated by micro neutralization, such as pg / and/or e . phage display selects antibodies in vitro, and antibodies expressed in phage system may not necessarily represent those in mammalian cells due to the difference in protein folding and post transcriptional modifications. it is difficult to draw direct conclusions and gain an understanding of the natural occurrence of antibody heavy and light chain selection based on phage display alone (zhang et al., ) . to avoid these limitations, other display methods have been developed to display antibodies on the surface of yeast or mammalian cells, which can be sorted by flow cytometry according to antigen specificity. this is known as yeast surface display, which has become a powerful engineering tool for displaying recombinant proteins on the surface of saccharomyces cerevisiae via genetic fusion (traxlmayr and shusta, ; mei et al., ; andreu and del olmo, ; sheehan and marasco, ; gera et al., ; boder et al., ) . yeast surface display is a eukaryotic expression system with the capability to induce post translational modifications on recombinant antibodies. each yeast cell expresses fusion recombinant proteins which allows for the application of cell sorting for a specific antigen. yeast display system was not only reported used for isolation of antibodies, but also reported to be used in displaying antigens and other recombinant proteins (srivastava et al., ; guo et al., ) . there is a panel of yeast display antigen library which has been constructed, including yeast display h n antigen epitope library, and yeast display siv antigen epitope library for epitope mapping. hiv- specific antibodies isolated by display techniques are less potent than those isolated by micro neutralization or single b cell sorting and cloning. however in recent studies, phage/yeast display showed great potentials in isolating single domain antibodies from recombinant libraries. single-domain antibodies are considered a separate class of antibodies composed of antibody fragments consisting of a single monomeric antibody domain. single-domain antibodies allow a broad range of applications due to their small molecular mass and size, efficient production, and high affinity. single domain antibodies can be labeled using fluorescent molecules for diagnostic purpose or biotechnological usage. with the conjunction of drug or toxin, single domain antibodies can also be used for therapeutic application. the most well-studied single domain antibodies are antibody heavy-chain variable domains. the dna sequence will be optimized in order to improve the stability. in addition to heavy-chain variable domains, there are other types of single domain antibodies which have been studied, including ch antibodies, and light-chain variable domain antibodies, and some other forms of nanobodies (duarte et al., ; li et al., ; gong et al., ; louis et al., ; lulf et al., ; bouchet et al., ; bouchet et al., ; boudet et al., ) . mammalian cell display is a powerful method for the isolation of antibodies in scfv format or full length igg with high affinity. it has been shown that single chain antibodies can be displayed on the surface of human hek- t cells and used for affinity maturation (soga et al., ; tomimatsu et al., ; beerli et al., ) . mammalian cell expression system ensures that all of the cellular components will be involved in process of antibody synthesis. isolation of human antibodies by mammalian cell display can benefit from the advantages of mammalian cell expression system.mammalian cell display relies on the transfection of antibody expression vectors. in comparison with the yeast display system, mammalian cell expression system allows multiple recombinant protein/antibody delivering vectors in one cell. thus a further modification, enrichment, and single clone isolation will be needed for panning purposes (fig. c ). an important advance in antibody screening and cloning technology is the development of single cell sorting and single cell reverse transcription pcr (ouisse et al., ; evans et al., ; battye et al., ) . human single b cells with different antigen specificities can be sorted by flow cytometer directly, and the original vh and vl pairing of the antibody from single human b cells can be amplified in a high efficient way, with the requirement of relatively few cell numbers. this methodology is quite efficient in obtaining antibody heavy and light chain from extremely rare and highly discrete b cell subpopulations (fig. d) . memory b cells and plasma cells are ideal cell types for the purpose of monoclonal antibody cloning due to the specificity of the cell types. this method is always combined with single cell cloning after flow cytometry based single cell sorting to sort the antigen specific b cells, thus accurate probe design for sorting is essential. the gp or gp proteins are not ideal probes, because they are reactive with many hiv- antibodies, including non-neutralizing but strong binding antibodies (wu et al., ; wu et al., ; zhou et al., ) . one successful example of probe design in fishing potent broadly neutralizing hiv- monoclonal antibodies is the agents' pair of rsc and Δrsc . rsc and Δrsc are computer-assisted designed for sorting of individual b cells expressing cd bs antibodies. the resurfaced stabilized core (rsc ) is a functional structure core of the cd binding site with loop deletion. rsc preserved the antigenic structure of the cd binding site and eliminated other antigenic regions by substitution with simian immunodeficiency virus (siv) or non-hiv- residues. Δrsc contains one amino acid deletion (at position ) in rsc . this single amino acid mutation can knock out the function of cd binding site. cd binding site antibodies like vrc and b can bind efficiently with rsc but fail to bind with Δrsc . potent broadly neutralizing antibody vrc is isolated through the single b cell positive sorting by rsc and negative sorting by Δrsc . vrc neutralized up to % of the tested pseudoviruses with an ic below μg/ml, and % of the tested viruses with an ic values below ug/ml (wu et al., ; wu et al., ; mccoy and burton, ) . bg , ioma are another two successful bnmabs isolated by reasonable probe design and single cell sorting/cloning (gristick et al., ; freund et al., ) . n-glycans on the trimeric envelope glycoprotein (env) can be accommodated by broadly neutralizing antibodies. an engineered crystal structures of the hiv- env trimer with an exposed native glycan shield of high-mannose and complex-type n- z. sun et al. virus research ( ) - glycans was used to fishing out the bnmabs. ioma, a new cd -binding site (cd bs) antibody was thus defined. the heavy chain of ioma derives from vh - * , which is considered the germline gene of vrc class bnabs, but its light chain lacks the short cdrl which defines vrc -class bnabs. bg , bg , and nc are three bnabs isolated by single igg+ b cell sorting by using four different fishing agents in four separate sorts: cc core, gp yu , a : mixture of gp ug . (clade a) + gp cza (clade c), and bg sosip. (sok et al., ; dey et al., ; yang et al., ; sanders et al., ; kovacs et al., ) . antibody bg (vh - and vl - ) targeted the glycan-v portion of envelope, can neutralize % of viruses in a virus panel tested with a geometric mean ic of . mg/ml. antibodies nc (vh - / - , vk - ) and bg showed geometric mean ic of . and . mg/ml respectively. nc can bind to a quaternary trimer epitope, the core of which overlaps with the cd bs, and bg binds to the v v region. antigen baiting and fishing has proven quite successful in isolating hiv-specific human monoclonal igg expressing b cells and igm expressing b cells from infected donors using cleverly engineered antigen probes. flow cytometry based single cell sorting and cloning can also be applied in understanding the natural selection of antibodies by cloning of antibodies in b cells from different infection stage from one donor or b cells in the same stage from different donors. deep sequencing allows for the study of the maturation of a specific antibody population and corresponding b cell subpopulations by the stimulation of virus isolates in progressingstages of infection (morris et al., ; banerjee et al., ; carter et al., ; karlsson hedestam et al., ; soto et al., ; stramer et al., ; zhu et al., ; zhu et al., ) . the difficulty in eliciting bnabs in immunized animals has been concluded (walker et al., ; sanders et al., ; sok et al., ) . the enormous antigenic diversity of the envelope glycoprotein and the n-linked glycan shield are considered as two major points. recent study shows that success of rapid elicitation of broadly neutralizing antibodies to hiv- by immunization in cows. jr-fl gp and bg sosip are immunized to cows, and strong immune response is observed. and broadly neutralizing antibodies were isolated from peripheral blood mononuclear cells (pbmcs) by sorting with biotinylated bg sosip. the broadly neutralizing antibody nc-cow displayed a % neutralization breadth with a potent median ic of . μg /ml on a -virus panel tested.this study gives lights in investigating the generation of antibodies against pathogens that have evolved to avoid human antibody responses in animals. in recent decades, the technology advances have allowed for specific human antibodies to be isolated directly from human b cells and/ or from recombinant libraries. these isolated human monoclonal antibodies can be used in therapy because of their safety, efficiency, specificity and tolerance in humans; thus are a powerful tool in fighting against pathogens. among the developed antibody isolation methods over the last decade, it is difficult to highlight one as the sole preferred method. different methodology has different advantages. display techniques basing on recombinant library can be used for isolating single domain antibodies with enlarged library size. but this technique cannot provide us the information of the original pair of heavy chain and light chain of a specific antibody. single b cell sorting and culturing facilitate the rapid cloning of potent neutralizing antibodies with the requirement of large labor cost. reasonable design of probes and the development of native-like stabilized trimers have greatly improved efficiency in screening of functional envelope-binding b cells. thus it is hard to conclude the best way to generate novel bnabs. combined methods can be used in screening and isolating potent neutralizing antibodies in order to maximize the advantages of different methods. meanwhile, the development of novel methodology of broadly neutralizing antibodies isolation and screening are encouraged to broaden our knowledge and enlarge the recourses of antibody classes. a purposed method basing on single cell sorting and viral neutralization instead of fishing agents should provide a direct and efficient way to minimum the workload. in addition to passive treatment and presentation, another important purpose of isolation and characterization of potent broadly neutralizing antibodies is the functional study of neutralizing epitopes which provides key knowledge for efficient vaccine design. human broadly neutralizing antibodies serve as a potential source of discovering neutralizing epitopes that can be targeted to fight against many infectious diseases and can thus facilitate the design of the vaccines. antibody structure studies can provide new insights into the mechanism of recognition of immune evasion epitopes at the atomic level, and how antibodies mature to capture the virus mutant (morales et al., ; wibmer et al., ; rujas et al., ) . however in recent reports, few immunogen candidates were reported that can elicit potent broadly neutralizing antibodies in several immunization studies. native-like hiv- envelope trimers bg sosip. and b sosip. are reported to induce neutralizing antibodies against the tier- virus in rabbits, even though the potency is much weaker in comparison to the broadly neutralizing antibodies isolated from human long term nonprogressors (sanders et al., ; bradley et al., ) .pre-stimulation of b cells by heterogenous epitopes or antigens showed improved stimulation of b cells in generating potent neutralizing antibodies (yang et al., ) . it has also been reported that pgt -class germlinetargeting stabilized trimer immunogens primed pgt -like responses in pgt inferred-germline knock in mice, highlighting the hypothesis of prime-boosting and sequential immunization strategy (steichen et al., ) . all these observations highlight the neutralizing antibodies' role in hiv- prevention. altogether, understanding antibody generation and function mechanisms can help to guide efficient vaccine design and produce effective therapy. not applicable. not applicable. not applicable. no. not applicable. the authors claimed no interest. ammonium ions improve the survival of glutaminestarved hybridoma cells isolation and characterization of anti ror single chain fragment variable antibodies using phage display technique development of a new yeast surface display system based on spi as an anchor protein passive immunization with the anti-hiv- human monoclonal antibody (hmab) e and the hmab combination e / f / g global structure of hiv- neutralizing antibody igg b is asymmetric evaluation of a novel multi-immunogen vaccine strategy for targeting e / e neutralizing epitopes on hiv- gp membrane proximal external region improved efficiency of ebv transformation of b-lymphocytes single cell sorting and cloning isolation of human monoclonal antibodies by mammalian cell display antibody immunoprophylaxis and immunotherapy for influenza virus infection: utilization of monoclonal or polyclonal antibodies? hybridoma cell-culture and glycan profile dataset at various bioreactor conditions humanized monoclonal antibody alemtuzumab treatment in transplant hiv- -induced cell fusion is mediated by multiple regions within both the viral envelope and the ccr- co-receptor identification and characterization of uk- : a novel inhibitor that interferes with human immunodeficiency virus type gp processing engineering antibodies by yeast display anti-human immunodeficiency virus type human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries inhibition of the nef regulatory protein of hiv- by a singledomain antibody single-domain antibody-sh fusions for efficient neutralization of hiv- nef functions single peptide and anti-idiotype based immunizations can broaden the antibody response against the variable v domain of hiv- in mice structural constraints of vaccine-induced tier- autologous hiv neutralizing antibodies targeting the receptor-binding site cross-group neutralization of hiv- and evidence for conservation of the pg /pg epitopes within divergent groups diverse hiv- subtypes and clinical: laboratory and behavioral factors in a recently infected us military cohort generation of human monoclonal antibodies against hiv- proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization emergence of monoclonal antibody b -resistant human immunodeficiency virus type variants during natural infection in the absence of humoral or cellular immune pressure production of lacz inducible t cell hybridoma specific for human and mouse hiv- neutralizing antibody response and viral genetic diversity characterized with next generation sequencing human recombinant antibodies specific for hepatitis c virus core and envelope e peptides from an immune phage display library antibody humanization by structure-based computational protein design cell-to-cell transfer of hiv- via virological synapses leads to endosomal virion maturation that activates viral membrane fusion binding interactions between soluble hiv envelope glycoproteins and quaternary-structure-specific monoclonal antibodies pg and pg three-dimensional structure-activity analysis of a series of porphyrin derivatives with anti-hiv- activity targeted to the v loop of the gp envelope glycoprotein of the human immunodeficiency virus type fluorescent protein-tagged vpr dissociates from hiv- core after viral fusion and rapidly enters the cell nucleus structure-based stabilization of hiv- gp enhances humoral immune responses to the induced co-receptor binding site a nonself sugar mimic of the hiv glycan shield shows enhanced antigenicity two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of hiv envelope generation of immunity against pathogens via single-domain antibody-antigen constructs measuring t cell-to-t cell hiv- transfer, viral fusion, and infection using flow cytometry activity of broadly neutralizing antibodies: including pg , pg , and vrc , against recently transmitted subtype b hiv- variants from early and late in the epidemic assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging broadly neutralizing hiv antibodies define a glycan-dependent epitope on the prefusion conformation of gp on cleaved envelope trimers phage display: a powerful technology for the generation of highspecificity affinity reagents from alternative immune sources coexistence of potent hiv- broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller antibodies vrc and e neutralize hiv- with high breadth and potency even with ig-framework regions substantially reverted to germline protein selection using yeast surface display specific determination of influenza h n virus based on biotinylated single-domain antibody from a phage-displayed library selection of recombinant anti-hud fab fragments from a phage display antibody library of a lung cancer patient with paraneoplastic encephalomyelitis natively glycosylated hiv- env structure reveals new mode for antibody recognition of the cd -binding site simian immunodeficiency virus infection evades vaccine-elicited antibody responses to v region successful treatment with humanized anti-interleukin- receptor antibody (tocilizumab) in a case of aa amyloidosis complicated by familial mediterranean fever peptide paratope mimics of the broadly neutralizing hiv- antibody b the quest for an antibody-based hiv vaccine effect of medium composition on hybridoma growth and antibody production generation of murine monoclonal antibodies by hybridoma technology a human hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by pseudomonas aeruginosa exotoxin a broad and potent neutralization of hiv- by a gp -specific human antibody the meaning of the anti-cancer antibody cln-igg (pritumumab) generated by human x human hybridoma technology against the cyto-skeletal protein: vimentin, in the course of the treatment of malignancy defucosylated anti-ccr monoclonal antibody exercises potent adcc-mediated antitumor effect in the novel tumor-bearing humanized nod/shiscid: il- rgamma(null) mouse model continuous cultures of fused cells secreting antibody of predefined specificity evolution of b cell analysis and env trimer redesign ebv lytic infection enhances transformation of b-lymphocytes infected with ebv in the presence of t-lymphocytes production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors ebv-b cell interactions: immortalization, rescue from apoptosis, tumorigenicity (a short review) hiv- envelope trimer elicits more potent neutralizing antibody responses than monomeric gp versatility of using major histocompatibility complex class ii dextramers for derivation and characterization of antigen-specific, autoreactive t cell bcl cooperates with cd stimulation and loss of p function to rapidly transform primary b cells corrigendum: generation of stable monoclonal antibodyproducing b cell receptor-positive human memory b cells by genetic programming immunotherapy with human monoclonal antibodies: fragment a specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin microrna signatures associated with immortalization of ebvtransformed lymphoblastoid cell lines and their clinical traits human antibodies for immunotherapy development generated via a human b cell hybridoma technology a single-domain antibody-linked fab bispecific antibody her -s-fab has potent cytotoxicity against her -expressing tumor cells structural analysis of the glycosylated intact hiv- gp -b antibody complex using hydroxyl radical protein footprinting immunogenicity of constrained monoclonal antibody a -human immunodeficiency virus (hiv) env gp complexes compared to that of recombinant hiv type gp envelope glycoproteins effect of interleukin (il)- and il- on activated natural killer (ank) and antibody-dependent cellular cytotoxicity (adcc) in hiv infection a rapid method to characterize mouse igg antibodies and isolate native antigen binding igg b cell hybridomas binding of hiv- gp -directed neutralizing and non-neutralizing fragment antibody binding domain (fab) and single chain variable fragment (scfv) antibodies to the ectodomain of gp in the pre-hairpin and six-helix bundle conformations generation of immortalized human na#xp#ve b cell libraries by optimized ebv transformation structural basis for the inhibition of hiv- nef by a high-affinity binding single-domain antibody effect of human adenovirus on antibody-dependent cellular cytotoxicity (adcc) in chickens inhibition of hiv- infectivity and epithelial cell transfer by human monoclonal igg and iga antibodies carrying the b v region extracting human antibody sequences from public databases for antibody humanization: high frequency of species assignment errors enhanced monoclonal antibody production in hybridoma cells by lps and anti-migg identification and specificity of broadly neutralizing antibodies against hiv ebv-directed t cell therapeutics for ebv-associated lymphomas neutralization profiles of newly transmitted human immunodeficiency virus type by monoclonal antibodies g : f , and e application of modified yeast surface display technologies for non-antibody protein engineering requirement of calmodulin binding by hiv- gp for enhanced fas-mediated apoptosis the hybridoma revolution: an offshoot of basic research towards a structure of the hiv- envelope glycoprotein gp : an immunochemical approach fragments of the v /v domain of hiv- glycoprotein engineered for improved binding to the broadly neutralizing pg antibody effectiveness of intranasal immunization with hiv-gp and an hiv- env ctl epitope peptide (e ) in combination with the mucosal adjuvant lt(r g) mabgel : first phase trial of the anti-hiv- monoclonal antibodies f , e and g as a vaginal microbicide differential antibody responses to conserved hiv- neutralizing epitopes in the context of multivalent scaffolds and native-like gp trimers reappraisal of ebv in diffuse large b-cell lymphoma (dlbcl): comparative analysis between ebv-positive and −negative dlbcl with ebv-positive bystander cells humanization of mouse ons-m antibody with the aid of hybrid variable regions tabhu: tools for antibody humanization antigen-specific single b cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies crystal structure of pg and chimeric dissection with somatically related pg : structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv- administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv- challenge generation and characterization of a novel fusion partner cell line for the production of human macrophage hybridoma bisfabs: tools for rapidly screening hybridoma iggs for their activities as bispecific antibodies use of broadly neutralizing antibodies for hiv- prevention identification of human anti-hiv gp monoclonal antibodies that make effective immunotoxins design and in vivo characterization of immunoconjugates targeting hiv gp immunogenomic engineering of a plug-and-(dis)play hybridoma platform influence of the envelope gp phe cavity on hiv- sensitivity to adcc responses evolution of phage display technology: from discovery to application peptides derived from the cdr -homologous domain of the cd molecule are specific inhibitors of hiv- and siv infection, virus-induced cell fusion, and postinfection viral transmission in vitro. implications for the design of small peptide anti-hiv therapeutic agents subtle alteration of residues including nlinked glycans in v loop modulate hiv- neutralization by pg and pg monoclonal antibodies cross-neutralization of human immunodeficiency virus type and and simian immunodeficiency virus isolates fusion of higg -fc to in-anti-amyloid single domain antibody fragment vhh-pa h prolongs blood residential time in app/ps mice but does not increase brain uptake structural basis for broad neutralization of hiv- through the molecular recognition of e helical epitope at the membrane interface cd is a potential marker of favorable prognosis in patients with diffuse large b-cell lymphoma treated with immunochemotherapy antibody humanization methods − a review and update a next-generation cleaved, soluble hiv- env trimer, bg sosip. gp , expresses multiple epitopes for broadly neutralizing but not nonneutralizing antibodies hiv- vaccines: hiv- neutralizing antibodies induced by native-like envelope trimers sequence and structural convergence of broad and potent hiv antibodies that mimic cd binding hiv-specific b cell response in patients with broadly neutralizing serum activity global gene expression changes of in vitro stimulated human transformed germinal centre b cells as surrogate for oncogenic pathway activation in individual aggressive b cell lymphomas phage and yeast display large-scale expansion of dendritic cell-primed polyclonal human cytotoxic t-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy mammalian cell surface display as a novel method for developing engineered lectins with novel characteristics recombinant hiv envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows developmental pathway of the mper-directed hiv- -neutralizing antibody e identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic h n influenza virus hiv vaccine design to target germline precursors of glycandependent broadly neutralizing antibodies preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging two human immunodeficiency virus type cases in us blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case immortalization of ebv-infected b cells is not influenced by exogenous signals acting on b cell proliferation. effects of mutant el- thymoma cells and transforming growth factor-beta reconstitution and characterization of antibody repertoires of hiv- -infected elite neutralizers isolation and characterization of hiv- envelope glycoprotein specific b cell from immortalized human naive b cell library use of hca in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns a human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface naturally occurring substitutions of conserved residues in human immunodeficiency virus type variants of different clades are involved in pg and pg resistance to neutralization in vitro glycoengineering of igg and its effect on fc receptor binding and adcc activity efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning a rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies hybridoma technologies for antibody production an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus directed evolution of protein thermal stability using yeast surface display humanization of a chicken anti-il- monoclonal antibody broad and potent neutralizing antibodies from an african donor reveal a new hiv- vaccine target broad neutralization coverage of hiv by multiple highly potent antibodies optimizing adoptive polyclonal t cell immunotherapy of lymphomas: using a chimeric t cell receptor possessing cd and cd costimulatory domains hiv- gp epitope recognition is influenced by immunoglobulin dh gene segment sequence the hiv- envelope glycoprotein structure: nailing down a moving target resistance of human immunodeficiency virus type to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites structure and recognition of a novel hiv- gp -gp interface antibody that caused mper exposure through viral escape immunodominance of antibody recognition of the hiv envelope v region in ig-humanized mice interaction of chemokine receptor ccr with its ligands: multiple domains for hiv- gp binding and a single domain for chemokine binding mechanism of human immunodeficiency virus type resistance to monoclonal antibody b that effectively targets the site of cd attachment rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv- maturation and diversity of the vrc -antibody lineage over years of chronic hiv- infection characterization of stable: soluble trimers containing complete ectodomains of human immunodeficiency virus type envelope glycoproteins identification of non-hiv immunogens that bind to germline b predecessors and prime for elicitation of cross-clade neutralizing hiv- antibodies broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library identification and characterization of a new cross-reactive human immunodeficiency virus type -neutralizing human monoclonal antibody identification of a novel cd i human monoclonal antibody fab that neutralizes hiv- primary isolates from different clades selection of a novel gp -specific hiv- neutralizing human antibody by competitive antigen panning cross-reactive human immunodeficiency virus type -neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp and lacks reactivity against self-antigens identification and characterization of a broadly cross-reactive hiv- human monoclonal antibody that binds to both gp and gp structural basis for broad and potent neutralization of hiv- by antibody vrc multidonor analysis reveals structural elements: genetic determinants, and maturation pathway for hiv- neutralization by vrc -class antibodies structural repertoire of hiv- -neutralizing antibodies targeting the cd supersite in donors somatic populations of pgt - hiv- -neutralizing antibodies identified by pyrosequencing and bioinformatics de novo identification of vrc class hiv- -neutralizing antibodies by next-generation sequencing of b-cell transcripts a novel human antibody against human immunodeficiency virus type gp is v : v , and v loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin g b we wish to thank dr. mei-yun zhang, dr. zhiwei chen (the university of hong kong), and dr. paul zhou (institute pasteur of shanghai cas) for helpful discussion.z. sun et al. virus research ( ) - key: cord- -mprsdi e authors: zhu, zhongyu; bossart, katharine n; bishop, kimberly a; crameri, gary; dimitrov, antony s; mceachern, jennifer a; feng, yang; middleton, deborah; wang, lin-fa; broder, christopher c; dimitrov, dimiter s title: exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: mprsdi e we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg(hev)). one of these antibodies, m , which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sg(hev). one of the selected antibody fab clones, m . , had affinity of binding to sg(hev) that was equal to or higher than that of the other fabs; it was converted to igg and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with % inhibitory concentrations below . and . μg/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg(hev) and sg(niv). m . bound a soluble form of niv g (sg(niv)) better than it bound sg(hev), and it neutralized niv better than hev, despite being originally selected against sg(hev). these results suggest that m . has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg hev ). one of these antibodies, m , which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavychain variable domain and panning against sg hev . one of the selected antibody fab clones, m . , had affinity of binding to sg hev that was equal to or higher than that of the other fabs; it was converted to igg and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with % inhibitory concentrations below . and . g/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg hev and sg niv . m . bound a soluble form of niv g (sg niv ) better than it bound sg hev , and it neutralized niv better than hev, despite being originally selected against sg hev . these results suggest that m . has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent. hendra virus (hev) and nipah virus (niv) are highly pathogenic paramyxoviruses that have recently emerged from flying fox populations to cause serious disease outbreaks in humans and livestock in australia, malaysia, singapore, bangladesh, and india [ ] . hev emerged in queensland, australia, in , killing human and horses [ ] and the virus was responsible for at least other sporadic outbreaks involving horses and humans between and [ ] . the closely related niv emerged in - in peninsular malaysia, resulting in the death of more than people and the culling of more than one million pigs [ ] . since then, several outbreaks of niv infection have been recorded in bangladesh and india [ , , ] . several important observations have been made during these most recent outbreaks, such as a higher incidence of acute respiratory distress syndrome, higher rates of person-to-person transmission, and higher case fatality rates ( %- %), compared with the malaysian outbreak (with case fatality rates of ϳ %) in which the virus was initially discovered [ - ] . there are currently no therapeutic modalities for treating niv or hev infections, and a vaccine for prevention of disease in human or livestock populations does not exist. the first antiviral antibody-based drug approved by the u.s. food and drug administration is a humanized antibody against respiratory syncytial virus (manufactured by medimmune), which has proven to be highly successful in reducing respiratory syncytial virus infection in infants and immunocompromised patients; this antibody has been recently improved [ ] . in this context, the development of neutralizing human mabs (hmabs) against henipaviruses could have important implications for prophylaxis and passive immunotherapy. one of the challenges in the development of human antibodies for antiviral applications is the heterogeneity and mutability of rna viruses. it is, therefore, important to select antibodies that recognize epitopes that are highly conserved among different virus variants. previously, we reported the isolation and characterization of potent neutralizing recombinant hmabs that targeted the viral envelope glycoprotein g by use of a highly purified, oligomeric, soluble hev g glycoprotein (sg hev ) [ ] as the antigen for the screening of a large, naive human phagedisplayed antibody library [ ] . one of these antibodies, m , exhibited cross-neutralizing activity against both hev and niv. in this article, we report the identification and characterization of a novel antibody, m . , derived from m by light-chain shuffling and heavy-chain variable domain random mutagenesis. this antibody exhibits exceptional potency against both, niv and hev, and being fully human antibody, it could be directly used for prophylaxis or treatment of humans exposed to or infected by hev or niv. such an antibody could also be used for improved diagnosis and as a research tool for better understanding of virus-host interaction. fine mapping of the hmabdefined epitope may also provide information useful for the rational development of candidate vaccines and small molecule drugs. hela-usu cells have been described elsewhere [ , ] . vero cells were provided by the australian animal health laboratory. the human glioblastoma cell line u -mg was provided by adam p. geballe, fred hutchinson cancer research center, seattle. hela-usu and u cells were maintained in dulbecco's modified eagle medium (dmem [quality biologicals]) supplemented with % cosmic calf serum (hyclone), and mmol/l l-glutamine (dmem- ). vero cells were maintained in eagle minimal essential medium (emem) (invitrogen) supplemented with % fetal calf serum (invitrogen australia pty. ltd), mmol/l hepes (invitrogen), and mmol/l l-glutamine (invitrogen) (emem- ). all cell cultures were maintained at °c in a humidified % co atmosphere. generation of phage-displayed fab libraries and selection of affinity-matured fabs. the original human fab phage display library, from which the antibodies m -m were identified [ ] , was used as the source for the light-chain variable domain (vl) repertoire in the shuffled library. the phagemid preparation from the original library was first digested with ncoi and spei, followed by electrophoresis on an agarose gel to delete the entire heavy-chain variable domain (vh) repertoire. the gene encoding the vh of m was amplified by error-prone polymerase chain reaction (pcr) (stratagene) to introduce random mutations and then fused with the ch gene fragment by use of splicing by overlapping extension pcr. the fused fragment was digested with ncoi and spei and purified from gel; it was then ligated into the purified backbone vector to create the vls-shuffled fab repertoire. escherichia coli tg cells were transformed with the ligation mixtures via electroporation. the transformed tg cells were plated on ϫ yeast extract-tryptone ( yt) agar plates containing g/ml ampicillin and % glucose ( ytag). after incubation overnight at °c, all of the colonies grown on the plates were scraped into ml of ytag medium, mixed with . ml of % glycerol (final concentration, %), aliquoted, and stored at Ϫ °c as the library stock. two rounds of biopanning were performed on sg hev conjugated magnetic beads as described for the original library panning [ ] . eight clones were identified as affinity-maturated antibodies, and m . was selected for further characterization. selected fabs and m . igg expression and purification was performed as described elsewhere [ ] . elisa binding assay. the sg hev glycoprotein ( ng per well) was coated in a -well plate in l pbs. serially diluted antibodies were added into the well after washing with ϫpbs with . % tween . after incubation for hour at room temperature and washing, horseradish-peroxidase conjugated secondary antibody was added and incubated for another hour. after washing, plates were developed and read at nm in an elisa reader. stable cell line construction and fermentation. linearlized m . pdr construct was transfected into cho k cells with polyfectin in accordance with the protocol from qiagen. a stable antibody-producing cell line was selected by screening in glutamine acid-free culture medium with mol/l methionine sulphoximine. it was adapted to grow in suspension in serum-free medium. the antibody was produced by fermentation in a -l fermentor and purification was performed with protein a. expression and immunoprecipitation of alanine hev g mutants. alanine mutations were introduced at specific residues in myc-tagged hev g using site-directed mutagenesis (stratagene). expression and immunoprecipitation of all hev g mutants were as performed as described elsewhere [ ] . binding and competition analysis using multiplex microsphere assays. multiplexed microsphere assays were performed as described elsewhere [ ] . cell fusion assays. the inhibition assay of cell fusion by fabs and iggs was performed as described elsewhere [ ] . virus neutralization assays. all live virus experiments were conducted under strict biocontainment procedures in a biosafety level laboratory. a total of ϫ vero cells were added per well with l emem- in -well plates and incubated overnight at °c in a humidified % co atmosphere. the foci assay was performed as described elsewhere [ ] . antibody pharmacokinetics in plasma and biological activity. four juvenile (Ͻ months), male ferrets were anesthetized by mask induction with isofluorane and maintained on % isofluorane and % oxygen. a baseline blood sample was collected from an axillary vein, and a -g intravenous catheter was placed in the left jugular vein. the antibody m . was administered via the catheter by slow infusion over minutes; ferrets received mg of m . and ferrets received mg of m . . the catheter was withdrawn, and animals were allowed to recover from the anesthetic. subsequently, the ferrets were anes-thetized on days , , , , , , , , and by intramuscular injection with ketamine (ketamil; ilium) and medetomidine (domitor; novartis) (reversed with atipamazole [antisedan; novartis]). blood was collected from an axillary or jugular vein while animals were under anesthesia. after collection of the final blood sample, the animals were euthanized by means of an intravenous barbiturate overdose. for all samples, serum was aliquoted and stored at Ϫ °c. ferret serum was diluted : and assayed by use of the binding multiplex microsphere assay described above. an m . standard curve ranging from ng/ml to . ng/ml and all ferret serum samples were assayed simultaneously. ferret serum m . concentrations were extrapolated from the standard curve using nonlinear regression analysis (graphpad software; graphpad). half-lives were estimated from the slopes of the natural logarithms of the antibody concentration as function of time by using the formulas ta ϭ . /a[ln ( ) respectively, a and b are measured as the slopes of ln (m . concentration, g/ml) dependent on time. serum collected on days , , and was evaluated in virus neutralization assays. all sera were diluted : , : , : and : and assayed in duplicate using the foci assay. complete neutralization was defined as no viral foci in either well at a particular dilution. previously, we reported the isolation of henipavirus-neutralizing recombinant hmabs by screening of a large nonimmune phage-displayed fab library against a soluble form of the hev g glycoprotein (sg hev ). one of these antibodies, m , exhibited the highest level of crossreactivity and relatively better binding to niv g than to hev g. we reasoned that improving m 's binding to hev g could further increase its cross-reactive neutralizing activity by increasing its affinity to hev g. to mature in vitro m , we constructed an antibody library-which contained approximately ϫ clones-by light-chain shuffling combined with heavychain vh random mutations introduced by error-prone pcr. two rounds of panning against sg hev conjugated to magnetic beads demonstrated sufficient enrichment (data not shown), and random colonies were screened by phage elisa. the best binders were selected for sequencing analysis. they represented different clones, which were designated m . -m . . although there were no amino acid sequence changes, silent mutations occurred in the vh regions in of the clones (data not shown), indicating that the error-prone pcr had worked. seven of the different light chains were from v subfamily iii, which is the same as that of m ; clone (m . ) was from the v subfamily i. a sequence analysis of these light chains showed that all clones contained mutations in all complementarity-determining regions; of the clones from subfamily iii, m . had the largest number of mutations (data not shown). all clones were expressed as fabs, purified, and analyzed by elisa for binding to the selecting antigen sg hev . the elisa data confirmed that all fab clones displayed a higher level of binding to sg hev than did the parental m fab (figure ). clone m . , which had binding affinity equal to or higher than that of the other clones, was selected for further characterization and converted to an igg format. binding of igg m . to cognate antigens. to investigate the binding of igg m . to hev g and niv g and its ability to block receptor-g interactions, we used multiplex mi- crosphere assays that we recently developed [ ] . as shown in figure a , m . binds to both sg hev and sg niv in a dosedependent fashion. at relatively low concentrations (in the range of ng/ml or less), the binding reached % of its maximum, indicating strong binding to both soluble g proteins. furthermore, the multiplex assays also demonstrated that m . is highly efficacious in blocking the binding of ephrin-b ligand (efnb ) to both sg hev and sg niv (figure b). it is important to note that the increase in affinity did not alter the specificity of the fab; it could still bind to both g proteins very well. it is also interesting to note that m . binds sg niv better than sg hev , a result that was also reflected in its slightly better efficiency at blocking efnb -sg niv binding. these results suggest that m . is a cross-reactive, high-affinity binder to the soluble forms of both hev g and niv g glycoproteins. hev env-mediated cell fusion in both formats, as fab and as igg . as expected, fab m . was significantly more potent than fab m , and igg m . was more potent than fab m . ( figure ) . a comparison with a previously identified antibody, m , which is specific for hev and the most potent inhibitor of infectious hev [ ] , suggested that m . and m had comparable activity in both fab and igg formats ( figure b) . interestingly, and similar to the multiplexed results, the inhibitory activity against niv g-mediated cell fusion was higher than that against hev g-mediated fusion although m . was selected by using sg hev . these results suggest m . possesses exceptional cross-reactivity and potency against hev and niv env-mediated cell fusion and syncytia formation. potent cross-reactive neutralization of live viruses. igg m . exhibited exceptionally potent and cross-reactive inhibitory activity against infectious niv and hev with ic values fig. . immunofluorescence-based syncytia assay of hendra virus (hev) and nipah virus (niv) infection. vero cells were plated into -well plates and grown to % confluence. virus and antibodies were premixed for min at °c prior to addition to the cell monolayers. cells were incubated in the presence of antibody-virus mixtures for h, fixed in methanol, and immunofluorescently stained for p protein prior to digital microscopy. all images were obtained at an original magnification of ϫ. a, hev without antibody; b, hev with m at g/ml; c, hev with m at g/m; d, hev with . at g/ml; e, hev with . at g/ml; f, niv without antibody; g, niv with m at g/ml; h, niv with m at g/ml; i, niv with . at g/ml; j, niv with . at g/ml. below . and . g/ml, respectively (table ) . these data suggest a strong correlation between binding to the antigens, inhibition of fusion, and neutralization of infectious virus and confirm the exceptional potency and cross-reactivity of m . . igg m . was also evaluated in the sensitive vero cell-fociimmunostaining assay overnight side by side with igg m ( figure ) . in this case, not only was the amount of virus per well high ( tcid for hev and tcid for niv), but the antibody-virus mixtures were incubated on vero cell monolayers overnight. if no antibody was present (panels a and f), there was massive coalesced syncytia for both viruses. in the presence of m , hev was neutralized to localized foci at g/ml, which was further reduced to individual infected cells at g/ ml. importantly, if this assay was carried out as a standard cytopathic effect-based neutralization assay, these wells would essentially look uninfected. by comparison, m . neutralized % of hev and niv at either g/ml or g/ml. this extended neutralization window demonstrates the exceptional potency of m . and may have important implications for postexposure therapeutic efficacy. to characterize the epitope of the affinity-maturated m . -compared with m -on the hev g glycoprotein, a panel of hev g alanine-scanning mutants constructed in a previous study [ ] were expressed and tested for binding to m and m . by immunoprecipitation. the binding of hev g mutants d a, g a, k a, and k a to both m and m . was almost absent, whereas the mutations g a and d a significantly decreased the binding of both antibodies, although they did so to varying degrees ( figure ). interestingly, one mutation, k a, almost completely eliminated the binding of the hevspecific antibody m but did not have any effect on the binding of the cross-reactive antibody m . . bishop et al. [ ] showed that all mutations-d a, g a, k a and k a-that eliminated the binding of both mabs in this study are detrimental for the binding of hev g to the receptor ephrin-b . these results suggest that the m . epitope overlaps the receptor-binding domain of hev g and the epitope of m . in vivo plasma half-life and biological activity. as hmab m . has the potential to be a potent henipavirus therapeutic agent, we next assessed its in vivo half-life and toxicity. we chose to use ferrets for these studies because they have been shown to be susceptible to niv-mediated disease (k. bossart, j. bingham, and d. middleton; unpublished data) and have become important animal models for several other human respiratory viruses, including sars coronavirus and highly pathogenic avian influenza virus. ferrets received of different doses of m . ( or mg), as detailed above. animals were closely observed for at least hours after recovery, and no adverse effects were noted. blood samples were collected over a -day period, and antibody concentrations were measured. a typical antibody concentration over time is shown in figure , which shows slopes in a logarithmic scale. half-lives calculated from these slopes did not vary with antibody dose. average distribution and elimination half-lives of . days and . days, respectively, were calculated, with relatively small individual differences. to determine whether the relatively short elimination half-life was the result of immune responses, we tested the ferret serum for anti-m . ferret antibodies. we were not able to detect such antibodies in ferret serum samples after administration of m . (data not shown). to demonstrate that m . measured in plasma was biologically active, serum collected on days , , and was evaluated using virus neutralization assays, as described above (data not shown). importantly, for all ferrets, niv was completely neutralized by : diluted serum collected on day after antibody administration. when serum samples collected on day were as- sayed, samples demonstrated complete virus neutralization, and a third serum sample demonstrated % neutralization. although negative on day , the fourth serum sample showed % neutralization on day . taken together, these data demonstrate that m . can remain biologically active in vivo for at least days. the major finding of this study is the identification of a novel, exceptionally potent, cross-reactive neutralizing hmab, m . . this antibody has significantly improved potency, compared with the parent antibody m and with other hmabs identified and characterized in our previous study [ ] . importantly, the substantial gain in potency was achieved without decreasing cross-reactivity. to our knowledge, this is the first fully human antibody that is capable of potently neutralizing both infectious hev and niv. an interesting observation made during the present study was that m . had better binding to sg niv than to sg hev , despite the fact that sg hev was used as the selector antigen during the original library screening [ ] and in the maturation panning procedures. the better binding to sg niv correlated with better neutralizing activity against niv, compared with hev. further studies are required to understand the mechanism underlying this unexpected observation. although the epitope mapping by alanine-scanning mutagenesis indicated that m . and m share most of the residues on the hev g glycoprotein that affect their binding, a dramatic difference was observed for the k a mutation, which completely abolished the binding of m to hev g, but did not affect m . 's binding. it was previously shown by bishop et al. [ ] that k a had no effect on the binding of hev g to the henipavirus receptors ephrin-b and ephrin-b . thus, the m epitope may contain contact site(s) located outside the receptor binding site. because the m . epitope does not include this site, one could hypothesize that it overlaps the receptor binding site to larger extent than the m epitope does. such a hypothesis is in agreement with m . 's much higher observed degree of cross-reactivity, compared with that of m . thus, one could further hypothesize that the m . epitope closely mimics the receptor binding site and, therefore, that the generation of escape mutants in the presence of this antibody would be less likely, compared with m and indeed any other known hmab. we have previously made similar observation for our potent cross-reactive neutralizing hmab m , which is predicted to be effective against all sars coronavirus isolates with known sequences [ ] . the m binding site overlaps extensively with the receptor binding site on the sars coronavirus spike (s) glycoprotein, as shown by the crystal structure of the m -s complex [ ] . thus, targeting the conserved and functionally important receptor binding site that is critical for virus entry into cells is a promising strategy for the development of cross-neutralizing antibodies. the igg m . was well tolerated in ferrets, and no any adverse effects were noted for the relatively short time ( days) of the experiment. the antibody pharmacokinetics consisted of phases. the estimated distribution half-life of ϳ . days is typical for iggs. the elimination half-life was significantly shorter than that for human iggs in humans (typically - weeks). we hypothesized that this could be the results of immune responses, specifically the elicitation of anti-human igg antibodies, which typically develop after - weeks. however, our attempts to detect such ferret antibodies against m . did not result in any measurable quantities above the background (data not shown). this could be because of the low levels of such antibodies during the relatively short period of observation. further studies are required to clarify the answer to this question. we also found that m . demonstrates reasonable stability and retains its biological activity in vivo. it has been previously shown that serum from hamsters immunized with vaccinia viruses that expressed niv envelope glycoproteins can protect the animals from challenge with niv [ ] . this important study by guillaume et al. [ ] further supports the notion that biologically active m . would be able to protect animals and humans from henipavirus infections. in summary, m . appears to be close to an ideal candidate for further development into an immunotherapeutic agent for henipavirus infection because it possesses many of the properties desired in such a therapeutic modality. it is a fully human mab; it retains its biological activity in vivo; it does not cause toxicity in ferrets; it has a distribution half-life typical for iggs, and its elimination half-life is likely to be significantly longer in humans; it cross-neutralizes both hev and niv; it has a muchimproved potency, compared with m , its parental antibody; and it targets the g glycoprotein region, which largely overlaps with the receptor binding site. this mab may also prove useful in the development of diagnostics, small molecule drugs, and vaccines, and as a research reagent. hendra and nipah viruses: different and dangerous a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus nipah virus encephalitis reemergence emerging viruses: coming in on a wrinkled wing and a prayer emerging infectious diseases. nipah virus (or a cousin) strikes again fatal fruit bat virus sparks epidemics in southern asia nipah virus encephalitis reemergence nipah virus outbreak(s) in bangladesh person-to-person transmission of nipah virus in a bangladeshi community ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus potent neutralization of hendra and nipah viruses by human monoclonal antibodies ephrin-b ligand is a functional receptor for hendra virus and nipah virus identification of hendra virus g glycoprotein residues that are critical for receptor binding neutralization assays for differential henipavirus serology using bio-plex protein array systems potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody nipah virus: vaccination and passive protection studies in a hamster model we wish to thank tim hancock from the australian animal health laboratory for his help with the in vivo ferret work. key: cord- -y smbtd authors: crouch, c. f; oliver, s; hearle, d. c; buckley, a; chapman, a. j; francis, m. j title: lactogenic immunity following vaccination of cattle with bovine coronavirus date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: y smbtd abstract in order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between and weeks before calving. this was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. the overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. no abnormal local or systemic reactions were observed as a result of vaccination. it is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection. neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination [ , , ] . in calves, economic losses are suered as a result of mortality, which can reach up to %, and also veterinary costs and decreased productivity of survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been shown to be primary pathogens in calves [ , ] . although this syndrome is most overtly associated with young animals, subclinical infections are also common in adults which may therefore act as reservoirs for reinfection [ ] . passive immunity against enteric viral infections is dependent upon the continual presence of a protective level of speci®c antibody in the gut lumen. this can be achieved by allowing the neonate to ingest colostrum or milk containing these speci®c antibodies from its dam (lactogenic immunity). although most adult cattle are seropositive for both rotavirus and coronavirus antibodies [ ] , during the transition from colostrum to milk production there is a dramatic decline in antibody titres which partially explains the high incidence of infection in calves older than days. successful rotavirus vaccines aimed at increasing both the titre and duration of speci®c antibody in both colostrum and milk have been developed [ ] , however, similar success has not been reported with vaccines targeted against bovine coronavirus [ ± ] . this paper reports the ability of a new single dose vaccine to signi®cantly increase both the level and duration of coronavirus neutralising antibodies in the serum and milk of vaccinated cattle. bovine coronavirus was grown in mammalian cell culture. for antigen production, cell culture supernatant was harvested and clari®ed by low speed centrifugation. the virus was then inactivated by treatment with . m binary ethyleneimine for h at c. finally, virus was concentrated up to -fold using a hollow ®bre ®ltration system with a , molecular weight cut-o. bovine coronavirus antigen levels were determined by quantitative elisa. immulon removawell strips (dynex technologies) were coated with an optimal concentration (previously determined by titration) of a sheep polyclonal anti-bovine coronavirus antibody diluted in . m carbonate±bicarbonate coating buffer of ph . . after incubation at c for h, the wells were blocked using pbs containing . % bovine serum albumin and % tween for min at ambient temperature. ml of pbs containing . % tween was added to each well followed by ml of sample, standard or control. after incubation for h at c on a shaker incubator the wells were washed three times using pbs containing % tween . ml of optimally diluted mouse monoclonal antibody speci®c for the virus haemagglutinin was then added to each well, followed by incubation for h at c with shaking. after washing, ml of an optimal concentration of a horseradish peroxidase -conjugated goat anti-mouse igg antibody (nordic immunologicals) was added to each well and incubated with shaking for a further h at c. after washing, bound conjugate was visualised using o-phenylenediamine dihydrochloride (sigma) as the chromagen. after stopping the reaction using . m sulphuric acid, the optical density ( nm) was proportional to the amount of bovine coronavirus antigen in the sample. full quantitation up to a range of antigen units per ml was achieved by including a set of six standards in each assay. the vaccine comprised of rotavirus and e coli k antigens from rotavec k (schering plough animal health) plus inactivated bovine coronavirus antigen and was adjuvanted using a mineral oil based adjuvant. the aqueous and oil phases were prepared separately and combined prior to emulsi®cation using a silverson homogeniser. twelve maiden heifers of mixed breeds that had not previously been treated with vaccines containing coronavirus were housed indoors, bedded with straw, although access was given to a grass paddock for exercise. the animals were fed hay, silage and a standard cattle ration (quinns of baltinglass), water was available ad libitum. the health of all the animals was monitored by daily observation throughout the study. all the animals were bled days prior to vaccination, serum prepared and the coronavirus antibody titre determined. the animals were ranked by descending antibody titre, and allocated into four groups of three animals each based on random assignment of one animal from each third of the ranking. animals in groups to were immunised with ml of the appropriate vaccine. group , vaccine a containing . vaccinated animals received a single ml injection intramuscularly into the neck. all animals were bled before vaccination, , and days post vaccination, heifers in groups and were also bled at , and days post vaccination. blood samples were stored for h to allow clotting to occur, the serum separated by centrifugation, and stored at À c prior to testing for bovine coronavirus (bcov) antibodies by virus neutralisation and haemagglutination inhibition. these were carried out on four farms, two (studies a and b) were beef suckler herds and two (studies c and d) were dairy herds. a double blind trial design was used. thirty cows on each farm were paired according to their expected calving dates and then randomly allocated to either vaccinate or placebo treatment groups ( cows per group). animals were included in the study on the basis that their expected calving dates fell between and weeks later than the day of vaccination. animals were excluded only if unhealthy or where they were known to have been previously vaccinated against coronavirus. all animals received a single injection of ml intramuscularly in the neck. animals in the vaccinate group were immunised with a preparation containing antigen units of coronavirus per dose, whilst animals in the control group were vaccinated with a saline/oil emulsion placebo containing none of the vaccine antigens. rectal temperatures were taken from all animals either days before vaccination (studies a and b) or immediately before vaccination (studies c and d), approximately h after vaccination and days after vaccination. on all sites, injection sites were examined for local reactions day, days and days after vaccination. calves from the beef suckler herds (studies a and b) were allowed unrestricted access to their dams for suckling. calves from the dairy herds (studies c and d) were fed twice a day by hand with approximately . l of colostrum or milk from their own mothers for at least the ®rst days of life. blood samples were collected from all cows at intervals from pre-vaccination to post-calving as indicated in the results section. in studies a and b, milk samples from the dam and blood samples from the calf were collected at day intervals from the day of calving (post-suckling) to days post-calving, whilst in studies c and d samples were only taken on the day of calving (post-suckling) and three days post-calving. blood samples were processed as described above. colostrum or milk samples were stored at À c prior to testing for the presence of bcov neutralising antibody. prior to testing, all sera were thawed, heat-treated at c for min, sub-aliquoted and stored frozen at À c until required. colostrum and milk samples were thawed, centrifuged at  g for min and the heavy liquid fraction removed aseptically by a pipette to a clean sterile container. sterile rennet solution was then added to a concentration of % v/v and incubated for h at c. the mixture was then centrifuged at  g for min and the upper liquid fraction (the whey) aseptically removed by pipette and dispensed into subaliquots of at least . ml before storage at À c. bovine coronavirus neutralising antibody responses were quanti®ed using a modi®cation of a standard plaque reduction assay [ ] . viral plaques were visualised by haemadsorption using % (w/v) rat red blood cells and the virus neutralising (vn) titre de®ned as the % end point following challenge with plaque forming units of bcov. bovine coronavirus haemagglutination inhibition (hai) antibodies were quanti®ed as follows. test sera were ®rst pre-treated to remove non-speci®c agglutinins and inhibitors by incubation with an equal volume of % (w/v) rat red blood cells overnight at ± c followed by treatment with . % (w/v) potassium periodate for min at room temperature. standard hai methodology was used, doubling dilutions ( ml sample + ml pbs containing . % bovine serum albumin) of test samples were carried out in`v' bottomed microwell plates. an equal volume of bcov antigen containing four haemagglutinating units was then added and incubated for h at room temperature. ml of . % (w/v) rat red blood cells was then added and the test plates incubated overnight at ± c. the haemagglutination antibody titre was de®ned as the reciprocal of the highest dilution giving % inhibition of agglutination, allowing for the pre-treatment dilution. all animals possessed some pre-existing antibody to bovine coronavirus as measured by both hai (range log ± and vn (range log x ± x ). no de®ned increase in antibody titre could be detected by either hai or vn in animals immunised with the lowest dose of bcov ( . antigen units). however, animals receiving r antigen units of bcov antigen showed an increase in mean hai antibody titre by day . peak hai titres of log x and log x were observed either days post-vaccination with antigen units per dose, or days post-vaccination with antigen units per dose, respectively. these animals were maintained for days and showed a steady drop in titre of approximately log x per month from day (fig. a) . the vn antibody response was similar but peaked earlier at day with titres of approximately log x x however, while vn antibody levels declined between days and in cattle receiving antigen units (fig. b) , those obtained from animals receiving the higher dose of units did not decline until after day . only the group immunised with antigen units of bcov still possessed a vn titre r log above the pre-immunisation titre at days post-vaccination (fig. b) . the sentinel group showed no increase in antibodies to bcov by either hai or vn during the study. as a result of this study, a bcov antigen load of antigen units per dose was selected for further study on the basis that it was likely to produce an optimal antibody response in terms of antibody titre and duration. vaccines for the cow/calf studies were therefore formulated at this level. the vaccine was well tolerated and no signi®cant local or systemic side eects were observed. the antibody results from these studies are shown in fig. . since virus neutralisation is the key parameter involved in protection by lactogenic immunity, antibody titres were only determined by vn. with the exception of study b, the serum vn antibody levels of the placebo groups did not change signi®cantly during the course of the experiment. in study b however, % ( / ) of placebo treated animals showed an increase in serum vn antibody levels of log x ± x during the experiment suggesting that`wild type' bcov was circulating within the herd. after immunisation, serum vn antibody levels in the vaccinated groups showed a signi®cant p` x increase in serum vn antibody levels compared with those observed in the placebo control groups. mean serum vn antibody levels were increased by at least log x by days post-vaccination and these levels were typically maintained until calving. associated with this increase in serum vn antibody levels, ®rst day colostrum vn antibody was also signi®cantly higher log x ± x in the test vaccine groups compared with the placebo control groups. similar dierences were maintained in milk antibody throughout the period of study (up to day on the dairy farms and up to day on the beef suckler farms). the smallest increase occurred in study b where sero-conversion amongst the placebo treated animals was observed (fig. b) . serum taken from calves allowed to suckle vaccinated dams showed that levels of circulating vn antibody were up to ten-fold higher than those obtained from calves suckling placebo-vaccinated cows. the eect of the interval between vaccination and calving on the increase in milk vn antibody titre can be seen in table where data from studies a and b have been combined into ®ve sets, each set comprising cows (three vaccinate and three placebo cows from each study) with mean vaccination/calving intervals of , , , and days, respectively. for placebo treated animals, irrespective of the vaccination/calving interval, day colostrum possessed vn titres of ap- proximately log x , falling rapidly to levels of approximately log x (background) in day milk. in contrast, for the vaccinated cattle, day colostrum possessed vn titres of approximately log x , with a much slower reduction in antibody level, only approaching background levels in -day milk. the duration of the milk antibody response seen in the vaccinated animals where the vaccination/calving interval was days was less marked than where this interval was longer. this re¯ects a reduced response to vaccination seen in approximately % of the vaccinates in the -day set. bcov antigen input into the formulated vaccine was measured by elisa, since measurements based on infectivity do not quantitate total antigenic mass, only viable virus particles and therefore could not be used post-inactivation. the elisa procedure used was primarily targeted towards the viral haemagglutinin which has been shown to contain a key virus neutralising epitope [ ] . antigen levels could therefore be tracked throughout the formulation processes. the results of the dose response study indicate that in the presence of pre-existing antibody, relatively large amounts of immunogen (> antigen units) are needed to stimulate a signi®cant increase in bcov serum antibody titre. there appear to be some minor dierences in the pattern of the antibody responses determined by either hai or vn in that the hai responses peak later than the vn responses. this may re¯ect dierences in antibody anity or sub-class requirements between the two methods of antibody measurement. in terms of neutralising antibody, the major eect of increasing antigen content appears to be in the duration rather than the magnitude of the response. with a vaccine dose containing antigen units of antigen the response was rapid, reaching a plateau approximately days post-vaccination which was maintained until at least days post-vaccination. with a dose containing antigen units of antigen, the virus neutralisation response was equally rapid and of similar magnitude, although in contrast declining rapidly to days post-vaccination. selection of the correct antigen load therefore appears critical in ensuring an optimal window in terms of the interval between vaccination and calving. an additional advantage in using a higher antigen dose was also demonstrated in cow / calf study b where elevated antibody responses were still observed even in the face of circulating infection. this suggests that vaccination could still play a signi®cant role in enhancing overall herd immunity even in an active disease situation. fig. . mean bcov vn antibody results in sera and milk from vaccinated (q) and placebo treated (q) cows and sera from their respective calves. studies were carried out on four farms, two beef suckler herds (a and b) and two dairy herds (c and d) and samples were obtained at vaccination (v), calving (c) or at the number of days indicated after the event. error bars denote % con®dence limits. the main source of antibody in bovine mammary secretions is serum igg . this is selectively transferred from serum throughout lactation, albeit at a reduced level in milk compared to colostrum [ ] . the initial serology data therefore suggested that with a bcov antigen load of approximately antigen units, successful vaccination could take place at any time between and weeks pre-calving. this hypothesis was supported by the observations on the magnitude of the antibody titres found in the colostrum and milk of cows vaccinated at dierent times pre-calving with a vaccine containing antigen units of bcov. signi®cant responses in colostrum and milk were observed at both ends of the vaccination window, although not all cows vaccinated approximately days pre-calving appeared to show an optimal response. individually, there was a large variation in the levels of speci®c bcov antibody present in both vaccinate and placebo control groups throughout the study. this variability was particularly noticeable in colostrum and milk samples, and presumably arose from the dierential levels of pre-existing bcov antibodies observed in the animals. although this would be expected to adversely aect the statistical testing of the results, signi®cant dierences p b x between test and control groups were found on the majority of days tested. this contrasts strongly with previously reported studies wherein increases in bcov antibody titre in serum or milk following vaccination were either minimal or non-existent [ ± ]. the reasons for the improved responses observed with the test vaccine are related both to the concentration of bcov antigen present and the use of an oil adjuvant, which have generally been reported to be highly eective in the enhancement of rotavirus antibody titres in mammary secretions [ ] . the magnitude of the antibody response observed following a single vaccination may be the result of the use of pre-primed (as a result of previous infection) cattle. bovine coronavirus is ubiquitous and in the ®eld situation it is extremely dicult to ®nd a seronegative individual. this observation is supported by the data presented in this study where a total of heifers and cows, with no previous bcov vaccination history, were tested prior to treatment and all were found to have substantial levels of pre-existing antibody. a major decline in speci®c antibody levels present in mammary secretions was observed in the ®rst few days after calving. this is consistent with previously reported research [ , ] , which showed that the switch from colostrum to milk was associated with a decrease in immunoglobulin concentration. enhanced antibody levels are maintained for to days post-calving, although as bcov speci®c antibody levels decrease calves will become more susceptible to sub-clinical infection. such infections will stimulate active immunity and hence confer long term protection upon the calf. in the beef suckler situation, where the calf is allowed to suckle naturally from its dam, it is relatively easy to ensure the continual presence of protective antibody in the gut lumen. in the dairy situation, typically the calf is allowed access to colostrum but is prevented from suckling milk and thus fails to ensure the presence of protective antibody in the gut lumen beyond the ®rst few days after birth. a modi®ed feeding regimen is therefore required whereby colostrum table the eect of the interval between vaccination and calving on the magnitude of the bcov vn antibody response in mammary secretions mean log bcov vn antibody titre in colostrum/milk following vaccination at the speci®ed time before calving feeding is continued as part of the calves' diet for at least the ®rst weeks of life. the levels of speci®c antibody present in the colostrum samples from the dairy herds (studies c and d) showed a marked drop in titre over days, this was associated with the switch from colostrum to milk but may be further exacerbated by a dilution eect related to the larger volumes of milk produced by dairy cattle compared with beef cattle. these antibody levels were however signi®cantly higher than those in the placebo treated group and equivalent to those seen in milk from beef cattle at days post-calving. continued feeding of colostrum pooled from collections obtained during the ®rst days post-calving should therefore provide an enhanced level of protection to the calf over this time period. it is accepted that by analogy with rotavirus infections in the calf [ ] and transmissible gastro-enteritis virus infections in the piglet [ ] , lactogenic immunity should protect against bcov infection in the calf [ ] , and this has been con®rmed experimentally (crouch c.f., unpublished observation). there are however sig-ni®cant problems in assessing the ecacy of vaccines where protection is based on boosting lactogenic immunity. this is a result of the number of variables that interact to eect the apparent level of protection obtained. such variables include the management system for the target animal, the titre of speci®c antibody achieved in both colostrum and milk, the duration of the enhanced antibody response, the volume and timing of antibody ingested and the nature (amount and pathogenicity) of the challenge. of these, the key parameters able to be in¯uenced directly by the design of the vaccine are the titre and duration of speci®c antibody achieved in colostrum and milk. this report indicates that a single dose of coronavirus vaccine administered to the pregnant heifer to weeks before calving is capable of signi®cantly increasing the titre and duration of speci®c antibody present in colostrum and milk. work is currently underway to con®rm the minimum level of speci®c antibody required to protect the calf from challenge and thus establish the duration of immunity. acute undifferentiated neonatal diarrhea in beef calves . occurrence and distribution of infectious agents can pathogenic relationships of rotavirus, escherichia coli, and other agents in mixed infections in calves pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhea pathology of neonatal calf diarrhea induced by a reo-like virus pathology of neonatal calf diarrhea induced by a corona-like virus prevalence of rotavirus and coronavirus antigens in the faeces of normal cows radioimmunological (ria) and enzymimmunological (elisa) detection of coronavirus antibodies in bovine serum and lacteal secretions vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic vaccination of dams with a combined rotavirus± coronavirus vaccine to protect newborn calves against diarrhea antibody levels in milk of vaccinated and unvaccinated cows against organisms of neonatal diarrhoea incidence of diarrhoea and of rotavirus-and coronavirus-shedding in calves, whose dams had been vaccinated with an experimental oil-adjuvanted vaccine containing bovine rotavirus and bovine coronavirus new polyvalent vaccine against intestinal infections in newborn calves enhancement of passive immunity with maternal vaccine against newborn calf diarrhea preliminary studies of a bovine coronavirus (bcv) antigen responsible for neutralization monoclonal antibodies to bovine coronavirus glycoproteins e and e : demonstration of in vivo virus neutralizing activity synthesis and distribution of immunoglobulins comparison of dierent antigen preparations as substrates for use in passive hemagglutination and enzyme-linked immunosorbent assays for detection of antibody against bovine enteric coronavirus passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunised or nonimmunised cows antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus enteric vaccines for farm animals and horses we thank k. webb, s. binnie and i. darby for their technical assistance in the preparation of the trial vaccines and dr. h. specter for performing the statistical analysis. key: cord- -g a ik authors: mukherjee, siddhartha title: before virus, after virus: a reckoning date: - - journal: cell doi: . /j.cell. . . sha: doc_id: cord_uid: g a ik the lasker awards, a celebration of one of the most prestigious international prizes given to individuals for extraordinary contributions to basic and clinical medical research, pubic health, and special achievement, was cancelled because of the covid- pandemic. typically, essays on the awardees and their scientific and medical contributions are solicited and published in cell in collaboration with the lasker committee. this year, the lasker committee commissioned an essay to reflect on the historic contributions that scientists and physicians have made to our understanding of immunology and virology, and future directions in medical and basic research that have been highlighted by covid- pandemic. the lasker awards, a celebration of one of the most prestigious international prizes given to individuals for extraordinary contributions to basic and clinical medical research, pubic health, and special achievement, was cancelled because of the covid- pandemic. typically, essays on the awardees and their scientific and medical contributions are solicited and published in cell in collaboration with the lasker committee. this year, the lasker committee commissioned an essay to reflect on the historic contributions that scientists and physicians have made to our understanding of immunology and virology, and future directions in medical and basic research that have been highlighted by covid- pandemic. ''if you think research is expensive, try disease.'' -mary lasker in the summer of , a russian professor of zoology, elie metchnikoff (also called ilya mechnikov) quarreled with his colleagues at the university of odessa. he was a temperamental man with a depressive streak, with scientific interests that ranged from the embryology of cuttlefish to the digestive system of flatworms. but he was often in conflict with his colleagues, and in ' , he moved to sicily, where he set up a private laboratory (gordon, ) . in messina, where the warm, shallow, windy beaches yielded a constant wealth of marine animals, metchnikoff began to experiment with starfish. alone one evening-his wife and children had gone to watch the local circus-metchnikoff devised an experiment that would change our understanding of immunity. the starfish larvae were semi-transparent; he had been watching cells move about in the bodies. he was particularly interested in the movement of the cells after injury. what if he stuck a thorn in one of the starfish's feet? he spent a sleepless night and returned to the experiment the next morning. a group of motile cells-a ''thick cushion layer''had accumulated busily around the thorn. he had, in essence, observed the first steps in inflammation and immune response: the recruitment of immune cells to the site of injury. the immune cells moved toward the site of inflammation actively-i.e., on their own. '' [t] he accumulation of mobile cells round the foreign body is done without any help from the blood vessels or the nervous system,'' he wrote, ''for the simple reason that these animals do not have either the one or the other. it is thus thanks to a sort of spontaneous action that the cells group round the splinter'' (mechnikov, ) . by the mid- s, the splinter of the idea-immune cells being recruited actively to inflammatory sites to launch a response-led to a series of monumental experiments. the immune cells, he found, tried to ingest-eat-the infectious agent or irritant that had accumulated at the site. the phenomenon was called ''phagocytosis''-or eating (of an infectious agent) by an immune cell (metchnikoff, ) . in an extraordinary series of papers published in the mid- s-a body of work that would eventually win him the nobel prize (table )-metchnikoff described the relationship between an organism and its invaders as ''kampf''-a ''drama unfolding within organisms'' that was like a perpetual struggle. he wrote, ''a battle takes place between the two elements [i.e., the microbe and the phagocytic cells]. sometimes the spores succeed in breeding. microbes are generated that secrete a substance capable of dissolving the mobile cells. such cases are rare on the whole. far more often it happens that the mobile cells kill and digest the infectious spores and thus ensure immunity for the organism'' (mechnikov, ) . as i write this, we are in mid-struggle against a miniscule, deadly pathogen that has swerved the course of human history. what words does one use-what phrases-to adequately capture the difference in living in the bv versus the av-before virus and after virus? to witness the sights and sounds of this struggle is to realize that life has been pushed off its known orbit forever: the constant beeping of alarms in the wards that eventually merged together into a mind-numbing wall of sound; the terror and confusion written across the brow of a (masked) cancer patient who was told that he had the virus; and, above all, the hideous damnation of dying alone, with a handheld camera as the only fragile connection with your family-''dying on iphone,'' as one doctor friend described it. this is not a moment to celebrate, but to reflect and recalibrate; it is a moment of introspection, perhaps even of revision. we need to look back to move forward. and so this essay looks back at history-of virology, vaccinations, and immunology-and asks: what have we learned, and what must be revisited? we knew about immunity long before we knew about the immune system. as early as , medical healers in china had realized that those who survived smallpox did not catch the illness again (survivors of the disease were enlisted to take care of new victims) and inferred that the exposure of the body to an illness must protect it from future instances of that illness. chinese doctors ground smallpox scabs into a powder and insufflated it into a child's nose with a long pipe (jannetta, ) . vaccination with live virus was a tightrope walk: if the viral inoculum in the powder was too large, the child, instead of acquiring immunity, would acquire a full-fledged version of the disease-a devastation that occurred about one in a hundred times. if all went well, the child would have a mild, local experience of the disease, and be immunized for life. in the seventeen-sixties, traditional healers in sudan practiced tishteree el jidderee (''buying the pox''); a healer, typically a woman, haggled with a mother over the price of her sick child's ripest pustules (bayoumi, ) . it was an exquisitely measured art: the most astute among the healers recognized the lesions that were likely to yield just enough viral material, but not too much. the differing sizes and shapes of the pustules led to the european name for the disease: variola, from variation. the process of immunizing against the pox was called ''variolation. '' in may , a young physician named edward jenner proposed a safer approach to smallpox vaccination. he used material from pustules of cowpox-a disease caused by a virus related to smallpox-harvested from a young dairymaid, sarah nelmes, and inoculated the son of his gardener, an -year-old boy named james phipps, with it. in july that year, he inoculated the boy again, but this time with material from a smallpox lesion. although jenner had breached virtually every boundary of ethical human experimentation (there is, for instance, no record of informed consent, and the subsequent ''challenge'' with live virus might well have been lethal to the child), it apparently worked: phipps did not develop smallpox. after facing initial resistance from the medical community, jenner increased his vaccination efforts and became broadly celebrated as the father of vaccination (even the word ''vaccine'' carries the memory of jenner's experiment; it is derived from ''vacca,'' latin for cow) (riedel, ) . yet even this story, retold and recycled in textbooks, is riddled with misattributions (history, too, has its revisions). the virus carried in sarah nelmes' pox lesions may have been horsepox, not cowpox (even jenner acknowledged the fact: ''the disease makes its progress from the horse [as i conceive] to the nipple of the cow, and from the cow to the human subject,'' he wrote). nor, perhaps, was jenner the first vaccinator: in , benjamin jesty, a prosperous farmer from yetminster village in dorset, convinced by the stories of dairymaids who frequently got cowpox and seemed immune to smallpox, supposedly harvested lesions from the udder of an infected cow, and inoculated his wife and two sons. jesty became an object of ridicule among physicians and scientists-but his wife and children survived the smallpox epidemic without catching the disease (hammarsten et al., ) . but how did inoculation generate immunity, particularly longterm immunity? some factor produced in the body must be able to counter the infection and also retain a ''memory'' of the infection over multiple years. in , the biochemist paul ehrlich (ehrlich, ) was traveling to egypt when he heard an extraordinary (and possibly apocryphal) story of a snake-charmer who, having been repeatedly bitten by a cobra during his childhood, had become resistant to subsequent attacks by cobra venom. ehrlich believed that an ''antivenin'' substance must have been generated in the snake-charmer's body. in , in berlin, emil von behring and kitasato shibasaburo launched a series of experiments to understand how immunity to toxins and venoms might arise. among the most dramatic of these experiments was the demonstration that the serum of an animal exposed to tetanus, or to diphtheria toxin, could be transferred to another animal and confer immunity to tetanus or diphtheria (behring and kitasato, ) . in a rather desultory footnote to the diphtheria paper, von behring first used the word ''antitoxisch''-or anti-toxin-to describe the activity of the serum (lindenmann, ) . in , in a wide-ranging, speculative paper entitled ''experimental studies on immunity'', ehrlich pushed scientists to imagine the material nature of this ''activity.'' he boldly coined the word ''anti-korper''-anti-body. the word ''korper''-from corpus, or body-signaled his growing conviction that an ''antibody'' was an actual chemical substance-a ''body'' generated to defend the body. where did these antibodies come from? in the s, the danish physiologists mogens bjørneboe and harald gormsen and their swedish colleague, astrid fagraeus (fagraeus, ) , showed that the serial inoculation of rabbits with vaccines or toxins caused a particular cell type, called plasma cells, to expand and secrete antibodies. the origin of these plasma cells was traced back to a particular class of white blood cell called a b cell (bjørneboe and gormsen, ) . drawing on this early work, max cooper, a young biologist working with robert good in minnesota, followed the trail of a report first published in a poultry journal and demonstrated that in chickens, b cells were generated in an organ called the bursa of fabricius, found near their cloaca (the organ had been described by the medieval anatomist hieronymus fabricius). when cooper removed the bursa in irradiated hatchlings, there were no b cells, and no antibodies. in humans, though, there was no bursa (cooper et al., ) . instead, b cells were eventually found to originate in white-blood progenitors, typically found in the bone marrow. but the puzzle of how a plasma cell might learn to produce a specific antibody to bind an antigen-a biological molecule that was a yang to an antigen's yin-remained unsolved until the late s. in the s, ehrlich had proposed a magnificent theory. every cell in the body, he argued, displayed an immense set of unique proteins-''side chains,'' as he called themattached to its surface. the side chains were shaped in the form of cognate opposites, or inverted shapes, to the toxin or antigen-like a lock to a key, or a mold to a statue. when a toxin or pathogenic substance bound to one such side chain in a cell, the cell increased the production of that side chain. with repeated exposures to the antigen, ehrlich speculated, the side chain was ultimately released into the blood, thereby producing an antibody. but the theory required every immune cell to come pre-loaded with side chains carrying an inverted universe of all molecules-a mind-boggling cosmos of antibodies that had to be present in every immune cell. decades later, the chemist linus pauling proposed an even more rococo theory: the specificity of an antibody for its cognate antigen was created by an antibody folding around an antigen and acquiring the inverted shape of the antigen. the antigen, in short, was like a mold that ''instructed'' an antibody how to form around it. but the ''instruction'' and the ''infinite side chain'' theory were both conceptually implausible: proteins couldn't be made to fold around antigens, like medieval drapery, nor could a cell display an infinite variety of side chains, awaiting release. the most plausible solution to the conundrum of how antibodies were generated, and how they became antigen specific, was eventually proposed in an obscure paper published in in the australian journal of science by a melbourne scientist, frank macfarlane burnet (burnet, ) , who drew on earlier work by niels jerne and david talmage. what if, burnet reasoned, every b cell expressed only one antibody? in short, a massive ''repertoire'' of antibodies was already present in the immune cells of the body, and it was the antibody-expressing cell-not the antibody itself-that was selected, and grew, when it bound the antigen. '' [i]t is tempting to consider that one of the multiplying units in the antibody response is the cell itself,'' talmage had written. '' [but] only those cells are selected for multiplication whose synthesized product has affinity for the antigen injected.'' (the italics are mine.) burnet, following this line of thought, reasoned that it was this clonal proliferation of an immune cell-a cell stimulated by the binding of an antigen-that enabled the antibody response. at oxford, james gowans discovered that the ''burnetian repertoire'' (as it came to be called) was carried by circulating small lymphocytes that divided rapidly in response to antigens. when he transferred these active lymphocytes-later found to be b cells-from an antigen-exposed animal to a naive animal (an ingeniously simple experiment), gowans found that he could transfer antibody-mediated immunity as well. as the geneticist joshua lederberg wrote with remarkable prescience (yet without experimental evidence), ''do antigens bear instructions for antibody specificity [as pauling had argued] or do they select cell lines [that are specific for the antigen-i.e. by clonal selection]''? lederberg clearly favored the second theory (lederberg, ) . the molecular ''shape'' of an antibody was also soon solved: between and , gerald edelman (edelman and poulik, ) and rodney porter (porter, ) , working at the rockefeller university in new york and oxford university (refs), respectively, discovered that most antibodies are y-shaped molecules (some subclasses of antibodies have modifications to this shape). the two outer tines of the y bind to the antigen, each acting like a prong. the shaft, or the stem, of the y, serves many functions. macrophages use shaft to capture antibodybound microbes, viruses, and peptide fragments and swallow them, much like the shaft of a fork is used to pull food into the mouth. this, indeed, is one mechanism of ''phagocytosis''cells eating microbes-the phenomenon that metchnikoff had observed. the shaft or stem of the y has yet other purposes: it also attracts a cascade of toxic immune factors to attack microbial cells. the genetics of how immune cells make such a diverse repertoire of antibodies-a unique antibody type per cell-was worked out, piece by piece, by susumu tonegawa, leroy hood, and phil leder. it involved the regulated shuffling of dna within the b cell-the recombination of genetic modules, followed by more mutations to create a ''mature'' antibody-a strategy that lederberg had loosely, and presciently, proposed years earlier. in , a thirty-year-old phd student in london, jacques miller, discovered the function of a human organ that most scientists had long forgotten. the thymus-named because it vaguely resembles the lobe-shaped leaves of the thyme plant-was, as galen described it, ''a bulky and soft gland'' that sat above the heart. even galen noted that it slowly involuted as humans grew older. and when the organ was removed from adult animals, nothing significant happened. a dwindling, dispensable, involuting organ; how could it possibly be essential for human ll lives? scientists began to think of the thymus as a vestigial detritus left behind by evolution-an appendix or a tailbone hanging, incidentally, above the heart. but might it have a function during fetal development? using minute forceps and the thinnest silk sutures, miller removed the thymus from neonatal mice about sixteen h after birth. the effect was unexpected and dramatic. the lymphocytes in the blood-the white cells in the blood that were not macrophages or monocytes-dropped dramatically, and the animals became increasingly susceptible to common infections. b cells dropped in number, but some other white cell-some previously unknown type-was even more dramatically diminished. many of the mice died of the mouse hepatitis virus; many had bacterial pathogens colonize their spleens. by the mid- s, miller had realized that the thymus was the site of maturation for a different kind of immune cell-not a b cell, but a t cell, from the word ''t-hymus'' (max cooper, working independently, had also established that two kinds of lymphocytes existed, and that the thymus was the maturation site for t cells). but if b cells generate antibodies to kill microbes, what do t cells do (miller, ) ? in the s, rolf zinkernagel and peter doherty, immunologists working in australia, provided the first clue. they began with so-called killer t cells: these t cells would recognize the virus-infected cells, perforate their cell membranes, and douse them with toxins, forcing the infected cells to shrivel and die, thereby purging the virus within the cell as a result. these t cells would be eventually known as cytotoxic (i.e., ''cell killing'') t cells, and they carried a marker on their surface: cd (zinkernagel and doherty, ) . but the peculiar thing about these cd -positive t cells, zinkernagel and doherty discovered, was that they had a capacity to recognize viral infections only in the context of the ''self''-i.e., only if the t cell and the infected cells came from the same strain of mouse. it was as if the t cell was capable of computing a kind of dual logic. first: does the cell that i am surveying belong to my body? and second: is it infected with a virus or a bacterium? using genetic techniques, zinkernagel and doherty tracked the detection of the ''self'' to a molecule called major histocompatibility complex (mhc) class i-a protein that comes in thousands of variants. each of us carries a unique combination of mhc class i genes. it is this ''self'' mhc that the t cell first detects. it is as if the mhc protein is a frame. without the right frame, or context, the t cell cannot even see the picture. the zinkernagel-doherty experiments had solved one half of the logic problem. but how does a cd cell find a self-cell with a virus embedded within it? my doctoral mentor, alain townsend, first at mill hill in london, and then at oxford, took up this question in the s. townsend began his experiments with cd killer t cells and influenza virus. some of these killer t cells elicited by flu infection, researchers had found, were detecting the presence of the influenza protein, called np, inside a flu-infected cell (townsend et al., ) . but that's where the mystery began. ''that protein, np, never makes it to the cell surface intact,'' townsend told me recently. we were sitting in a london taxi cab, returning from a lecture. it was london dusk, with its mix of smog and rain and sudden shards of oblique english light, and the streets, as we sped through them-old bond, bury street-were full of houses with partially lit windows and closed doors. how could you detect a resident inside one of these houses, unless the resident happened to poke his head outside? ''np is always inside the cell,'' alain continued. he performed the most sensitive tests-assay upon assay, week upon weekto find the np protein on the flu-infected cell's surface, where a t cell might detect it. but it wasn't there. ''as far as cell surface proteins are concerned, there is nothing for a np-detecting t cell to see. it's invisible on the cell surface-it isn't even there-and yet it's perfectly visible to the t cell'' (a. townsend, personal communication) . how, then, was the t cell detecting np? the crucial discoveries came in late s. the cd killer t cells, alain found, was not recognizing intact np, poking its face outside the cell. rather, the cells were detecting viral peptides-small pieces, or fragments, of the viral protein, np. and crucially, these peptides had to be ''presented'' to the t cells in the right ''frame''-in this case, carried, or loaded, by the class i mhc protein-the very protein that zinkernagel and doherty had implicated in the killer t cell response. the class i protein was actually a carrier, a peptide-bearer-and thus the ''frame'' required for the recognition by a cd t cell. in the s, working in parallel, emil unanue began to explore the immune detection of microbes that are internalized by cellsa la metchnikoff. once phagocytosed, the microbes and their debris are targeted to compartments, such as the lysosome, chock-full of degrading enzymes, that can chop the proteins into peptides. and analogous to what townsend had found, these peptide fragments from the microbes are bound by a related class of protein carriers-called class ii mhcs-that present the peptides, as if on a special molecular platter, to the t cell (harding and unanue, ) . but it's here that the immune response diversifies and forks; it assumes a second wing of attack. a second subclass of t cells, called cd positive cells, senses these mhc-ii carrier-mounted peptide fragments. instead of killing the infected cell, the cd t cell incites b cells to start synthesizing antibodies. it secretes chemical substances, including cytokines, that amplify the macrophage's capacity to become mobile and phagocytose; it causes an upsurge of local blood flow and summons yet other immune cells to challenge the infection. in the absence of the cd cell, the transition between the detection of a pathogen and antibody production by b cells falls apart. for all these properties-and especially for supporting the b cell antibody response-this type of cell is called the ''helper'' t cell. there's a final type of immune cell that deserves mention. in , ralph steinman, working at the rockefeller university in new york, looked down a microscope and found cells in lymph nodes that ''assume a variety of branching forms, and constantly extend and retract many fine cell processes''-like a mobile, many-branched tree. ''dendritic cells,'' as steinman named them (after the greek work for ''tree'') are professionally designed to present antigens to t cells and jumpstart an immune response (steinman and cohn, ) . in a sense, the discovery of dendritic cells brings us back, full circle, to the kampf between pathogens and the immune system and to the origins of immunology. the history of immunology forms a strange circle: it returns to rediscover its origins. the ll cell , october , century that followed metchnikoff's discovery of macrophages-from the s to s-was dominated by antibodies, b cells, and t cells. these responders to infection are ''adaptive'', i.e., they arise, on command, to attack specific pathogens. but in evolutionary terms, this adaptive immunity is a relative newcomer. amidst the buzz and excitement of b and t cells, a more ancient wing of the immune system-the so-called ''innate'' system-was largely forgotten and ignored. dendritic cells and macrophages, among several other cell-types, are part of this innate immune system. these cells possess receptors, including a family called tolllike-receptors, or tlrs, that do not recognize specific pathogens but molecular ''patterns'' common to pathogens in general. these patterns are chemicals carried or released by viruses and bacteria when they enter the body or infect a cell, including components of the bacterial cell wall or forms of viral rna (these pathogen-induced, pattern-recognition receptors and the signals activated by them were described and discovered by many scientists. among them, bruce beutler, jules hoffman, charles janeway, and ruslan medzhitov deserve special mention). prompted by signals from these pattern recognition receptors, the cells of innate immune system release specific signals and chemicals-interferons, among them-to stir up an anti-viral and inflammatory response. they are the first responders to infections-and yet, ironically, among the last to be fully acknowledged, or understood, as essential parts of the organismal physiology of the immune response. i am an immunologist-turned-virologist-turned-internist-turnedoncologist-turned-writer-turned-historian (which is to say: i have mastered the science of lack of expertise). but i am also a new york doctor who experienced the devastating brunt of the sars-cov- epidemic through the stories of my patients, nurses, and colleagues. i present this history-cursory, abbreviated, and familiar, perhaps, to many readers-with due humility to capture two contrasting points. first: to illuminate how richly the past century of immunological research has contributed to our understanding of the typical response to viruses and some pathogens. but second, and conversely: to highlight how poorly we understand the physiological consequences of the immune response to sars-cov- . the power of science lies in its ability to dissect physiological phenomena into their component pieces. but the sars-cov- pandemic has illustrated that reassembling those pieces to understand immune physiology at an organismal level remains elusive, particularly for this virus. take, for instance, just three of the many mysteries of sars-cov- infection that we are still trying to solve. first: what determines the strength and durability of an immune response to the virus? it's a question of seminal importance to vaccine developers, and yet, definitive answers are missing. in a paper published in nature, michel nussenzweig and his colleagues dissected the immune response to sars-cov- infection (robbiani et al., ) . nearly one-third of infected patients, they found, produced very low amounts (or ''titers'') of neutralizing antibodies to the virus. i asked nussenzweig, one of the most knowledgeable immunologists in the field, about the relevance of these sluggish antibody responses. do individuals with low-titer antibodies have fewer memory b cells to combat a future infection? can they be re-infected-and if so, would they suffer milder disease? and could such re-infected individuals carry enough virus to infect the immunologically naive population? or take an even more basic question that has enormous epidemiological and public policy significance: is there a level, or threshold, of viral load that a patient must carry in order to infect others? in other words, is there a difference between the infected and the infectious (if so, more stringent isolation protocols might be deployed on those that are infectious until they clear the virus)? nussenzweig doesn't know-and nor, of course, does the whole field. and what about t cells? some of the vaccines currently in latephase trials elicit t cell responses, while the nature and strength of the t cell response for some vaccine candidates remains unknown. does it matter? does it influence the efficacy or durability of the vaccine? we don't know. and there's an odd finding that keeps cropping up: some people-up to forty percent in some studies-possess t cells that ''cross-recognize'' sars-cov- infected cells because these people have been previously infected by other, related common-cold coronaviruses that share genetic similarities. could these people be partially protected? we don't know. more generally, why do infections by some viruses, or inoculation with some vaccines, precipitate durable, long-term responses, while the immunity to others wanes over time, causing re-infections, and requiring ''boosters'' for continuous immunity? we don't know. despite decades of research on the immune response to viruses, fundamental questions about vaccine development, immune durability, and the physiology of the anti-viral response in the human organism, remain unsolved. second: why do some people recover from infection, while others progress to a fulminant, deadly disease? are there host factors that predict severe disease? an intriguing dutch study implicated one gene: tlr . this x-linked gene was mutated in two pairs of brothers who suffered an atypically severe form of covid- for their age (one pair was found to have a deletion of the gene, while the other pair had a single amino acid change) (van der made et al., ) . tlr is one of the receptors involved in the innate immune response to viruses. when cells from the peripheral blood of these brothers were challenged with chemical signals that activate tlr , the production of interferons (particularly a subtype termed type i), and interferon-related genes, was blunted, especially in the pair of brothers with the deletion in tlr . a separate study from a team in paris converged on similar results (hadjadj et al., ) . the team profiled fifty virus-infected patients and eighteen controls. and again, in patients with most severe forms of the disease, the expression of type i interferon was blunted, while the blood levels of other inflammatory cytokines, such as interleukin and tumor necrosis factor a, were increased. akiko iwasaki's group at yale also profiled a large cohort of patients with moderate or severe infection and compared them to healthy controls (lucas et al., ) . the sustained activation of certain patterns of chemokines and cytokines was correlated with severe illness-a phenomenon that iwasaki has termed ''immunological misfiring.'' a more recent paper, published in cell, also implicated dysfunctions in innate immune cells, particularly myeloid cells such as neutrophils and monocytes, in patients with severe covid infection (schulte-schrepping et al., ) . to read these papers is to glimpse a code, or a pattern, behind them-but to be unable to find the code-breaking algorithm. the rosetta stone is missing. one possibility is that type interferons produced by lung cells (possibly by lung-resident immune cells, including dendritic cells) are necessary for initial resistance. a blunted response fails to control the virus and predicts worse disease. once the infection progresses, though, innate cells such as monocytes produce the dysfunctional cytokine storm-the immunological misfiring that iwasaki describes. i asked iwasaki and medzhitov to reconcile these various studies. ''there appears to be a fork in the road to immunity to covid- that determines disease outcome,'' iwasaki told me. ''if you mount a robust innate immune response during the early phase of infection, you control the virus and have a mild disease. if you don't, you have uncontrolled virus replication in the lung that result[s] in misfiring of the immune response that fuels the fire of inflammation leading to severe disease'' (a. iwasaki, personal communication). but overall, the data suggest that innate cells, interferons, and a dysregulation of the intricate networks of signals that connect immune cells are somehow involved. again, though, these studies illustrate the fact that our understanding of the organismal physiology of this viral infection lacks the detail and resolution that are required to understand sars-cov- infection at a granular, mechanistic level. finally: what about the diffuse, systemic manifestations of sars-cov- infection? there are systemic physiological effects of cov- infection that remain mysterious. some infected children experience an autoimmune illness similar to kawasaki's disease (jones et al., ) . why? we don't know. microstructural changes have been found in the brains of some affected patients (filatov et al., ) ; there are cardiac, vascular, and autoimmune sequelae of the infection that we don't understand. many infected adults have blood clotting disorders that require the use of anti-clotting medicines (al-samkari et al., ) . the pandemic has energized us, yes, but it has also provided a necessary dose of humility. it has also been a call to action. it is time, as mary lasker would have it, to return to research, to reflection, to revision (''[i]f you think research is expensive, try disease''). we have learned so much. we have so much left to learn. this article was commissioned and paid for by the lasker committee. the paragraph on the practice of the chinese and sudanese practice of variolation is adapted from the new yorker (mukherjee, ) and will appear in a forthcoming book by s.m. s.m. is a co-founder of vor, myeloid, immuneel, faeth, and cura therapeutics and serves on the boards of frequency, trialspark, equillium, cellenkos, and puretech. covid- and coagulation: bleeding and thrombotic manifestations of sars-cov- infection the history and traditional treatment of smallpox in the sudan ueber das zustandekommen der diphtherie-immunitä t und der tetanus-immunitä t bei thieren experimental studies on the role of plasma cells as antibody producers a modification of jerne's theory of antibody production using the concept of clonal selection delineation of the thymic and bursal lymphoid systems in the chicken studies on structural units of the g-globulins experimentelle untersuchungen ü ber immunitä t plasma cellular reaction and its relation to the formation of antibodies in vitro neurological complications of coronavirus disease (covid- ): encephalopathy. cureus elie metchnikoff: father of natural immunity impaired type i interferon activity and inflammatory responses in severe covid- patients who discovered smallpox vaccination? edward jenner or benjamin jesty? quantitation of antigen-presenting cell mhc class ii/peptide complexes necessary for t-cell stimulation the vaccinators: smallpox, medical knowledge, and the opening of japan covid- and kawasaki disease: novel virus and novel case do antigens bear instructions for antibody specificity, or do they select cell lines that arise by mutation? origin of the terms 'antibody' and 'antigen' longitudinal analyses reveal immunological misfiring in severe covid- on the present state of the question of immunity in infectious diseases ueber eine sprosspilzkrankheit der daphnien. beitrag zur lehre ü ber den kampf der phagozyten gegen krankheitserreger. archiv fü r pathologische anatomie und the early work on the discovery of the function of the thymus, an interview with jacques miller how does the coronavirus behave inside a patient? the new yorker the hydrolysis of rabbit y-globulin and antibodies with crystalline papain edward jenner and the history of smallpox and vaccination convergent antibody responses to sars-cov- in convalescent individuals deutsche covid- omics initiative (decoi) ( ). severe covid- is marked by a dysregulated myeloid cell compartment identification of a novel cell type in peripheral lymphoid organs of mice. i. morphology, quantitation, tissue distribution the epitopes of influenza nucleoprotein recognized by cytotoxic t lymphocytes can be defined with short synthetic peptides presence of genetic variants among young men with severe covid- restriction of in vitro t cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system key: cord- - yrf ofy authors: finlay, william j. j.; bloom, laird; grant, joanne; franklin, edward; shúilleabháin, deirdre ní; cunningham, orla title: phage display: a powerful technology for the generation of high-specificity affinity reagents from alternative immune sources date: - - journal: protein chromatography doi: . / - - - - _ sha: doc_id: cord_uid: yrf ofy antibodies are critical reagents in many fundamental biochemical methods such as affinity chromatography, enzyme-linked immunosorbent assays (elisa), flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry techniques. as our understanding of the proteome becomes more complex, demand is rising for rapidly generated antibodies of higher specificity than ever before. it is therefore surprising that few investigators have moved beyond the classical methods of antibody production in their search for new reagents. despite their long-standing efficacy, recombinant antibody generation technologies such as phage display are still largely the tools of biotechnology companies or research groups with a direct interest in protein engineering. in this chapter, we discuss the inherent limitations of classical polyclonal and monoclonal antibody generation and highlight an attractive alternative: generating high-specificity, high-affinity recombinant antibodies from alternative immune sources such as chickens, via phage display. the rapid expansion of the genomics, proteomics , and biotechnology fi elds has led to a growing demand for affi nity reagents that can specifi cally recognize proteins, peptides, carbohydrates, and haptens . affi nity reagents of high specifi city are routinely required for diverse protein drug targets, members of newly discovered biochemical pathways, posttranslationally modifi ed proteins, protein cleavage products, and even small molecules such as drugs of abuse and toxins. individual biomedical researchers will often need to monitor, quantify, and purify proteins of interest via affi nity chromatography , but there may not be any commercially available antibody reagents to allow them to do so [ ] . indeed, even in situations where there are commercially available antibodies, these reagents are often expensive, poorly characterized, and/or simply not appropriate for demanding applications. compounding this problem, the technical diffi culty of monoclonal antibody generation by the untrained researcher and the high cost (~$ , ) of a commercial monoclonal antibody generation program leads many researchers to the default solution of producing polyclonal hyperimmune sera in hosts such as rabbits. the net result of this is that researchers often settle for reagents that lack the necessary specifi city to perform the applications for which they were intended. in this review, we will outline the limitations of classical antibody generation technologies and illustrate an attractive alternative: the use of phage display libraries of recombinant antibodies built on immunoglobulin repertoires from nonmammalian animals. in particular, we will highlight the advantages of libraries derived from the domestic chicken gallus gallus , which offers a relatively inexpensive and technically accessible route to high-quality monoclonal reagents [ ] . if, like many people, you have purchased (or paid to generate) a costly and "specifi c" antibody, but subsequently found that it is actually polyreactive and of dubious quality, phage display from immunized chickens may offer an attractive alternative. hyper-immune sera from rabbits, sheep, or other mammals may be produced in large quantities, but they do not offer the consistency of monoclonal antibodies and need to be regularly replenished and recharacterized. serum antibodies are also polyclonal and frequently polyspecifi c, even when purifi ed over an antigen column, rendering them suboptimal for the specifi c recognition of a single component in a complex matrix. one illuminating study has demonstrated that when used to probe a comprehensive yeast proteome chip, unpurifi ed polyclonal antibody preparations could recognize up to different proteins, with some monoclonal antibodies and antigen column-purifi ed polyclonal antibodies also recognizing multiple proteins (related and unrelated) [ ] . the arrival of monoclonal antibody technology [ ] was a major step forward in generating high-specifi city reagents, but the reliance on the murine immunoglobulin system frequently leads to a number of practical diffi culties: ( ) monoclonal antibodies are raised on the basis of an ineffi cient fusion of splenic b-cells to an immortalized mouse myeloma line, followed by limiting dilution of the cell population. target-specifi c antibodies are randomly identifi ed, often by a simple direct elisa , where few preconditions can be set to determine which antibodies are identifi ed and one must "take what one can get" during the screening process. ( ) it is often desirable to have multiple monoclonal antibodies with specifi city for different epitopes on the same target molecule, but the diffi culty in sequencing monoclonals does not allow the rapid identifi cation of unique clones early in the screening process. ( ) humans and rodents are relatively closely related phylogenetically. many proteins of interest are highly conserved among mammals and this can frequently lead to thymic tolerance, restricting the antibody response after immunization. ( ) when an immune response to a human protein is raised in mice, the large regions of sequence similarity between murine and human proteins may lead to a restricted number of immunogenic epitopes. ( ) to generate antibodies that cross-react with orthologues from multiple species of mammal is particularly tricky, as the common epitopes among mammals are the very ones that are unlikely to provoke a strong immunoglobulin response in the mouse. ( ) tolerance issues can become even harder to circumvent when the protein of interest is from a mouse or rat. creating "knockout" mice, in which the endogenous copy of the gene for the target protein has been disabled, can often break tolerance, but this is a highly laborious and time-consuming process that few laboratories have the resources to undertake. these factors all hinder the generation of high-quality antibody reagents and thereby limit one's experimental options when developing antibodies for purifying or tracking novel proteins. to bypass the limitations in polyclonal and monoclonal antibody generation, several groups have turned to in vitro display technologies such as phage display, ribosome display, or yeast display libraries to generate recombinant antibodies. the more recent technologies of yeast and ribosome display are becoming highly established, but phage display is currently the most robust, well characterized, and reliable of these methods. antibodies derived from these technologies are cloned in microbial hosts and are therefore monoclonal from the start, with their production being easily scaled up. critically, selection and screening efforts can be directed toward specifi c epitopes or species cross-reactivity and away from polyspecifi c binding. phage display allows a researcher to do in a single tube what would be unfathomable in traditional hybridoma work: interrogate libraries of millions to tens of billions of antibodies on the basis of their binding specifi cally to a target of interest. all of this is possible due to the ingenious concept of "genotypeto-phenotype linkage," which is exemplifi ed by phage display. phage display was originally described as a rapid method for cloning gene fragments that encode a specifi c protein [ ] . by cloning gene fragments into the genome of a fi lamentous e. coli bacteriophage, smith et al. were able to generate libraries of gene fusions with the key phage coat protein p . after transformation into e. coli , the viral replication system packaged the genome (and therefore the cloned gene) into a highly stable complex carrying the gene product on the tip of the phage particle, as a fusion protein with p . by subsequently selecting expressed virions on an immobilized antibody with specifi city for a known protein that had been cloned in the phage genome, they were able to show -fold enrichment of the gene product. this set of experiments showed that "genotypeto-phenotype linkage" could be achieved and thereby defi ned the basis of all display technologies developed since. the subsequent development of a method for the effective cloning of antibody v-gene sequences via pcr allowed the capture of antibody sequences in a recombinant form [ ] , removing the need to immortalize b-cells via hybridoma fusion as required in traditional monoclonal antibody generation. this discovery was combined with the phage display process to make an effi cient method of isolating antibodies and their corresponding gene sequences simultaneously [ ] . in the antibody phage display process, libraries of diverse v-gene sequences are cloned into an appropriate expression vector in e. coli , creating an in-frame fusion with the p protein or, as favored in most recently described libraries, a truncated form of p . the phage display of protein libraries was originally performed using "phage" vectors (i.e., built upon the phage genome itself), but due to practical diffi culties in handling these libraries, more recent phage methods have mostly used so-called phagemid expression vectors. in this case, the dna backbone is a stable, small plasmid such as puc, and the p gene plus f phage packaging origin are the only phage-derived dna sequences [ ] . these phagemid libraries are more easily handled and more stable than phage libraries, as the plasmid is incapable of causing phage production by itself. the libraries can therefore be more simply cloned, expanded, and controlled than phage libraries. by infecting "helper" phage (based on m ) into a growing culture of e. coli harboring a phagemid library, the phage propagation machinery is provided, but due to a mutation introduced in the origin of replication on the helper phage genome, preferential packaging of the phagemid dna and p fusion occurs during phage replication [ ] . phage production is thereby induced, genotype-to-phenotype linkage is created, and the expressed phage particles are interrogated for the presence of useful protein sequences via target binding. this selective step may be performed by simply immobilizing the target protein on, for example, a protein-binding plastic surface such as an elisa plate, adding phage and allowing binding to occur via the antibody-p fusion proteins. nonbinding phages are removed by washing the immobilized surface, and the remaining bound phages are eluted. the eluted phages are then reinfected back into a fresh culture of e. coli to retrieve the selected gene sequences. this process is not perfect, however, and two to four rounds of selection/re-expression/selection are normally performed iteratively to remove all unwanted clones and enrich the binding population from the background of the library. nonetheless, this process can be spectacularly powerful, massively enriching specifi c antibodies in a single selection round [ ] . furthermore, the use of multiple forms of the target antigen in sequential selection rounds and the inclusion of competitor proteins can drive the selected pool toward a highly specifi c set of epitopes. much of the evolution and progression of phage display technology has been driven by the remarkable discovery that this method can be used to mine large libraries of combinatorial human antibody diversity, theoretically removing the need for animals in antibody production [ ] . by random recombination of v h and v l sequences from human lymphocyte cdna [ ] , or, by using degenerate oligonucleotides to create diversity of dna sequence in the loops associated with target binding [ ] , several groups have created very large libraries of antibody gene sequences for phage display. these libraries are analogous to the naïve antibody repertoire in an animal, and selecting from them can result in the identifi cation of antibody fragments that exhibit high specifi city and occasionally high affi nity for the target protein. several major studies have proven that with appropriate application of this technology, specifi c antibodies can be raised to proteins, peptides, haptens and even carbohydrates. in the most exemplary studies, antibodies have been raised with equivalent affi nities to those associated with a strong humoral immune response [ - ] . additionally, antibodies with suboptimal affi nities can provide useful starting molecules for the construction of mutagenized libraries which can be further screened for affi nity matured variants [ - ] . in addition to the affi nity maturation process, phage display also provides a useful tool for the production of ultra-humanized antibodies from nonhuman sources. it is widely accepted that there are intrinsic diffi culties associated with the humanization of nonhuman antibodies using cdr grafting. frequently, such grafted antibodies suffer from immunogenicity associated with high non-germ-line amino acid content, the propensity for v-domain destabilization, and very often downstream expression and formulation issues. in a recent study, ultra-humanized antibodies from three nonhuman sources were generated via singlestep complementarity-determining region (cdr) germ-lining in a process termed " augmented binary substitution" (abs) [ ] . this process utilizes phage display for selection from germline targeting combinatorial libraries which results in the identifi cation of cdr residues amenable to human germ-lining without compromising on specifi city and affi nity. a further degree of complexity is incorporated into the library through the random substitution of cdr-h residues in tandem with the germ-lining process. this simple single pass process generates signifi cantly lower non-germline sequence content, on cdr grafts from nonhuman hosts, rendering them virtually indistinguishable from fully human antibodies. this process opens up new possibilities for the affi nity maturation and humanization of antibodies from alternative sources such as chickens. recombinant antibodies are typically displayed and expressed in "fragment" forms. the simplest and most commonly used fragment is the single-chain fragment variable (scfv) where a fl exible peptide sequence links the v-regions of antibodies between the c-terminus of one domain and the n-terminus of the other, thereby combining both v-domains into a single polypeptide [ ] . the scfv may be assembled in v l -v h or v h -v l orientations, with v h -v l being the most heavily used format historically. the fl exible linker helps to make the scfv simple to express, but must be sufficiently long and fl exible to allow effective association of the v-regions to form a functional antigen-combining site. as long as this is true, the classical hydrophobic pairing of the v-regions will stabilize the structure. by far, the most common linkers are based on glycine-serine repeat structures such as ggggs × . the second most commonly used recombinant antibody format is the fab (fragment antigen binding) molecule. this structure is a complete binding "arm" of an antibody and is comprised of the full immunoglobulin light chain, expressed in conjunction with the v h -c h region from the heavy chain [ ] . fabs obligately form predominantly monomeric, monovalent fragments. they are the most "natural" of the recombinant antibody fragments and it has been shown that the presence of the constant regions can often help to stabilize antibody variable regions [ ] . the fab format is the less commonly used of the two main recombinant antibody formats, however, as its dual polypeptide structure is generally more diffi cult to express and display in e. coli than the scfv [ ] . a recent engineered scfab platform however attempts to address some of the limitations associated with scfab expression [ ] . much of the under-use of phage display may be due to experiences with the early libraries derived from naïve or synthetic human antibody diversity, which were donated to academic laboratories that were not specifi cally invested in antibody engineering. unfortunately, these forays into display technologies have often left investigators somewhat disappointed. many people have accepted the viewpoints of recombinant antibody technology experts, that these libraries can yield useful high-affi nity antibodies to any form of antigen . in general, (mostly to those highly skilled in the fi eld), this is indeed true, but the average antibody generated from these libraries is often of disappointingly low affi nity to those who are used to high-sensitivity antibodies from immunized sources. human recombinant antibodies from naïve library sources can require technically challenging in vitro molecular evolution if they are to perform the demanding "real world" functions required of many reagent antibodies [ ] . molecular evolution is far from trivial to perform and is usually beyond both the scope and interest level of the average researcher. for such reasons, it is of little wonder then that most people either ignore, or at worst disparage, phage display technology itself. nevertheless, phage display can be a relatively simple technology to use and when employed to harness natural repertoires of antibodies from immunized animals, it can offer a rapid path to highly specifi c, high-affi nity antibodies against problematic antigens . while the most successful naïve antibody libraries contain over members and are often the domain of biotechnology companies, typical immune libraries are in the - range and are easily assembled by a single investigator [ , ] . when an immunized rabbit or sheep has raised a signifi cant serum immunoglobulin titer, the common end-point to the experiment is to exsanguinate the animal and harvest the serum. however, harvesting b-cell rich lymphoid tissues from the animal, such as the spleen and bone marrow, allows the isolation of total rna and the subsequent generation of cdna [ ] . this is a simple method with which many biomedical researchers are familiar, and commercial kits are available to simplify most steps of the process. the immunoglobulin gene sequences of many animals are now known and the cdna from immune tissues can subsequently be used for the rt-pcr amplifi cation and cloning of the animal's variable region sequence repertoire [ ] . these cloned variable region sequences can then be assembled into a display library format such as scfv or chimeric fab (using human c h and c k/λ regions) [ ] . these targeted immune libraries thereby offer a potentially huge advantage over monoclonal antibodies , as libraries of > variants may be built, allowing the effective sampling of a much broader range of antibodies than the hundreds (occasionally thousands) of clones usually examined in a monoclonal antibody screen. the resulting library can be interrogated for specifi c binding proteins via phage display and the retrieved antibody fragments expressed very simply in bacteria [ ] . this process has been used to successfully harness the antibody repertoires of a large number of immune host species, including mice [ ] , rabbits [ ] , sheep [ ] , camelids [ ], and sharks [ ] . of greater interest to us, however, is to exploit this approach to harvest the novel immunoglobulin repertoires of the domestic chicken ( gallus gallus ), which is as simple to use as mice and rabbits, but also highly phylogenetically distant from mammals. avians can circumvent many of the common problems encountered with mammalian immunizations described above. as a fully domesticated small animal, chickens are an attractive host for immunization as they are highly accessible, very affordable, and easily housed in a generic animal house. most importantly, however, the amino acid homology between the mammalian and avian orthologues of a given protein is typically lower than between the mammals commonly used for antibody generation, and indeed, some mammalian proteins may not even exist in avians. the immunoglobulin response of chickens to highly conserved mammalian proteins is reliably robust, generally exhibits high avidity, and potentially targets a broad spectrum of epitopes on protein immunogens [ - ]. chickens therefore have a potentially major advantage over other common immune hosts: they can produce a high-affi nity cross-reactive antibody response targeting an epitope that is conserved across multiple orthologues of a mammalian protein. this can lead to signifi cant savings in time and resources as, if a single, broadly applicable cloned reagent can be identifi ed, it can then be used to generate a single affi nity column for the capture of the target protein from multiple species. chicken immunoglobulins have also shown benefi cial biophysical properties: they exhibit high stability to changes in ph and temperatures up to °c [ , ] , provide functional coating on latex microspheres [ ] , and demonstrate functional direct covalent coupling to a dextran layer for the detection of serum proteins by surface plasmon resonance [ ] . furthermore, as chickens are small animals, very little protein immunogen is required to raise a strong immunoglobulin response. approximately μg/bird of purifi ed protein is suffi cient to carry out a full immunization regime [ , ] . these observations have led to the regular use of chickens as an immune host for production of the polyclonal antibody termed igy (egg yolk antibody), in both research and commercial settings. laying hens will export signifi cant quantities of polyclonal igy into the egg (~ mg of igy per yolk), in a process analogous to mammalian placental igg transfer, which allows direct screening of their antibody response without the need for serum sampling [ ] . once a strong immune response has been raised, large quantities of polyclonal antibody are easily prepared from the yolk. these polyclonal antibodies have been successfully applied in research immunochemistry [ ], diagnostics [ ] , and affi nity column purifi cation [ ] . indeed, immunodepletion resins based on chicken igy can be used to remove high abundance proteins from serum and are now commercially available (sigma-aldrich seppro ® igy spin columns). unfortunately, polyclonal igy does still suffer from the same issues of ill-defi ned specifi city that all polyclonal antibody preparations do. in addition, there have been several studies describing successful chicken hybridoma monoclonal antibody generation to antigens such as human peptides [ ], sporozoite proteins [ ], and prion protein [ ], but the low antibody expression and instability associated with chicken myeloma cell lines [ , ] led to the under-use of this species as a source of monoclonal antibodies. today however, the progress in chicken antibody phage display has circumvented these problems and made recombinant chicken antibody reagents readily accessible, as we describe below. the chicken immunoglobulin repertoire is almost ideally suited to antibody phage display, as chickens generate their immunoglobulin repertoire from a single set of v h and v l germ line sequences [ ] , with the majority of positions in the framework regions maintained as germline [ ] . diversity in the v-regions is created by both v-d-j recombination and somatic hypermutation, with the additional infl uence of "gene conversion," where multiple upstream pseudogenes are recombined into the functional sequence. this germline v-gene system means that the entire chicken antibody repertoire can be captured using only four pcr primers [ ], making chicken libraries highly representative of the induced immunoglobulin response. this is in direct contrast with immune hosts such as mice, which have diverse germline v-gene sequences and therefore require complex mixes of pcr primers [ ] . additionally, the two v-gene germline sequences found in chickens are highly homologous to the human v λ and v h germline families [ ] , which are both associated with creating v-domains with high stability and solubility. indeed, chicken scfvs can be stable in crude bacterial culture supernatants for up to month at room temperature [ ] . the initial work of davies et al. [ ] showed that a simple recombinant chicken antibody library could be displayed on phage. while this small library was nonimmune and derived from the bursa cells of a single young chicken, the group was able to select target-specifi c scfv sequences recognizing lysozyme , serum albumin and thyroglobulin. the potential of chicken recombinant antibodies was further highlighted by a study [ ] which used an scfv library derived from the spleens of immunized chickens and successfully generated highly specifi c scfv antibodies that targeted both mouse and rat serum albumins, where tolerance issues limit the ability to generate murine monoclonal antibodies . in addition, an in-depth analysis of the avian immunoglobulin v h repertoire demonstrated that naïve repertoire features were fully replicated in the resulting target-selected, phage display repertoire [ ] . a major study [ ] subsequently demonstrated that chickens could be a useful source of scfv and chimeric fab antibodies with specifi city for hapten molecules. however, none of these early studies characterized the antibodies for their affi nity, or their function as practical reagents. more recent studies have shown that scfv antibodies derived from immunized chickens are highly effective reagents in diverse settings such as diagnostic elisa for infectious bursal disease virus [ ], the diagnosis of prion disease [ ], immunodetection of haptenic shellfi sh toxins [ ], immunostaining of sars-infected cells [ ], biosensing of cardiac biomarkers [ ] , and the measurement of apob protein in mouse and human sera [ ] . raats et al. [ ] have also illustrated that antiidiotype scfvs from an immune chicken scfv library were of considerably higher sensitivity than those derived from a human antibody library in the same study. the generation of highly selective scfvs toward the prp protein, which is highly conserved in mammals demonstrates the advantage of the chicken as an immune model, as isolated scfvs were shown to react with murine, ovine, and bovine orthologues of the protein [ ] . the cloning of chicken antibodies via phage display has also allowed the precise dissection of the specifi city and affi nity of the chicken immunoglobulin response. high-throughput affi nity measurements for panels of chicken scfvs to the infl ammatory biomarker c-reactive protein have identifi ed clones that preferentially recognize the multimeric and monomeric forms of the protein [ ]. in the same study, clones with affi nities as high as pm were generated from an immune phage display library of only × total clones. in addition, chicken anti-prp scfv have been reported to have affi nities up to pm, making them among the highest affi nity scfvs reported to date [ ] . what may be of particular practical interest to many researchers is that chickens can serve as a host for simultaneous immunization with multiple proteins of interest, with as many as eight proteins being used successfully in a single immunization scheme [ , , ] . the target proteins of interest are mixed in a single adjuvant preparation and each immunized animal receives all multiplexed proteins simultaneously. spleen and bone marrow tissues from the immunized animals are then used to generate relatively small phage display libraries and specifi c antibodies are derived via selection of the library separately on the individual proteins originally used for immunization [ ] . the immunized chickens appear to react to the proteins fully independently, as the phage display libraries generate individual scfv antibody clones that are fully specifi c by western blot and elisa , showing no reactivity to their co-immunogens [ , ] . this approach has major benefi ts practically and ethically, as it allows the use of a single library to derive high-affi nity antibodies to a group of proteins of interest. multi-immunization methods also simultaneously minimize animal use and raise the likelihood of success in generating an immediately useful reagent [ , ] . multi-target immunization regimes should be designed with one of two objectives in mind. firstly, the simplest scenario combines multiple unrelated proteins, which leads to unrelated b-cell responses after immunization. to derive antibodies of greatest specifi city during a multi-target immunization of this kind, it is important to ensure that each of the protein immunogens is highly purifi ed and that no closely related proteins are co-immunized into a single animal. secondly, to derive antibodies that are cross-reactive to orthologues of a conserved protein from multiple species, it is likely to be benefi cial, but not necessarily essential, to include each orthologue in the mix of immunogens given to each animal. iterative selection rounds that change orthologue each time can then be used to bias toward the isolation of cross-reactive antibodies. the generation and selection of chicken recombinant antibodies is extremely reliable using the methods described in detail in the accompanying chapter. the subsequent identifi cation and sequencing of antibodies displaying the characteristics desired can also be performed simply. in general, scfvs isolated from immunized chicken libraries exhibit high affi nity and can be assayed via a direct elisa , using crude periplasmic extracts from the protein expressing e. coli clones. the level of further downstream analysis carried out on positive hits identifi ed during the binding elisa depends on what the end user requires. specifi c binding function may suffi ce for scfvs that are to be used simply as reagent antibodies for in vitro analysis of samples via elisa or western blotting . for antibodies to be used in affi nity chromatography , the antibody fragments must be purifi ed and tested for their function after being coupled to a solid matrix and for their specifi city during purifi cation. few studies have been performed using antibody fragments in affi nity chromatography, but several potential approaches have been described. for the antibody binding site(s) to be fully solventexposed and active, the antibody fragment must be directionally captured onto the solid matrix. even for full-length igg , nonspecifi c adsorption or covalent coupling onto solid supports can lead to denaturation, reducing or negating antigen-binding function [ ] . antibody fragments may be slightly more prone to chemical or physical denaturation than full-length immunoglobulins , but scfv and fab have been used successfully in affi nity chromatography, and in the creation of spr sensing surfaces which can go through serial rounds of binding and regeneration [ ] . mcelhinney et al. [ ] created a simple scfv-based affi nity column for the concentration and cleanup of microcystin toxins from environmental samples, by transiently coupling the his-tagged scfv to a disposable nickel chelate column. the scfv was thereby coupled directionally, maximizing the functional antibody content on the column, and the analyte for purifi cation was co-eluted with the scfv before quantifi cation by reverse-phase hplc. however, this method can only be used under a limited number of conditions, as the interaction of the his-tag with nickel is noncovalent and ph dependent. other possibly useful low affi nity expression tags include e. coli maltose binding protein and glutathione s -transferase , which have both been successful in protein purifi cation [ , ] ( see also chapter for a discussion on protein tagging ). high affi nity, highly stable linkage via affi nity tagging may also be achieved by site-specifi c biotinylation of antibody fragments and their immobilization onto a matrix that has been passively or covalently coated with avidin. bacterial expression vectors are now available which introduce biotin into specifi c peptide tags ( avitag ), which can be produced on the termini of recombinant proteins. a similar method was proven to be effi cient in the production of fabs that are specifi cally biotinylated in vivo during bacterial expression, via c-terminal fusion of the fabs to the e. coli acetyl-coa carboxylase [ ] . importantly, these biotinylated fabs were successfully used to purify recombinant tnf-alpha from bacterial lysates, via a streptavidinated column. the peptide tagging method has also been used successfully to label both fab and scfv antibodies for their oriented immobilization and use as capture antibodies in clinical diagnostic elisas [ , ] . these studies suggest that biotinstreptavidin coupling is a simple and rapid method for the stable, directional capture of recombinant antibody fragments. while the covalent coupling of recombinant antibody fragments via their reactive lysine side chains is likely to be disruptive to their function, some alternative covalent coupling methods have been identifi ed. in the simplest example, the disulfi de bonds linking the two constant regions of a fab can be reduced using a mild agent to expose cysteine thiols. these thiol groups can then be used to covalently couple the fragment to a thiol-activated surface [ ] . more elegant versions of this approach have expressed antibody fragments with a c-terminal cysteine group, then gently applied the same chemistry, to preferentially reduce the exposed disulphide groups [ ] . the exposed terminal thiols are again an effi cient reactive group for covalent attachment. it is also possible to express scfv fused to the constant regions of human igg light chains as another source of usable cysteine residues external to the v-regions [ ] . whether any of the above attachment methods are appropriate for a given affi nity purifi cation application may be decided upon by the individual investigator. in cases where stable linkage has been achieved and the column is to be reused, it is prudent for the investigator to examine multiple clones for their stability under repeated cycles of elution and regeneration. while chicken scfvs are built upon naturally stable frameworks, the stability of different clones cannot be taken for granted. in cases where stability remains an issue, the appropriate chicken v-regions can be cloned into an fc-fusion [ ] or igg [ ] expression vector to produce full-length antibody in mammalian, yeast, and even plant culture systems [ ] . in conclusion, this chapter demonstrates that phage display is a powerful technology that can be used to generate highly specifi c reagents from immune sources. it is an attractive alternative to classical antibody generation technologies that can be employed in many fi elds for reliable antibody and antibody fragment generation . antibody microarrays: promises and problems fundamental characteristics of the immunoglobulin vh repertoire of chickens in comparison with those of humans, mice, and camelids analyzing antibody specifi city with whole proteome microarrays continuous cultures of fused cells secreting antibody of predefi ned specifi city filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface cloning immunoglobulin variable domains for expression by the polymerase chain reaction phage antibodies: fi lamentous phage displaying antibody variable domains multi-subunit proteins on the surface of fi lamentous phage: methodologies for displaying antibody (fab) heavy and light chains selecting and screening recombinant antibody libraries human antibodies from v-gene libraries displayed on phage semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem human antibodies with sub-nanomolar affi nities isolated from a large non-immunized phage display library fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides generation of high-affi nity human antibodies by combining donor-derived and synthetic complementarity-determiningregion diversity high-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries picomolar affi nity antibodies from a fully synthetic naive library selected and evolved by ribosome display affi nity maturation of a humanized rat antibody for anti-rage therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both inside and outside the complementarity-determining regions in vitro selection and affi nity maturation of antibodies from a naive combinatorial immunoglobulin library augmented binary substitution: single-pass cdr germ-lining and stabilization of therapeutic antibodies protein engineering of antibody binding sites: recovery of specifi c activity in an anti-digoxin single-chain fv analogue produced in escherichia coli human monoclonal fab fragments derived from a combinatorial library bind to respiratory syncytial virus f glycoprotein and neutralize infectivity domain interactions in the fab fragment: a comparative evaluation of the singlechain fv and fab format engineered with variable domains of different stability an improved single-chain fab platform for effi cient display and recombinant expression reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system high affi nity scfvs from a single rabbit immunized with multiple haptens methods for the generation of chicken monoclonal antibody fragments by phage display the isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep high throughput ranking of recombinant avian scfv antibody fragments from crude lysates using the biacore a determination of lox- -ligand activity in mouse plasma with a chicken monoclonal antibody for apob generating recombinant anti-idiotypic antibodies for the detection of haptens in solution humanization of chicken monoclonal antibody using phagedisplay system multipleantigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens profi ling receptor tyrosine kinase activation by using ab microarrays oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-lr by phage display and its use in the immunoaffi nity concentration of microcystins from water advances in recombinant antibody microarrays comparison of affi nity tags for protein purifi cation in vivo biotinylated recombinant antibodies: construction, characterization, and application of a bifunctional fab-bccp fusion protein produced in escherichia coli in vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail use of an in vivo biotinylated single-chain antibody as capture reagent in an immunometric assay to decrease the incidence of interference from heterophilic antibodies sitedirected immobilisation of antibody fragments for detection of c-reactive protein recombinant antibody expression vectors enabling double and triple immunostaining of tissue culture cells using monoclonal antibodies production of anti-prion scfv-fc fusion proteins by recombinant animal cells whole igg surface display on mammalian cells: application to isolation of neutralizing chicken monoclonal anti-il- antibodies plant expression of chicken secretory antibodies derived from combinatorial libraries key: cord- -bb lyvhy authors: nan title: monoclonal antiprothrombinase ( d . ) prevents mortality from murine hepatitis virus (mhv- ) infection date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: bb lyvhy the induction of monocyte/macrophage procoagulant activity (pca) has been implicated in the pathogenesis of murine hepatitis virus strain (mhv- ) infection and disease. previously, we have shown that induction of pca by mhv- correlated with resistance/susceptibility to infection in different mouse strains. in this study, all balb/cj mice that were infected with ( ) plaque-forming units of mhv- developed severe liver disease and died within - h. examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of pca by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. splenic mononuclear cells recovered from these mice expressed high concentrations of pca with time after infection. infusion into mice of a high-titered monoclonal antibody that neutralized pca ( d . ) attenuated the development of hepatic necrosis and enhanced survival in a dose- dependent manner. all of the animals receiving micrograms, and % and % of the animals that received and micrograms per day, respectively, survived for d and made a full recovery. administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, pca expression as detected by direct immunofluorescence staining and by a functional assay. in animals treated with high concentrations of antibody, titers of antibody to pca fell from +/- micrograms/ml to +/- ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing pca. surviving animals, when rechallenged with mhv- , had a % mortality, consistent with the known rates of metabolism of immunoglobulin. this further suggested that protection was by a passive mechanism. the results reported here demonstrate that a neutralizing antibody to pca protects animals from fulminant hepatitis and death associated with mhv- infection, and supports the notion that pca is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from mhv- infection. . furthermore, using recombinant inbred strains of mice, we showed that genetic linkage between resistance/susceptibility to mhv- infection and induction of pca was controlled by two non-h - inked recessive genes ( ) . after infection with mhv- , disturbances of the hepatic microcirculation associated with sinusoidal thrombosis occurred coincident with the rise in pca ( , ) . intravenous infusion of macrophages, induced to express high amounts of pca by mhv- , resulted in rapid ( - min) death from disseminated intravascular coagulation in both susceptible and resistant mice (our unpublished observations). together, these observations suggest that coagulative necrosis occurring as a result of induction of pca may be a crucial feature of mhv- -induced hepatic injury. we have recently produced a panel of mabs to mhvinduced macrophage pca ( ) . the antibodies did not react with purified viral proteins nor did they inhibit viral replication. one of these mabs ( d . ) , an igg ak, strongly inhibited pca expression in a one-stage dotting assay and inhibited conversion of prothrombin to thrombin ( ) . this antibody had no reactivity with murine, rabbit, or human tissue factor. the monoclonal bound to a -kd protein that is distinct from murine and human tissue factor ( kd) ( , ) . the purpose of this study was to determine whether treatment with this specific murine antiprothrombinase would modify the morbidity and/or mortality associated with murine hepatitis virus infection. virus. the origin and growth of mhv- have been previously described ( ) . mhv- , obtained from the american type culture collection (kockville, md) (atcc-vr ), was plaque purified on monolayers of dbt cells. stock virus was grown to a titer of . x pfu/ml in cl cells. the virus was harvested by one cycle of freeze-thawing and clarified by centrifugation at , g for h at ~ virus was then assayed on monolayers of l cells in a standard plaque assay as previously described ( ) . to induce mhv- infection, each mouse received pfu of mhv- by intraperitoneal injection. mice. balb/cj mice - wk of age, were obtained from charles river laboratories, (st. constant, quebec). animals were housed in the d class facility at the university of toronto fed a diet of standard chow and water ad libitum. mice were killed on days , , , , , and after infection. blood was obtained by axillary bleeding. splenic mononuclear cells were harvested and assayed for pca both by immunofluorescence staining and in a onestage clotting assay. livers were harvested for viral titers, histopathology, and pca by immunofluorescence staining as described below. all mice used were screened by an elisa for exposure to mhv, and were found to be negative ( ) . anti-pca mat~ the preparation of mab d . has been previously described ( ) . after injection of hybridoma cells into pristane-primed caf mice, ascites was harvested. animals were treated with ascitic fluid containing , , or #g of mab daily for d preinfection and d postinfection (p.i.) by intraperitoneal injection. as a standard control, ~ of ascites from animals that were infected with sp myeloma cells was injected into six separate mice. all ascitic fluid whether from sp -or d . injected mice had no reactivity with mhv- either in the elisa or plaque reduction assays ( ) . to ensure that the protective effect of ascites containing mab to pca was specific, mab to pca ( d . ) was isolated from hybridoma supematants by affinity chromatography using goat anti-mouse igg immobilized on sepharose b (pharmacia, montreal, quebec). immunoblot analysis of the column eluate confirmed that igg was the only ig present. subclass analysis by elisa, as previously described ( ) , confirmed that igg ak was the only subclass present. #g of the purified mab was injected into mice for d preinfection and for d p.i. as described above. histology. histology was assessed by a blinded observer as previously described ( ) . briefly, li~r were cut into . x . -cm blocks and fixed by immersion into % formalin in . molar phosphate buffer, ph . . after fixation, the tissue was dehydrated in graded alcohols and xylene, then embedded in paraffin. -#m sections were cut, stained with harris' hematoxylin for min, and counterstained with eosin y for s. the sections were then washed with distilled water, dehydrated in graded alcohols and xylene, and mounted with parmount. for each group five animals were used. to quantitate the effect of the mab on liver histology, a digitalized image analysis system (hp- ; hewlett packard co., ltd., mississauga, ontario) with customized software was used. this constitutes a modification of a technique described previously ( ) . the areas of necrosis were encircled as well as the entire section yielding a percentage figure representing the proportion of diseased liver present in that particular section. for each animal, three random sections were assayed in this fashion, and the mean +_ sd was calcuhted. hbrin deposition was assessed by the morris-lendrum hcro-mallory stain as previously described ( ) . viral titers. livers that had been snap frozen at - ~ were homogenized in dmem supplemented with % fcs and mm glutamine as a % homogenate at ~ as previously described ( ) . viral titers of liver homogenates were then determined on monolayers of l cells in a standard plaque assay ( , ) . spleens were harvested aseptically and cells teased from splenic tissue and suspended in ml of dmem as previously described ( ) . smnc were isolated over ficoll-hypaque gradients (density, . ) (pharmcia) by centrifugation at , g for rain at ~ cells at the interface were collected. viability was > % as assessed by trypan blue exclusion. cells were washed three times and resuspended in dmem at a concentration of x smnc/ml. procoagutant activity. samples of frozen thawed smnc were assayed for the capacity to shorten the spontaneous clotting time of human citrated platelet-poor plasma in a one-stage clotting assay as previously described ( ) . equal volumes ( # ) of the cellular homogenate were admixed with citrated normal human plateletpoor plasma, and then # of mm caclz was added at ~ to start the reaction. the time in seconds for the appearance of a fibrin gel was then recorded. to establish arbitrary units, a rabbit brain thromboplastin standard at mg dry mass/ml (dade division, american hospital supply, miami, fl) was assigned a value of , mu. the assay was used over the range of - , mu, and the results were linear with normal human plasma substrate. data were converted to pca per splenic macrophages and expressed as the mean and standard deviation from six mice done in triplicate. media and buffers were all without activity in this assay. inhibition of viral replication. mab purified from hybridoma cell cultures containing d . and ascites were assayed for their ability to inhibit replication of mhv- in a standard plaque reduction assay as previously described ( ) . briefly, pfu of virus was admixed with dilutions of purified antibody or ascites, media as a negative control, or a high titered anti-mhv- -neutralizing antibody as a positive control, for min at ~ the mixture was then added to a monolayer of l cells in culture medium, overlayed with % agarose, and incubated at ~ in a % co environment for an additional h. the effect of antibody on viral replication was assessed by reduction of viral plaques ( ) . immunofluorescence. blocks of liver tissue were snap frozen in liquid nitrogen. cryostat sections ('~ #m thick) were fixed for rain in acetone and air dried for h as previously described ( ) . unoccupied sites were then blocked with % horse serum in pbs, ph . , for h. mab d . was conjugated with fitc (sigma chemical co., st. louis, mo) according to the method of thi and feltkamp ( ) . the fluoresceinated antibody did not react with normal liver or uninduced normal peritoneal macrophages. tissues were then stained with fitc-conjugated mab d . for h at room temperature, washed three times, mounted in % glycerol in pbs, and viewed on a phase-epifluorecence microscope equipped with a x fluotar objective (e. leitz, inc., rockleigh, nj). elisa for anti-pca antibody. titres of antibody to pca were determined in a standard elisa as previously described ( ) . well enzyme immunoabsorbant assay (eia) plates (dynatech, mclean, va) were coated with /~l/well of mhv- -stimulated pca-positive macrophage membranes or unstimuhted pcanegative membranes ( x ~ macrophage/ml) at ~ overnight. the plates were then washed three times with pbs, ph . , conraining . % tween (washing buffer), and the unoccupied sites were blocked with /zl of % ig-free horse serum (flow laboratories, mississauga, ontario) that had been dissolved in washing buffer for h at room temperature. -/~l/well dilutions of sera from treated animals were then added to the plates and incubated for h at ~ after washing three times, #l/well of alkaline phosphatase-conjugated goat anti-mouse ig in pbs containing . % bsa and . % tween was added for i h at ~ after three washings, p-nitrophenyl phosphate in . m -amino- -methyl- , -proponediol buffer, ph . (zymed laboratories, san francisco, ca), substrate was then added. the plates were then incubated at ~ for h and read at nm with a plate reader (titertek mcc/ ; icn/flow, mississauga, ontario). antibody levels were expressed (/~g/ml) by comparison to a standard curve. statisticalanalysis. statistical analysis was carried out using analysis of variance and the wilcoxon ranked sum test. a p value of . % or less was considered statistically significant. suwival. mice infected with , pfu ofmhv- (n = ) all succumbed to the infection within d (fig. ) . this was consistent with previous data reported by our group as well as others ( , ) . in contrast, there was survival in some animals that were treated with ascites containing antibody to pca (fig. ). animals treated with ascites containing /~g/d had a % survival; % of those treated with ascites containing #g/d and % of animals treated with /~g/d survived. all animals survived when treated with /~g of monodonal anti-pca (igg ak), which had been purified from hybridoma superuatant, confirming that the beneficial effect of ascites was due to the monoclonal anti-pca. mice immunized with /~ of ascites from sp injected animals and infected with mhv- all died within d (data not shown). liver histology. mhv- -infected mice that did not receive antibody developed histologic evidence of severe liver disease. by h p.i., small, discrete loci of necrosis with a sparse pmn infiltrate could be seen. at h, these lesions became both more pronounced and more numerous (fig. a) , and by - h, confluent liver necrosis was apparent (fig. b) . in contrast, mice infected with mhv- but treated with antibody to pca showed a marked reduction in liver disease in all groups ( , , and /.r ( were a few small loci of inflammatory cells with no necrosis (fig. d) . morphometric image analysis showed that the proportion of the liver that was necrotic was significantly different between the mhv- -infected, anti-pca-treated, and untreated groups at , and h (fig. ) . the histolog of livers from all survivors at and d postinfection appeared normal. fibrin deposits were seen in hepatic sinusoids as well as in areas of necrosis of untreated and mhv- -infected mice (fig. ) . in animals treated with and /~g of anti-pca, a marked reduction in fibrin was noted. no fibrin was seen in the livers of infected mice treated with /~g of mab. treatment with anti-pca alone resulted in no detectable histological evidence of liver disease in nonirlf~ed animals. viral titers. by h p.i., large amounts of infectious virus were recovered from liver homogenates of mhv- -infected and untreated animals, and these persisted until the death of the animals (fig. ) . in animals treated with and /~g of antibody to pca, there was no significant difference in viral titers from those observed in untreated mice at days and . viral titers remained high at day , however, viral titers decreased by day and no virus could be detected after day . in animals treated with the highest dose of antibody to pca ( #g/d), viral titers were markedly reduced (p < . ) and approached those seen in resistant a/j mice ( , ) . using the method of reed and meunch, the mhv- recovered from infected and antibody-treated animals was as pathogenic as stock mhv- or virus recovered from infected and untreated mice (data not shown) ( ) . pca. in untreated, but mhv- -infected animals, a sharp increase in splenic macrophage pca was noted at h p.i. (fig. ) . maximal pca was seen at h and pca remained elevated until the animals' death on day . animals treated with low concentrations of antibody to pca ( and /zg/d) expressed high amounts of pca at early time points, but by day , pca levels fell and only basal levels of pca were detected in smnc by day . in contrast, no expression of pca could be detected in animals treated with /zg of antibody to pca during the course of infection (fig. ). immunofluorescence. by direct immunofluorescence staining, pca could be detected at h in livers from mhv- - infected and untreated mice. pca was seen in areas of inflammation and necrosis, and also in hepatic sinusoids, localized primarily in endothelial cells and kupffer cells, but not expressed by hepatocytes (fig. ) . livers from animals treated with antibody to pca had significantly less pca expression, although small amounts of pca could be seen in macrophages and endothelial cells in hepatic sinusoids in animals treated with high-dose antibody to pca ( ~tg/d), even as late as day (fig. ) . antibody to pca. sera were collected from animals at all time points and analyzed in an elisa for the presence of antibody to pca as previously described ( ) . no antibody to pca could be detected in sera from normal control animals or in animals infected with mhv- who had not received antibody to pca (data not shown). sera from animals treated with ascites containing - /zg of antibody to pca contained large amounts of anti-pca before mhv- infection. after mhv- infection, the concentration of circulating antibody fell, but remained at > ng/ml in animals treated with /~g/d. in animals that were treated with and /zg of antibody, by day , antibody was undetectable (< ng/ml) (fig. ) . antibody to pca either in ascites or purified from supernatants did not neutralize mhv- in a standard plaque reduction assay as previously described ( ) to determine whether treated animals that had survived the acute infection developed long-term resistance to mhv- infection, mice previously infected with mhv- and treated with /~g of antibody (n = ) were infected with , pfu of mhv- , d after their last exposure to virus. pca antibody titers in this group were ~ % of that present during antibody therapy, consistent with known rates of disappearance of igg. the mhv-rechallenged mice experienced a % mortality rate, suggesting that protection was by passive immunization of antibody, not an acquired, active immune process (fig. ). in addition, no antibody to mhv- was detected in these mice either before or after rechallenge with virus. mhv- infection produces fulminant hepatic failure and death in balb/cj mice ( , ) . the availability of a mab to pca that neutralized acceleration of coagulation in vitro ( ) provided us an opportunity directly to examine the role of pca in vivo in murine hepatitis virus strain infection. all balb/cj mice which were infected with pfu of mhv- developed severe liver disease and died within - h. administration of the mab to pca attenuated the hepatic necrosis, fibrin deposition and enhanced survival in a dosedependent manner. all of the animals receiving /~g, and % and % of the animals that received and /~g/d, respectively, survived for d and made a full recovery. furthermore, the same protective effect was seen in mice treated with igg ak purified from hybridoma culture supernatants ( d . ). the increased survival of the treated animals was specific for anti-pca, since ascites from mice injected with sp cells alone, which contained no antibody to pca, failed to protect mice from mhv- infection. although antibodytreated, mhv- -infected animals demonstrated clinical evidence of viral hepatitis early in the course of the infection, by - d their behavior appeared normal and liver sections showed little or no apparent disease. examination of the livers of antibody-treated animals showed marked reduction in hepatic necrosis and inflammatory cells (neutrophils and mononuclear cells), each of which are prominent features of mhv- infection ( , , ) . pca expression is a feature of mhv- infection in this mouse strain ( ) ( ) ( ) , and in untreated mhv- -infected mice, pca was detected by immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. both macrophages and endothelial cells expressed detectable pca, but hepatocytes did not. it is likely that the hepatic necrosis is secondary to ischemic changes resulting from induction of pca, leading to the deposition of fibrin. administration of mab resulted in a dose-dependent reduction in the deposition of fibrin and in expression of pca as detected by immunofluorescence staining in the liver and by a direct functional assay in smnc. previously, we have shown that mhv- infection of peritoneal macrophages in vitro results in the production of pca, tnf, leukotriene b ( ) , and il- ( ) . thus, expression of pca by endothelial ceils might either be due to direct induction by mhv- or due to induction by il. and/or tnf, which have previously been shown to induce pca in endothelial cells in vitro ( , ) . treatment of mice with /~g of mab to pca not only increased survival and reduced hepatic necrosis, but also resulted one possible explanation for the decrease in viral replication observed in mice treated with antibody d . is that this antibody reacts with the mhv receptor recently described by holmes and coworkers ( , ) . we regard this explanation as unlikely, since d . does not show any neutraliza- tion of viral infectivity in a plaque reduction assay, whereas antibody to the mhv receptor does inhibit infectivity. furthermore, the mhv receptor has a molecular mass of kd, considerably different than that of the pca molecule ( kd). a second possible explanation for the effect of anti-pca antibody on mhv- growth in mice is related to the normal cleavage of the mhv s protein. it has been shown that cleavage of s by proteases is necessary to activate the membrane-fusing properties of the s protein ( ) . this fusion property facilitates the spread of virus to uninfected cells by cell-cell fusion and also increases the specific infectivity of mhv when compared with virus in which s has not been cleaved ( ) . it is possible that in infected macrophages, pca, a serine protease, mediates at least in part the proteolytic cleavage of s into $ and $ , and thereby activates the fusion properties of this molecule. consistent with this idea is the observation that infection of a/j macrophages, which do not produce pca in response to mhv infection, does not result in the appearance of syncytial giant cells (our unpublished observations). thus, antibody to pca could inhibit the spread of virus to uninfected cells by decreasing the activation of the fusion properties of s. although the mechanism by which anti-pca treatment protects the susceptible animals is not clear, the use of antibody to pca neutralizes pca, thereby preventing activation of the coagulation system and inhibiting fibrin formation. the antibody could also result in complement-mediated destruction of mhv- -infected cells that express membranebound pca or promote macrophage activation with restriction of viral growth. in animals treated with #g of antibody for d before infection and d p.i., concentrations of antibody to pca in sera fell from /~g/ml before mhv- infection to ng/ml during the active phase of disease (day ), consistent with sequestration perhaps due to binding of the ig to cells expressing pca. furthermore, in these mice, levels of pca in smnc remained at basal levels. in animals treated with lower amounts of antibody ( or #g/d) to pca, hepatic necrosis and survival were only partially attenuated, and antibody was not detected (< ng/ml) after day p.i. together, the data strongly support the notion that anti-pca antibody neutralizes pca in vivo during the infection and that this may be the basis for its protective effect. in a previous report we have demonstrated that dimethyl prostaglandin e inhibited procoagulant activity and prevented fulminant viral hepatitis, yet all animals still succumbed to the infection ( ) . in pge-treated mice, viral replication proceeded at a rate similar to that in untreated animals. recently, we have demonstrated that although pge inhibited functional pca, antigenic expression of pca was not altered as determined by western immunoblotting ( ) . we have proposed that pca may exert its effect through activation of the coagulation system with microvascular and macrovascular thrombosis ( , ), but the present results, in concert with the inhibition studies of pca by pge , suggest that pca has other sites of action as well. the protective effects of the mab to pca occurred even though the balb/cj mice failed to mount an antiviral humoral response. our ob-servations are consistent with a recent report by korner et al. ( ) and support the notion that the murine antiviral antibody response may not be required for protection from acute viral infection. cytokines can play a potent role in the course of inflammatory injuries in vivo, and interference with their action can alter the course of certain inflammatory diseases ( , ) . treatment of rats with recombinant antibody to tnf has been shown to protect animals from the hypotension, hypothermia, and mortality of gram-negative sepsis ( ) , and treatment of rabbits with an il- r antagonist reduced the mortality associated with endotoxin shock ( , ) . our studies support the notion that induction of pca during mhv- infection in mice is an integral and potentially central step in the disease. a previous report by taylor et al. ( ) demonstrated that lethal escherichia coli septic shock can be prevented by blocking tissue factor (a distinct procoagulant) with mab, demonstrating the importance of cellular coagulants in the pathophysiology of other infectious diseases. we conclude that pca is a potent inflammatory mediator that plays a pivotal role in hepatic injury resulting from mhv- infection. the biology and pathogenesis of coronaviruses induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice the pathobiology of viral hepatitis and immunologic activation of the coagulation protease network activation of the immune coagulation system by murine hepatitis virus strain susceptibility/resistance to murine hepatitis virus (mhv- ) and monocyte procoagulant activity (mpca) are genetically linked and controlled by non-h- linked genes acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type the effects of mouse hepatitis virus type on the microcirulation of the liver in inbred strains of mice monoclonal antibody analysis of a unique macrophage procoagulant activity induced by mufine hepatitis virus strain infection complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation protease cascade molecular cloning of the cdna for tissue factor, the cellular receptor for the initiation of the coagulation protease cascade a sensitive radioimmunoassay for the detection of antibodies to mhv- induction of resistant hepatocytes as a new principle for a possible short-term in vivo test for carcinogens morris-lendrum picro-mallory stain for fibrin dimethyl prostaglandin e prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain infection lymphocyte cooperation is required for amplification of macrophage procoagulant activity, f ex f med conjugation of fluorescein isothiopropriate to antibodies. i. experiments in the conditions of conjugation a sensitive radioimmunoassay for the detection of antibody to mvh- mechanism of protective effect of prostaglandin e in murine hepatitis virus strain infection: effects on macrophage production of tumor necrosis factor, procoagulant activity and leukotriene b susceptibility to mouse hepatitis virus strain in balb/cj mice: failure of immune cell proliferation and interleukin production interleukin- activation of vascular endothelium. effects on procoagulant activity and leukocyte adhesion the role of endothelial cells in inflammation monoclonal antibody to the receptor for murine coronavirus mhv-a inhibits viral replication in vivo purification of the -kilodalton glycoprotein receptor for mouse hepatitis virus (mhv)-a from mouse liver and identification of a nonfunctional, homologous protein in mhv- resistant sjl/j mice. f virol proteolytic cleavage of the e glycoprotein of murine coronavirus: host dependent differences in proteolytic cleavage and ceu fusion effect of eicosanoids on induction of procoagulant activity by murine virus strain in vitro nucleocapsid or spike protein-specific cd + t lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of cd + t cells, f immunol shcokd and tissue injury induced by recombinant human cachectin cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-alpha in rodents treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension and hypothermia of gram-negative sepsis interleukin- receptor antagonist reduces mortality from endotoxin shock lethal e. coli septic shock is prevented by blocking tissue factor with monodonal antibody key: cord- - ahmyicn authors: renegar, kathryn b. title: passive immunization: systemic and mucosal date: - - journal: mucosal immunology doi: . /b - - / - sha: doc_id: cord_uid: ahmyicn nan the passive transfer of maternal immunity is responsible for keeping all mammalian species alive. the process of evolution developed effective mechanisms for the passive transfer of both systemic and mucosal immunity from the mother to her offspring. experimental passive transfer of systemic immunity via serum antibody is well established, but the experimental passive transfer of mucosal immunity has only recently been accomplished. this chapter addresses the contributions of both natural and experimental mechanisms to the study of passive immunization. the transfer of systemic immunity (igg) from mother to offspring occurs prenatally via the placenta or yolk sac and after birth via the colostrum. species vary in the contribution each route makes to the transfer of immunity (waldman and strober, ) and can be grouped into three categories: prenatal transfer only, combined prenatal and postnatal transfer, and postnatal transfer only. this group includes primates, rabbits, and guinea pigs. transport of igg in primates occurs almost exclusively through the placenta. igg transfer occurs via a receptormediated transcytosis across the syncytiotrophoblast and a transcellular pathway through the fetal endothelium (leach et al., ) . human placental transfer of protective igg antibodies to a number of pathogens, including hepatitis b (hockel and kaufman, ) , measles (lennon and black, ) , and group b streptococcus (baker et al., ) , has been reported. this process suggests an effective method of neonatal immunization, i.e., immunization of the pregnant mother in order to protect the neonate. prenatal transfer of igg in the rabbit occurs via the yolk sac, and in the guinea pig, via both the yolk sac and fetal gut (waldman and strober, ) . this group includes rats, mice, cats, and dogs. prenatal transmission occurs via the yolk sac/placenta and the fetal gut in the rat (waldman and strober, ) . igg is bound rapidly to receptors on the surface of the yolk sac membrane (mucchielli et al., ) , is endocytosed in clathrin-coated vesicles, and, early in gestation, is stored in subapical vacuoles. by late gestation, the antibody has been hydrolyzed or transferred to fetal capillaries (jollie, ) . prenatal transmission in mice occurs by a similar mechanism (gardner, ) . although placental transfer occurs, studies in rodents have shown that most transport of antibody occurs postnatally from colostrum or milk (arango-jaramillo et al., ; barthold et al., ; heiman and weisman, ; kohl and loo, ; nejamkis et al., ; oda et al., ) over a period of to days, depending on the species. there is a gradual decrease in transmission over the last days (waldman and strober, ) , and transmission is limited to antibodies of the igg class (appleby and catty, ; hammerberg et al., ) . transport is a receptor-mediated process (simister and rees, ) . in rats, the receptor (fcrn) is found in enterocytes of the proximal intestine during the early postnatal period but is absent after weaning (jakoi et al., ) . fcrn is specific for igg and its fc fragment and consists of two similar polypeptides of , to , daltons (p ) in association with β microglobulin (jakoi et al., ; simister and mostov, ) . the fc binding subunit (p ) has three extracellular domains and a transmembrane region that are all homologous to the corresponding domains of class i major histocompatibility complex (mhc) antigens (simister and mostov, ) . junghans and anderson ( ) have shown that disruption of the fcrn in knockout mice also destroys the receptor (fcrp) necessary for the prolonged half-life of serum igg in adults, suggesting that the same receptor protein that mediates transient igg transport across the neonatal gut functions as the fcrp throughout life. this group includes ruminants (cattle, sheep, goats), horses, and pigs (reviewed in tizzard, ) . transport of colostral proteins from the lumen of the ileum in ruminants is largely nonspecific, but in the horse and the pig, igg and igm are preferentially absorbed. proteins are actively taken up by epithelial cells through pinocytosis and passed through these cells into the lacteals and intestinal capillaries (tizzard, ) . intestinal absorption occurs for only the first to hours after birth. following this, the "open gut" closes down, and no further transfer from milk or colostrum occurs (ellis et al., ; francis and black, ; tizzard, ; waldman and strober, ) . newborn piglets have also been shown to absorb colostral lymphoid cells during this period (tuboly et al., ) . it is unclear whether these cells are fully functional and capable of immune processes such as the transfer of delayed-type hypersensitivity (dth). absorption of colostral immunoglobulin is normally extremely effective, supplying the newborn with serum immunoglobulin (particularly igg) at a level approaching that found in adults (tizzard, ) ; however, failure of passive transfer (fpt) can occur and, when it does, can pose a considerable problem in animal husbandry. about % of newborn foals fail to obtain sufficient quantities of immunoglobulin (mcguire et al., ; tizzard, ) . in the mcguire study ( ) , two of nine foals affected by fpt died of infections within a few days of birth, and five of the remaining seven developed nonfatal respiratory infections between and weeks of age. mcguire et al. ( ) also reported fpt in calves, finding that % of calves less than weeks old dying from infectious diseases have significant hypogammaglobulinemia. although adequate methods to diagnose and treat fpt are available (bertone et al., ; tizzard, ) , the phenomenon remains a significant veterinary problem. mother's milk provides passive protection of the mucosal surfaces it contacts. this protection may be mediated either by specific immunity or by nonspecific factors found in milk, such as lactoferrin, lysozyme, fatty acids, and complement (reviewed in goldman et al., ) . the antibody composition of milk differs from that of colostrum (tizzard, ) , and the class of protective antibody in milk varies with the species and the route of immunization of the mother. with the exception of igg in rodents, these protective antibodies are not systemically absorbed by the suckling offspring, but exert their protective effect locally by neutralizing viruses or virulence factors and by binding to microbial pathogens and preventing their attachment to the mucosal surface (goldman et al., ) . secretory iga (s-iga) is especially suited to this protective role, because secretory component enhances its resistance to proteolytic enzymes and gastric acid (kenny et al., ; lindh, ; tomasi, ; zikan et al., ) , providing extra antibody stability in mucosal secretions. rodents have been a popular model for the study of passive transfer of maternal immunity via milk; however, this class of animals has a major drawback as a model for passive mucosal immunity. both rats and mice can actively transport igg from the gut into the serum for approximately weeks (see combined prenatal and postnatal transfer earlier in this chapter); thus, observed protection could be due either to antibody in the milk bathing the mucosal surfaces or to maternal antibody being transported into the serum and secretions of the offspring. this caveat should be kept in mind during evaluation of the many reports of milk-borne protection in these species. three rodent models in which protection of mucosal surfaces is due to milk-borne, not serum-derived, antibody are described next. the predominant immunoglobulin in mouse milk is igg, although significant levels of iga can also be present (ijaz et al., ) . protection of infant mice from colonization with campylobacter jejuni can be achieved by the consumption of immune milk at and after the time of bacterial challenge. infant mice were not protected by prior consumption of colostrum, showing that milk antibody was required in the gut lumen for protection to be observed (abmiku and dolby, ) . a similar requirement for antibodies active at the intestinal cell surface in immunity to primate rotavirus sa- was reported by offit and clark ( ) . protection of rats against dental caries by milk can be due to either igg or s-iga antibodies, depending on the route of maternal immunization. rat dams immunized intravenously with heat-killed streptococcus mutans developed igg antibodies in their colostrum, milk, and serum. their offspring demonstrated significant protection against s. mutans-induced caries formation. rat dams locally injected in the region of the mammary gland with heat-killed s. mutans or fed formalin-killed s. mutans developed s-iga antibodies in their colostrum and milk.their offspring were also protected against caries formation (michalek and mcghee, ) . caries protection in suckling rats could theoretically be due to bathing of mucosal surfaces and/or leakage of antibody into the saliva from the serum. nonimmune adult rats can be protected from s. mutans-induced caries by feeding on lyophilized immune bovine milk or on immune bovine whey containing specific igg (michalek et al., a; michalek et al., ) . since adult rats are unable to transport orally administered igg into their serum, protection must be from milk-derived antibodies bathing the oral cavity. in ruminants (sheep, cattle, goats), the predominant antibody in both colostrum and milk is igg. the predominant antibody in the colostrum of pigs and horses is also igg, but as lactation progresses and colostrum becomes milk, iga predominates (tizzard, ) . protection can be mediated by either antibody class. while bathing of the mucosal surfaces by milk-derived antibodies can provide passive immunity to some pathogens, the high rate of infections in fpt foals and calves shows that milk (mucosal immunity) alone cannot provide complete protection to neonates. in cattle and pigs, passive immunity against enteric infections with viruses such as rotaviruses and coronaviruses (transmissible gastroenteritis, or tge) is dependent on the continual presence in the gut lumen of a protective level of specific antibodies (crouch, ) . passive immunity against intestinal infection with the tge virus is generally more complete in piglets ingesting iga antibodies than in those ingesting igg antibodies, although both classes of antibody are protective. the class of antibody present in the sow's milk depends on the route of immunization (bohl and saif, ) . in cattle, passive immunity in calf scours (neonatal bovine colibacillosis caused by escherichia coli) correlates with the level of specific iga antibody in the mother's milk (wilson and jutila, ) . in primates, iga is the predominant immunoglobulin in both colostrum and milk (tizzard, ) . both lysozyme and s-iga in human milk remain functional in the digestive tract of the early infant (eschenburg et al., ) . human milk has been shown to contain s-iga antibodies to at least five viral and nine bacterial pathogens, as well as to fungi, parasites, and food antigens (goldman et al., ) . mucosal immunity to rotavirus, for example, was shown to be transferred to the infant by the s-iga in milk; there was a positive correlation between titers of secretory component (sc) in the mother's milk and the infant's feces vs. virus-specific iga in the infant's fecal samples (rahman et al., ) . in addition to providing passive mucosal immunity, human breast milk also stimulates the early local production of s-iga in the urinary and gastrointestinal tracts, thereby accelerating the development of an active local host defense in the infant (koutras and vigorita, ; prentice, ) . since the original demonstration of transfer of immunity by the injection of serum (von behring and kitasato, ), passive transfer of humoral immunity has been intensively investigated. the use of specific serum antibody (igg) to transfer protection to nonimmune individuals is now standard medical practice in, for example, the postexposure prophylaxis for rabies and tetanus and the treatment of snakebite (arnold, ; centers for disease control, a , while intravenous immunoglobulin (ivig) treatment has been shown to lower the incidence of pneumonia in patients with common variable immunodeficiency (busse et al., ) . local immunity has been correlated with the level of iga antibody in various secretions (reviewed in renegar and small, ) ; however, direct demonstration of the mediation of local immunity by injected iga could not occur until specific transport of passively administered iga had been confirmed. in rabbits, rats, and mice, polymeric iga (piga) is efficiently transported from the circulation into the bile via the liver (delacroix et al., ; koertge and butler, a; mestecky and mcghee, ; orlans et al., orlans et al., , .these species express pig receptor (pigr) on their hepatocytes (socken et al., ) and, in addition, have piga as the primary molecular form in their serum (heremans, ; vaerman, ) . serum iga is also efficiently transported into bile in cattle (butler et al., ) . in fact, most iga in ruminant bile may be of serum origin. transport of serum iga into bile in humans has been reported (delacroix et al., ; dooley et al., ) , although iga transport is about -fold less efficient than in rats and rabbits. the human biliary iga level is approximately % of the human serum iga level and, under physiologic conditions, only % of human biliary iga is derived from the serum (vaerman and delacroix, ) . even though transport is possible, passively administered iga does not reach high levels in human bile. in one study, less than % of intravenously injected radiolabeled piga was found in human bile at hours (vaerman and delacroix, ) . serum piga can be transported into saliva in dogs (montgomery et al., ) , monkeys (challacombe et al., ) , mice (falero-diaz et al., ) , and humans (delacroix et al., ; kubagawa et al., ) . in humans the amount of iga acquired from the plasma is low (only %) compared with the amount acquired from local production (delacroix et al., ) . transfer of piga from the plasma into canine or murine saliva is a selective process requiring the pigr (montgomery et al., ; falero-diaz et al., ) , while transport into oral fluids in monkeys appears to be by leakage from the plasma into the crevicular spaces surrounding the deciduous molars (challacombe et al., ) . in sheep, active transport of iga from the circulation into milk seems likely (sheldrake et al., ) ; however, studies on the transport of iga into murine milk have produced conflicting results. using radiolabeled iga, halsey et al. ( ) demonstrated that in the mouse, iga can be transported from the circulation into milk during early lactation. other investigators (koertge and butler, b; russell et al., ) , using assays based on antibody-binding activity, were unable to show transport of iga into murine milk. using radiolabeled iga, koertge and butler ( b) were able to show that the iga present in milk was degraded and suggested that the previous study (halsey et al., ) detected only iga fragments that had been transudated into the milk from the serum and not specifically transported iga. passively administered iga is not transported into the milk of rats (dahlgren et al., ; koertge and butler, b) . only a limited number of studies on the transport of antibodies into respiratory secretions have been reported, but the results have shown that selective transport of passively administered serum iga into the respiratory tract is possible in sheep and mice. because of their importance as background to the experiments demonstrating the passive transfer of local immunity by iga, these respiratory transport studies will be addressed in more detail. sheep. using the intravenous injection of radioiodinated ovine immunoglobulin, scicchitano et al. ( ) showed that % of the iga in the mediastinal lymph of sheep is plasmaderived. it was further demonstrated (scicchitano et al., ) by the simultaneous intravenous injection of radiolabeled iga and radiolabeled igg or igg that iga is selectively transported into ovine respiratory secretions. transport of iga was approximately . times greater than transport of igg, and the transported iga was intact in the secretions. biological activity of the transported iga was not determined. mice. mazanec et al. ( ) found that to hours after the intravenous injection of radiolabeled monomeric or polymeric iga anti-sendai virus monoclonal antibodies into mice, transport of piga into nasal secretions was three to seven times more efficient than transport of monomeric iga (miga), while piga transport into bronchoalveolar lavages was only one to three times more efficient. this difference may reflect an increased contribution of serum antibody due to the transudation of igg into alveolar fluids. transport of piga into the gut was four to five times more efficient than the transport of miga, as expected. the agreement of the nasal secretion and gut transport indices suggests that transport at these two sites could occur by a similar mechanism. the investigators were unable to demonstrate the presence of functionally intact piga in the upper respiratory tract. the piga transported into murine nasal secretions in the studies reported by renegar and small ( a) was, in contrast, functionally intact. to avoid problems associated with the quantification of intact vs. degraded radiolabeled iga in secretions (described by koertge and butler, b) , this study used an anti-influenza enzyme-linked immunosorbent assay (elisa) to evaluate the transport of monomeric or polymeric iga or igg monoclonal anti-influenza antibodies into the nasal secretions of mice. nonimmune mice were injected intravenously with influenza-specific miga, piga, or igg , and sacrificed at varying times between and hours postinjection. the peak nasal wash piga titer was reached hours after antibody injection and was approximately times greater than the nasal wash titer of either monomeric immunoglobulin. to determine whether piga was selectively transported relative to igg , the investigators injected a mixture of the two monoclonals intravenously into nonimmune mice and calculated a selective transport index for nasal antibody for each mouse. twenty-nine of the mice studied showed selective transport of iga relative to igg. using a similar model, steinmetz et al. ( ) determined that passively administered monoclonal piga isotype-switch variants, generated from igg hybridomas producing antibodies specific for bacterial respiratory tract pathogens, were selectively transported relative to igg into both the upper and lower respiratory tract secretions of mice. in agreement with the results of renegar and small ( a) , falero-diaz et al. ( ) showed that, in mice, parenterally administered (either intravenously or by "backpack" tumor growth) monoclonal piga acquired sc as it was transported from the serum into nasal or vaginal secretions or into the bile, while similarly administered igg did not, suggesting specific transport of the iga but not the igg. in contrast to steinmetz et al. ( ) but in agreement with mazanec et al. ( ) , the falero-diaz group found efficient transmission of igg but not of piga into the lungs of mice. in fact, they found that topical administration under light anesthesia of iga as nosedrops was a more effective method for the delivery of iga into the lungs than either parenteral method. thus, transport of serum iga into nasal secretions is possible in some species. the relevance of this transport to the passive transfer of local immunity will be addressed in the following section. studies of the passive transfer of local immunity can be classified into two categories. in the first are those studies in which the antibody is introduced into the local secretions exogenously or mixed with the target pathogen prior to host challenge. the second category includes those studies in which systemically administered piga must be physiologically transported by a pigr-mediated mechanism to its site of activity. the studies in this category have investigated the role of iga in mucosal immunity by feeding antibody or instilling it intranasally or intravaginally and then challenging, or by administering antibody-pathogen mixtures intranasally. oral antibody. offit and clark ( ) demonstrated the ability of milk-derived igg and iga to protect the murine intestine from infection with primate rotavirus sa- . suckling mice were protected by milk from dams that had been orally immunized with sa- virus. this protective activity was detected in both the igg and iga fractions, but the iga fraction was more potent in vivo than the igg fraction. in newborn mice from immune dams foster-nursed on seronegative dams, the presence of circulating systemic antirotavirus antibodies in high titer did not protect against sa- viral infection. thus, the specific antibody had to be present in the gut lumen to protect the intestinal cell surface from viral infection, and s-iga could mediate this protection. enriquez and riggs ( ) developed a series of dimeric iga monoclonal antibodies directed toward the sporozoite antigen p of cryptosporidium parvum, an important diarrhea-causing protozoan parasite. when administered orally prior to parasitic challenge, these antibodies were able to reduce the number of intestinal parasites in infected neonatal mice by up to %. these results extend the work of albert et al. ( ) , who successfully treated cryptosporidiosis in nude mice by the oral administration of rat bile containing c. parvum-specific iga, while czinn et al. ( ) protected germ-free mice from infection by helicobacter felis by incubating the bacteria with specific iga antibody prior to oral administration. the protective antibody was later shown to be directed against urease (blanchard et al., ) . a significant number of systemic infections in the human neonate originate from the gastrointestinal tract, especially in premature infants with immature gut barriers. maxson et al. ( ) fed rabbit pups human s-iga via intragastric gavage, then challenged them with e. coli k . iga-treated pups had significantly fewer bacteria translocated from the gut to the liver, spleen, and mesenteric lymph nodes. this neonatal rabbit model provides the first demonstration of control of bacterial translocation by iga and suggests that oral supplementation with iga may be beneficial for patients at risk for gut-origin sepsis. intranasal antibody. a number of studies have shown that exogenously administered iga can protect against intranasal challenge with a pathogen. bessen and fischetti ( ) showed that s-iga given by the intranasal route protected mice against streptococcal infection. live streptococci were mixed with affinity-purified human salivary s-iga or serum igg antibodies directed toward the streptococcal m protein. the mixture was administered intranasally to mice. the s-iga antibody protected against streptococcal infection, while the serum antibody had no effect. this study suggested that s-iga alone is capable of protecting the mucosa against bacterial invasion. demonstrated that iga can protect mucosal surfaces against viral infection. ascites containing iga anti-sendai virus monoclonal antibody was administered intranasally to lightly anesthetized mice before and after the mice were challenged intranasally with live virus. three days later, mice were sacrificed and lung viral titers were determined. animals treated with the specific monoclonal antibody were protected against viral infection. further work from the same laboratory showed that local immunity to sendai virus can also be mediated by intranasally administered igg (mazanec et al., ) . tamura et al. ( ) purified anti-influenza s-iga antibodies from the respiratory tracts of mice immunized with influenza hemagglutinin molecules. this iga, when given intranasally, protected nonimmune mice from influenza infection. protection was observed up to days after antibody administration, was proportional to the amount of iga administered, and was observed at iga doses equivalent to naturally occurring antibody titers. intravaginal antibody. zeitlin et al. ( ) were able to protect the mouse vagina from infection with herpes simplex virus (hsv- ) by the topical administration of either igg or iga monoclonal antibody directed against glycoprotein d of hsv- . these studies show that topically administered local iga or igg can protect against viral or bacterial infection of the mucosa. they do not show that physiologically transported (secretory) iga or serum-derived igg actually does so. for that demonstration, antibody must be administered parenterally and transported into the mucosal secretions by a physiologic mechanism. the studies presented in the next section satisfy that criterion. the definitive studies in this category have involved the respiratory and gastrointestinal tracts, although passive transfer of uterine immunity by piga has also been observed (renegar and small, ; cotter et al. ; pal et al. ), and leher et al. ( ) demonstrated the protection of chinese hamsters and pigs against acanthamoeba castellanii-mediated keratitis by intraperitoneally administered antigen-specific monoclonal iga. work with the respiratory and gastrointestinal tracts will be presented in more detail. respiratory tract. the respiratory tract can be separated into an upper region (nose and trachea) and a lower region (lungs and bronchi) with immunity at each site involving different elements of the immune system. numerous studies have shown that passively administered serum anti-influenza antibody (igg) can prevent lethal viral pneumonia (barber and small, ; kris et al., ; loosli et al., ; ramphal et al., ; palladino et al., ) . serum antibody, however, does not prevent influenza infection of the upper respiratory tract (barber and small, ; kris et al., ; ramphal et al., ) . protection of the nose correlates with an increased nasal secretion iga antibody level (reviewed in renegar and small, ) , making influenza an excellent model in which to investigate the hypothesis that nasal immunity is mediated by s-iga. the first demonstration of the passive transfer of local immunity by physiologically transported s-iga was reported by renegar and small ( a) in the murine influenza model. they showed, as described above, that intravenously administered piga is transported into nasal secretions. to determine whether intravenously administered piga antiinfluenza monoclonal antibody could mediate protection against local influenza virus challenge, passively immunized mice were challenged intranasally while awake with influenza virus. twenty-four hours later the mice were sacrificed and the amount of virus shed in their nasal secretions was determined. of the saline-injected control mice, shed virus into the nasal secretions, while only of the piga injected mice shed virus, and those that did shed virus had a low titer. the observed protection was significant ( p < . ). passive immunization with influenza-specific piga therefore conferred complete protection against viral infection in % of the mice and partial protection in the remaining %. serum igg was found to confer only limited protection against nasal influenza infection (minimal reduction in viral shedding [ p < . ], with only one of eight mice intravenously injected with influenza-specific igg not shedding virus [ p < . ]). the passive protection studies showed that iga can mediate local immunity. to confirm that iga is the mediator of local immunity, mice passively immunized with piga were given nose drops of anti-iga antibody minutes before and hours after they were challenged intranasally with influenza virus suspended in anti-iga antiserum. anti-iga treatment abrogated iga-mediated protection in the passively immunized mice (renegar and small, a) . to show that the passive transfer of local immunity by iga was a reflection of the natural situation, the abrogation technique was extended to mice convalescent from influenza infection (renegar and small, b) . nonimmune mice and convalescent mice, i.e., mice that had recovered from an influenza virus infection to weeks earlier and were therefore naturally immune, were treated intranasally with antiserum to iga or igg or with a mixture of antisera to igg and igm and then challenged while awake with influenza virus mixed with antiserum. intranasal administration of antiserum was continued at intervals for hours. one day after viral challenge, the mice were killed and their nasal washes were assayed for virus shedding. nonimmune mice all became infected, regardless of whether the virus was administered in saline, normal rabbit serum, or anti-immunoglobulin antiserum. convalescent mice, as expected, were protected from viral infection. administration of influenza virus in either anti-igg or a mixture of anti-igg and anti-igm antisera did not affect protection, i.e., the convalescent mice were still immune. administration of virus in anti-iga antiserum, however, abrogated convalescent immunity. these results demonstrate that iga is a major mediator of murine nasal immunity and suggest that passive immunization mimics the role s-iga plays in natural immunity. these observations were extended by the work of philipon et al. ( ) . using the backpack method of monoclonal antibody administration, they demonstrated that monoclonal iga antibody directed against shigella flexneri serotype a lipopolysaccharide protects mice against intranasal challenge with s. flexneri. mbawuicke et al. ( ) , however, have used the iga knockout mouse model to challenge these findings. mice unable to produce iga antibodies were able to generate a protective anti-influenza response and to transport passively administered anti-influenza antibodies into both the upper and lower respiratory tracts. knockout mice, however, may not be the best model in which to study the role of iga in nasal immunity, because, with the congenital loss of iga, production of other classes of antibody such as igg may be increased as a compensatory mechanism. the data of mbawuicke et al. suggest that this may be the case, as influenza-specific igg levels were higher in immune knockout mice than in normal mice, and an altered igg subclass distribution in response to influenza infection was observed zhang et al., ) . furthermore, perturbation of mucosal iga transport in the pigr knockout mouse (johansen et al., ) led to increased mucosal leakiness and increased serum total igg levels, indicating a defect in the mucosal barriers. it is known (renegar et al., ) that at a dose high enough to give a serum titer seven times the normal anti-influenza igg titer of convalescent mice, intravenously administered influenza-specific igg can lower or eliminate the nasal secretion viral load; however, scanning electron microscopy has revealed that even this high igg dose does not prevent infection of the nasal epithelium. thus, the lowered nasal secretion viral titers reported in igg-protected mice may be due to neutralization of newly replicated virus by serum antibody leaking through the virally damaged epithelium. furthermore, the serum antiinfluenza igg antibody titer has to be several times higher than that normally observed for the igg effect to be observed, because serum antibody at a level comparable to that of normal convalescent mice neither depresses viral shedding nor prevents viral pathology in the nasal epithelium. since the publication of the iga knockout mouse work, a model has been reported in which mucosal iga levels are depressed in genetically normal mice while serum igg remains unaffected (renegar et al., a (renegar et al., , b . in mice, total parenteral nutrition (tpn) depressed nasal mucosal immunity to influenza virus, resulting in increased viral shedding from the noses of tpn-fed immune mice (fig. . a) . in these mice, nasal influenza-specific iga was severely depressed (fig. . b) , while the serum anti-influenza igg titer was unaffected (fig. . c) . thus, in genetically normal icr mice, serum igg alone is not capable of preventing viral infection of the nose. protection could be restored by the intravenous administration of influenza-specific piga monoclonal antibody (fig. . d) . this work strongly suggests that iga is required for the prevention of influenza virus infection in the noses of normal mice. gastrointestinal tract. additional evidence that s-iga is the mediator of local immunity comes from studies of the gastrointestinal tract. polymeric iga hybridomas against vibrio cholerae were generated and the resulting monoclonal antibodies were used to determine whether iga can mediate immunity toward a bacterial pathogen in the gut (winner et al., ) .the investigators selected a clone that produced dimeric monoclonal iga antibodies directed against an ogawa-specific lipopolysaccaride carbohydrate antigen exposed on the bacterial surface. these antibodies were able to cross-link bacterial organisms in vitro, suggesting that they might be effective in preventing mucosal colonization by the pathogen in vivo. to provide continuous physiologic (i.e., secretory-component-mediated) transport of specific antibody into the gut, hybridoma cells were injected subcutaneously into the backs of adult balb/c mice. these "backpack" tumors released monoclonal iga into the circulation, and the plasma iga was transported into the gut lumen. neonatal mice bearing these backpack tumors survived challenge with v. cholerae, while neonatal mice bearing backpack tumors of unrelated iga hybridomas and nontumor-bearing neonatal mice died. this ingenious model provided the first evidence that s-iga alone can mediate mucosal immunity to a bacterial pathogen. michetti et al. ( ) have since demonstrated that pathogen-specific monoclonal s-iga can protect mice from infection by salmonella typhimurium following oral challenge. the backpack model has also been used to treat rotavirus infections of the gastrointestinal tract of mice (burns et al., ) . non-neutralizing monoclonal iga antibodies directed against vp , a major inner capsid viral protein, were capable of both preventing primary and resolving chronic murine rotavirus infections. these findings are consistent with the hypothesis that in vivo intracellular viral inactivation by piga during transcytosis is a mechanism of host defense against rotavirus infection. the general approach of passive parenteral transfer of mucosal immunity has proven to be a useful research tool for determining the role of s-iga in protection against various mucosal pathogens. the possibility of therapy by passively administered iga antibody is more problematic. passive protection by injection is highly speculative because of both the questionable efficiency of transport to the targeted mucosal surface and the potential adverse effects of intravenous iga antibody. serum iga has been associated with both decreased complement activation (russell et al., ) and decreased immune lysis (griffiss and goroff, ) . systematically administered piga may also be suppressive of both specific humoral and cellular responses (renger and small, unpublished observations). a more thorough knowledge of the role iga can play in regulating the immune response is needed before intravenous passive mucosal immunization can become an acceptable means of therapy in man; however, the direct oral or respiratory application of antibodies to the mucosal surfaces is both practical and acceptable. control of dental carries is a feasible target for topical iga administration. one study is particularly intriguing. ma et al. ( ) generated a monoclonal secretory antibody in transgenic plants and showed that it survived up to days in the human oral cavity; furthermore, this antibody afforded specific protection against oral streptococcal colonization for at least months. the literature contains a number of reports of the passive transfer of immunity against gastrointestinal pathogens in humans (reviewed in bogstedt et al., , and in hammarström et al. ) . this immunity has been provided by the oral administration of purified human igg or serum iga and has been used as both a prophylactic and a therapeutic measure against rotavirus infection in children (barnes et al. ; guarino et al., ) and as a therapeutic measure against bacterial diarrheas (tjellstrom et al., ; hammarström et al., ) . oral administration of bovine antibodies has also been used successfully in the prophylaxis of bacterial and rotaviral diarrheas in man and in the treatment of human rotavirus infections (bogstedt et al., ; mitra et al., ) . chicken egg yolk antibody (igy) protects calves against bovine rotavirus (kuroki et al., ) . provision of passive immunity by the intranasal administration of antibodies has been reported in both human and three weeks later, they underwent surgical instrumentation and were randomized to iv tpn feeding (n = ) or chow feeding (n = ). following days on protocol, the mice were sacrificed. levels of influenza-specific iga in nasal secretions were determined by elisa and normalized to ng-specific iga/ μg nasal protein (chow vs iv tpn, p < . , anova). influenza-specific iga was undetectable in nonimmune mice. c, influenza-specific serum igg titers: immune mice (n = ) underwent instrumentation and were randomized to iv tpn feeding (n = ) or chow feeding (n = ). following days on protocol, mice were sacrificed and serum igg anti-influenza titers and nasal viral shedding were determined. antibody titers were the serum dilution with an elisa absorbance reading of . or greater. the igg titers were comparable in chow-fed and tpn-fed immune mice. all of the tpn-fed mice shed virus in their nasal secretions, while none of the chow-fed mice did so. d, passive protection: immune mice were fed on mouse chow or iv-tpn. following days on their respective protocols, the iv-tpn mice were divided into two groups. nonimmune controls, (n = ), the chow-fed immune mice (n = ), and one of the iv-tpn groups (tpn-nms, n = ) received μl ascites fluid containing mopc (does not recognize influenza) piga monoclonal ab iv. the second iv-tpn group (tpn-piga, n = ) received μl ascites fluid containing piga antiinfluenza monoclonal ab. four hours later, all mice were challenged i.n. with pr influenza virus. after hours, the mice were euthanized and their nasal secretions assayed for viral shedding. nonparametric statistical analysis: nonimmune vs. tpn-nms, p = ns; immune vs. tpn-piga, p = ns; nonimmune vs. immune chow-fed, p < . ; nonimmune vs. tpn-piga, p < . . panels a-c modified from renegar et al., b . panel d from renegar et al., a nonhuman primate models. human gamma globulin administered intranasally showed promise in challenge experiments with influenza or coxsackie a- viruses (fruchtman et al., ; buthala et al., ) . iga antibodies (igabulin), given as a nasal spray to the swedish ski team during the albertville winter olympic games, significantly reduced the level of upper respiratory tract infections (hammarström et al., ) ; however, igabulin treatment had no effect on the frequency of upper respiratory tract symptoms in elite canoeists studied during hard and moderate training regimens (lindberg and berglund, ) . igabulin nosedrop treatment of variable immunodeficiency patients who were chronic nasopharyngeal carriers of nonencapsulated haemophilus influenzae eliminated the carrier state in of patients and alleviated coughing in all (lindberg et al., ) . heikkinen et al. ( ) treated children aged to years intranasally with iga or a placebo and found a % reduction in rhinitis in the iga-treated group. thus, intranasal instillation of antibodies is feasible and may prove to be effective in the management of immunosuppressed patients. intranasal administration of specific antibodies may prove to be even more exciting. weltzin et al. ( ) treated rhesus monkeys with nose drops containing mouse monoclonal iga antibody against respiratory syncytial virus (rsv). treated monkeys had reduced viral shedding in the nose, throat, and lungs and developed neutralizing serum antibody to rsv, even in the absence of detectible viral replication. rsv is a major cause of lower respiratory tract disease in infants and young children, producing severe disease in children with underlying conditions of the heart or lungs. these results suggest that prophylactic administration of monoclonal antibody nose drops could provide effective protection against rsv infection in at-risk human infants. the mechanism of protection of infant mice from intestinal colonization with campylobacter jejuni treatment of murine cryptosporidiosis with anticryptosporidial immune rat bile transmission of immunoglobulin to foetal and neonatal mice newborn rats in the murine typhus enzootic infection cycle: studies on transplacental infection and passively 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in mice intranasal monoclonal iga antibody to respiratory syncytial virus protects rhesus monkeys against upper and lower respiratory tract infections experimental neonatal colibacillosis in cows: immunoglobulin classes involved in protection new model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin a from hybridoma tumors protects against vibrio cholerae infection comparison of an anti-hsv- monoclonal igg and its iga switch variant for topical immunoprotection of the mouse vagina immunoglobulin a-deficient mice exhibit altered t helper -type immune responses but retain mucosal immunity to influenza virus studies on human secretory immunoglobulin a. v. trypsin hydrolysis at elevated temperatures chapter passive immunization: systemic and mucosal key: cord- -qaagup d authors: flicker, sabine; zettl, ines; tillib, sergei v. title: nanobodies—useful tools for allergy treatment? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qaagup d in the last decade single domain antibodies (nanobodies, v(h)h) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. according to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type i allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence. type i allergy, an ige antibody mediated hypersensitivity disease, represents a common health problem affecting almost % of the population worldwide ( ) . the recognition of allergens by specific ige antibodies that are generated after sensitization is a key event for the initiation of allergic inflammation ( ) . allergic patients suffer from a variety of allergic symptoms including rhinoconjunctivitis and asthma ( ) but also food allergy and skin inflammation ( ) . these clinical manifestations cause a major burden by reducing the quality of life of affected persons ( ) . while anti-inflammatory treatment based on pharmacotherapy reduces allergic symptoms and is the most commonly prescribed medication for treatment of allergic patients ( ) , only allergenspecific immunotherapy (ait) represents a causative treatment of type i allergy. in fact, ait induces a protective immunity in allergic patients based on the modification of cellular and humoral responses to the disease causing allergen ( ) . besides the inhibition of ige binding to their specific allergen, the immune deviation from a th to th response, and the decreases in numbers of effector cells in target organs, the generation and maintenance of allergen-specific regulatory t and b cells and the involvement of their suppressive cytokines are essential for the induction of allergen tolerance ( ) ( ) ( ) . beyond doubt the improvement of allergic symptoms is further caused by ait-induced igg antibodies found in serum and nasal secretions ( , ( ) ( ) ( ) ( ) . for many years ait was conducted with aqueous natural allergen extracts and patients experienced considerable side effects due to the unpredictable composition and poor quality of the injected extracts ( ) . recent developments like next-generation forms of ait based on molecular approaches may overcome the limitations of current forms of ait ( , ) . the last generation of improved vaccines, i.e. peptide carrier vaccines, induces an igg response that targets ige binding sites on allergens. induced igg antibodies effectively block ige binding and are termed blocking antibodies ( , ) . however, the efficacy of such blocking antibodies was long questioned because it revealed to be cumbersome to isolate reproducible defined, i.e. monoclonal allergen-specific antibodies comprising the capacity to inhibit allergen-induced allergic reactions. a recent proof of concept study re-stimulated the idea to generate monoclonal allergen-specific antibodies and to evaluate their feasibility for allergy treatment. the authors could show that a single subcutaneous injection of a mixture of two human monoclonal allergen-specific igg antibodies significantly reduced allergic symptoms in allergic patients ( , ) . moreover, validated in a pca mouse model, the mixture of these two monoclonal antibodies proved to be more potent in inhibiting mast cell degranulation than igg antibodies purified from patients' sera who underwent successful ait ( ) . furthermore, these human monoclonal igg antibodies recently completed the phase ii clinical trial in treatment of cat allergic patients (https://clinicaltrials.gov/ct /show/nct ). these results proved for the first time that allergy treatment with monoclonal allergen-specific antibodies is a well-tolerated, rapid, and effective approach to reduce allergic inflammation and rekindled the blocking antibody concept ( , , ) . nevertheless, the generation and identification of blocking conventional human or humanized antibodies is connected with high costs for production, validation and application ( , ) . therefore, cost-effective alternatives are currently sought. the nanobody technology represents such an alternative implying a significant improvement to the laborious methods to obtain monoclonal blocking conventional antibodies. due to their beneficial properties of small molecules and monoclonal antibodies, nanobodies in general are an attractive agent for development of novel therapeutic strategies ( , ) . the ease of their generation and production, the single domain organization, their beneficial biochemical properties and their feature to recognize small cavities on the surface of antigens and hence bind to epitopes inaccessible for conventional antibodies ( ) have raised the particular interest of allergologists recently. can the nanobody technology provide enhanced opportunity to generate a panel of antigen-binding molecules with various epitope specificities for certain allergens different to conventional antibodies? will these identified allergen-specific nanobodies be more efficient in blocking than conventional igg antibodies due to their pronounced cleft recognition? will it be possible with this technology to find single nanobodies that are able to abrogate ige-mediated allergic inflammation? these questions and our wish to answer these questions attracted our attention. within this review, we focus on the powerful nanobody technology to generate allergen-specific nanobodies and report on their evaluation for prospective application for passive allergy treatment. if allergologists are asked why the search for effective protective allergen-specific monoclonal antibodies is complex and laborious, they will describe this issue by the typical quest for a needle in a haystack. through intense and precise molecular and immunological exploration of available allergen-specific monoclonal antibodies in the past it was proven that epitope specificity and affinity are decisive for their inhibitory potential to block ige binding and thus ige-mediated reactions ( , ( ) ( ) ( ) . the commitment to find and isolate monoclonal antibodies with specificity and high affinity for certain allergens and even more for certain epitopes always started with several fundamental decisions. amongst them the choice for the perfect source to gain dna coding for antibodies and the applied technology to generate allergen-specific antibodies are two of the most critical ones. regarding the dna source both animals, mainly mice, and humans served as blood, spleen, tonsils and even bone marrow donors in the last decades to isolate b cells or plasma cells and thus dna coding for antibodies ( ) ( ) ( ) . for the proof of principle, murine igg antibodies overlapping with human ige binding sites are valuable tools to investigate the effects to inhibit ige epitope recognition on allergens and consequently to contribute to the design of hypoallergenic derivatives suitable for ait ( ) . however, the direct therapeutic use of these murine monoclones in humans is limited by the high incidence of harmful immune responses against these administered foreign proteins ( ) . to mitigate this limitation numerous murine monoclonal antibodies have been re-engineered by chimerization and humanization technologies. these expensive procedures are justified for fatal diseases like different forms of cancer but were barely applied for allergen-specific murine antibodies so far with a few exceptions ( , ) . this was one of the main reasons why allergologists in the recent past endeavour to focus on human donors including allergic patients, ait-treated patients and even healthy individuals depending on the research question ( , , ) . various methods were utilized to generate allergen-specific genuine, i.e. native antibodies with the preservation of the natural vh and vl pairing including hybridoma technology, epstein-barr-virus (ebv) transformation, single b cell sorting and cloning and humab mice (transgenic mice that produce fully human antibodies) ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in parallel, versatile approaches were developed to generate non-genuine antibodies by random combination of vh and vl chains, i.e., combinatorial fab/scfv libraries or (semi-) synthetic libraries ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . based on pcr amplification as strong tool to depict large antibody repertoires and phage display to screen these large repertoires, many recombinant allergen-specific antibody fragments (fabs or scfvs) were isolated ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . all mentioned technologies have definitely contributed to the isolation and evaluation of monoclonal allergen-specific igg, ige antibodies and fragments thereof and furthermore to assess their feasibility for allergy treatment. nevertheless, all mentioned technologies are also reported to have some limitations. while the hybridoma technology and ebv transformation are generally unsuitable for a comprehensive screening of large antibody repertoires because of their inefficient fusion and transformation events, the single b cell sorting was long hampered by inadequate staining technologies to clearly identify allergen-specific antibody producing cells ( , ) . the main drawback of combinatorial libraries is that they usually rely on random combination and thus most likely unnatural vh and vl antibody pairings. additionally, it turned out independent of the applied technology to be very difficult to isolate monoclonal igg and ige antibodies with a broad epitope spectrum for each allergen. it also revealed that besides several blocking antibodies also many non-blocking or even enhancing antibodies were isolated ( , ( ) ( ) ( ) . while all three types of monoclonal antibodies were unambiguously supportive to study the structural requirements for efficient effector cell activation and hence contribute to elucidate the underlying mechanisms of type i allergy, non-blocking and enhancing antibodies were fully useless for the prospective application as protective antibodies. these insights forced allergologists to look beyond the conventional antibody horizon. about years ago, a group of belgian scientists made an unexpected discovery, which was patented and later presented to the scientific community in the form of the well-known discovery publication in the journal nature in ( ) . they found that a significant amount of non-canonical types of antibodies is naturally present in blood of camelidae in addition to conventional antibodies. this exceptional type of antibody called heavy chain-only antibody (hcab) lacks light chains and consists of a homodimer of shortened (without ch domain) heavy chains. the antigen-recognition region in hcabs is formed by only one variable domain (v h h) that is directly linked via a hinge region to the fc-domain ( ) . later on, similar non-canonical hcabs were found in some cartilaginous fishes such as sharks and ratfish ( ) ( ) ( ) . the antigen-binding variable domain of these antibodies was named vnar as opposed to v h h in camelids. a recombinant protein version of the v h h-or vnar-domain is usually called "single domain antibody" or "nanobody". the very popular term "nanobody" is the commercial name given by the belgian biopharmaceutical company ablynx, a pioneer in hcab-based therapeutic applications that was acquired by sanofi in . the nanobody generation technology was proven to be a very efficient machinery to generate nanobodies with required properties and offered crucial advantages compared to traditional techniques utilized to produce murine or human conventional antibodies. after the typical initial immunization (of camelids) step, the full repertoire of cdna coding for functional nanobodies can be efficiently cloned from peripheral blood lymphocytes of immunized animals using pcr amplification and then a panel of nanobodies of required specificity can be easily selected using phage (or other type of) display-based methods ( , ( ) ( ) ( ) . in addition, there are different in vitro affinity maturation approaches to improve features of initially selected nanobodies ( , , ) . in some cases, especially if the antigen of interest is toxic, unstable, non-immunogenic or not available in sufficient quantity, other types of libraries (naive, semisynthetic or fully synthetic libraries) can be efficiently used instead of immune libraries for generation of nanobodies ( ) ( ) ( ) ( ) ( ) . synthetic libraries can be made using special predesigned scaffolds such as humanized scaffolds optimized for intracellular stability ( ) or optimized for bacterial expression ( ) . non-immune libraries are typically much larger than immune libraries and a ribosome display was suggested for the initial selection round of such large libraries to work with higher concentrations of nanobody variants than in case of phage display ( , ) . synthetic libraries combined with different selection procedures were successfully used to obtain conformationally selective nanobodies against g protein-coupled receptors ( ), sybodies against very challenging targets such as the heterodimeric bacterial abc exportertm / ( ) or the intracellular kdel receptor ( ) to name a few examples from many others. nanobodies comprise unique features that distinguish them from classical antibodies. nanobodies are the smallest known antibody fragments ( × . x nm, - kda) of natural origin that are able to specifically bind their cognate antigens. due to their often extended cdr loop they can form unusual paratopes, i.e. finger-like extensions and thus recognize special native antigenic epitopes (small cavities, concave surfaces, conformational epitopes, active sites of enzymes) that are hidden for conventional antibodies (figure ) . indeed, nanobodies have proven to be useful tools for modulating the activity of enzymes ( , , ) . it could be therefore speculated that allergen-specific nanobodies that modulate or inhibit the proteolytic activity of certain allergens (e.g., phl p , der p ) might reduce their penetration capacity through mucosal surfaces. furthermore, nanobodies are able to bind small peptides with high affinity ( ) ( ) ( ) ( ) ( ) . their high affinity, solubility and stability over a wide range of temperatures and ph, ease of producing in bacteria or other expression systems make them convenient molecules for different applications, as well as for all possible engineering modifications e.g., development of complex constructs and conjugates. nanobodybased tools are therefore increasingly used for research, molecular visualization, diagnostics and development of new treatment options for various pathologies, including cancer and other socially significant diseases ( , , ( ) ( ) ( ) ( ) ( ) . so far, only one allergen-specific nanobody is described in the literature. this nanobody is reported to be specific for the major peanut allergen, ara h and was isolated from a synthetic library of humanized nanobodies via phage display ( ) . the interaction between ara h and the ara h -specific nanobody resulted in a dissociation constant of nm representing medium affinity binding and was further investigated by the structural determination of formed co-crystals ( ) . the authors acknowledged that additional work is needed to improve the affinity of the isolated nanobody to make it an attractive tool for the development of biosensors for peanut allergen detection. this finding clarifies that the selection procedure is only one part of the successful discovery of potent ige-blocking nanobodies, thus the evaluation of selected nanobodies is critical as well. nevertheless, we are confident that soon more allergenspecific nanobodies will arise to be studied for their potential to abrogate ige-mediated allergic inflammation. similar to the evaluation of conventional antibodies with the focus to identify effective protective monoclones, generated nanobodies have to be assessed first for their allergen specificity, epitope recognition, cross-reactivity to homologous allergens present in related species, for their affinity to their corresponding allergens and most importantly for their ability to inhibit patients´ige binding to these allergens (figures a-c) . after the allergen specificity of isolated nanobodies is confirmed, the proof for cross-reactivity (figure a) is of great importance because ige antibodies from allergic patients often displayed cross-reactivity to allergens from other allergen sources ( , ) . high affinity and slow dissociation of formed nanobody/allergen complexes will be critical prerequisites for allergen-specific nanobodies to be chosen as suitable candidate ( figure b) . however, the pivotal characteristics for an allergen-specific nanobody to be attractive for further processing will be the determination of its potential to block patients' ige binding and hence ige-mediated effector cell activation ( figure c) . additionally, specific nanobodies have to be tested as well for their cross-protectivity to homologous allergens. all these properties are crucial requirements for allergen-specific nanobodies to be selected for further essential investigations concerning half-life, clearance and safety. nanobodies are considered as proteins of weak immunogenicity due to the shared similarities with variable vh domains of human immunoglobulins (igg subclass), and they can be further improved by a humanization approach ( ) ( figure d) . consequently, no immune response against applied nanobodies was raised in mice or humans that were injected with nanobody-containing constructs ( ) ( ) ( ) . safety of nanobody-based drugs is confirmed by several completed phase and phase clinical trials ( ) and recent approval by the us food and drug administration (fda) and the european medicines agency (ema) of the first therapeutic nanobody, caplacizumab, a bivalent nanobody designed for the treatment of thrombotic thrombocytopenic purpura and thrombosis ( ) . though advantageous for in vivo imaging, the small size of nanobodies could be seen as a disadvantage for passive treatment of allergy due to a quick renal clearance of nanobodies from blood (approx. min). many different strategies to extend the in vivo half-life of nanobody-based construct have been developed ( ) . they include increasing the hydrodynamic radius of a protein by attaching highly flexible and hydrophilic molecules such as polyethylene glycol (peg) and carbohydrates or by genetic fusion with polypeptide chains mimicking the biochemical properties of peg, fusion of v h h to the fc region of igg, fusion or non-covalent binding to albumin ( ) ( figure d ). nanobodies can also be used as modules to engineer larger molecules with several valencies and/or specificities, such as multivalent ( ) ( ) ( ) ( ) , bispecific ( , ) , and other ( , ) constructs that may acquire considerably higher specificity, binding efficiency and biological activity ( , , ) . nanobodies were also considered as possible ligands to design new highly specific immunosorbents ( ) ( ) ( ) . different types of nanobody-based tools/approaches can be envisaged to be potentially profitable for an allergy treatment: a) bispecific nanobodies for topical application to capture allergens before they penetrate epithelial mucosa in airways, b) very stable nanobodies to capture food allergen in gastrointestinal tract, c) anti-idiotypic nanobodies mimicking allergenic epitopes as a possible replacement for a complex natural allergen for a new kind of ait vaccine development, d) multivalent nanobody-based constructs for systemical administration to efficiently block allergen interaction with ige on mast or basophil cells, e) efficient immunosorbents to remove ige from the blood by immune apheresis. correspondingly, different administration approaches for nanobody-based constructs can be developed: aerosol or topical applications, oral route or subcutaneous administrations. temporary blocking of allergen-ige interaction (i.e. by topical or systemic administration of specific nanobodies) or a subtraction of ige from the periphery blood (i.e. apheresis) may give a short-term treatment effect. for a long-term treatment effect we could hypothesize the use of anti-idiotypic nanobodies to ige. such nanobodies may represent "internal images" of an allergen and mimick hypoallergenic b cell epitopes. to efficiently induce igg response that targets ige binding sites on allergens, these nanobodies should be fused to a viral coat protein as it was described for next-generation forms of ait ( ) . the generation of allergen-specific nanobodies unambiguously represents a reasonable progress in the field of allergy. with their well-documented qualities including their ability to recognize unusual "hidden" epitopes, high affinity binding, solubility, extreme stability and low immunogenicity, nanobodies attracted the interest of allergologists to study their suitability for passive allergy treatment. the chance to find allergen-specific nanobodies with this powerful technology that ideally comprise high affinity and bind to epitopes partly or fully overlap with ige binding sites on allergens is tempting. however, so far no allergen-specific nanobody fulfilling these criteria was reported indicating that it might be rather difficult to raise allergenspecific nanobodies of sufficient affinities. whether the current lack of such nanobodies is owed to some inherent structural or functional properties of nanobodies and/or the camelid immune system or the simple reason that the current research focus in the allergy field is on ait and its improvement has to be resolved. if allergen-specific nanobodies are identified that competitively block allergen binding to ige and thus abrogate ige-mediated allergic inflammation, we assume that they will represent appropriate tools for future allergy treatment. their economic properties, i.e. low production costs encouraged researchers to elaborate antibody engineering of these single-domain antibodies for diverse applications in biotechnology and medicine. this gathered knowledge will facilitate the implementation of modified allergen-specific nanobodies tailored to the needs of allergy treatment. nanobodies can be easily formatted for a particular application e.g., modified as recognition modules in large constructs or as bi-or oligo-specific, bi-or oligovalent derivatives. 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method for the parallel and sequential generation of single-domain antibodies for the proteomic analysis of human blood plasma the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © flicker, zettl and tillib. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ofd ipvs authors: cheng, matthew p.; yansouni, cedric p.; basta, nicole e.; desjardins, michaël; kanjilal, sanjat; paquette, katryn; caya, chelsea; semret, makeda; quach, caroline; libman, michael; mazzola, laura; sacks, jilian a.; dittrich, sabine; papenburg, jesse title: serodiagnostics for severe acute respiratory syndrome–related coronavirus- : a narrative review date: - - journal: ann intern med doi: . /m - sha: doc_id: cord_uid: ofd ipvs accurate serologic tests to detect host antibodies to severe acute respiratory syndrome–related coronavirus- (sars-cov- ) will be critical for the public health response to the coronavirus disease pandemic. many use cases are envisaged, including complementing molecular methods for diagnosis of active disease and estimating immunity for individuals. at the population level, carefully designed seroepidemiologic studies will aid in the characterization of transmission dynamics and refinement of disease burden estimates and will provide insight into the kinetics of humoral immunity. yet, despite an explosion in the number and availability of serologic assays to test for antibodies against sars-cov- , most have undergone minimal external validation to date. this hinders assay selection and implementation, as well as interpretation of study results. in addition, critical knowledge gaps remain regarding serologic correlates of protection from infection or disease, and the degree to which these assays cross-react with antibodies against related coronaviruses. this article discusses key use cases for sars-cov- antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation. s ince the initial identification of severe acute respiratory syndrome-related coronavirus- (sars-cov- ) as the etiologic agent of coronavirus disease (covid- ) , there have been over . million confirmed cases and around deaths reported worldwide, according to the world health organization (who) ( ) . however, given the prevalence of asymptomatic or minimally symptomatic individuals ( , ) , the imperfect sensitivity of molecular assays performed at a single time point ( ) , and limited molecular testing capacity in several parts of the world, the true number of infections probably exceeds the who's estimate by several fold. in addition to scaling up molecular testing for diagnosis of active disease, several countries have incorporated serologic surveillance studies to their covid- pandemic response. these studies can help elucidate disease transmission dynamics and improve disease burden estimates by identifying persons who were previously infected, even if pauci-or asymptomatic ( ); assess transmission within and between subgroups in the population; and provide insight into the kinetics of humoral immunity after infection ( , ) . serologic testing may also serve as an adjunct to molecular methods for covid- diagnosis in certain clinical scenarios ( ) . despite a rapid increase in the number and availability of serologic assays to test for antibodies against sars-cov- ( ), most have undergone minimal or no external validation or have poorly described validation panels, which hinders assay selection and interpretation of results. in addition, interpretation of serologic assays is limited at present because of critical knowledge gaps. for example, no definite serologic correlates of protection from infection or disease have been identified in humans, and the degree to which these assays cross-react with antibodies against related coronaviruses is poorly described. we discuss key use cases for sars-cov- antibody detection tests and their application to serologic studies. we review currently available assays, highlight key areas of ongoing research, and propose potential strategies for test implementation. we searched the medline ovid database for articles on sars-cov- serologic assays (the appendix, available at annals.org, shows the search strategy). additional studies were identified by hand-searching references of selected articles, consulting international experts, and searching covid- and sars-cov- preprints on medrxiv and biorxiv. this search was last updated on may . innate responses. of note, infection prevalence in the population being tested must always be considered. in patients with clinical features of covid- , a highly specific test, such as sars-cov- polymerase chain reaction (pcr), has a high positive predictive value for true infection. conversely, if testing asymptomatic individuals when the true seroprevalence of a population is only %, an assay with a specificity of % would produce a false-positive rate of %. low specificity is particularly problematic in cases where incorrectly identifying an individual as immune could place them at significant risk-for instance, if they were to enter settings with high risk for exposure without appropriate personal protective equipment. the sensitivity of a serologic assay can be established by testing sera from patients who have been identified as infected on the basis of a reference standard. however, a single estimate of sensitivity to de-scribe test performance can be difficult to interpret when samples are collected at different time points since infection. sensitivity estimates will vary according to time since infection in the validation cohort. early (< days since symptom onset) and mid-stage ( to days) pcr-confirmed cases of covid- will have lower rates of seroconversion than in the later stage (> days); thus, antibody tests will have lower sensitivity to detect infection in earlier phases. likewise, antibody responses may be more easily detectable in severe cases (hospitalized patients) than in mild or asymptomatic infections ( ) . establishing the analytic specificity of sars-cov- seroassays presents a challenge because of potential for cross-reactivity with antibodies to related coronaviruses ( , ) . to address this, test reactivity thresholds used to define a positive result can be adjusted to optimize the tradeoff between sensitivity and specificity ( ). with higher thresholds, sensitivity decreases as cases with low serum antibody levels are categorized as negative, but specificity improves as low amounts of nonspecific antibody are no longer considered positive. physicochemical assay variables can also be modified so that less specific antibodies, with less "avidity" for the antigen, are excluded. this also improves specificity at some expense to sensitivity. tests that target igm, which by its nature can be nonspecific, will probably have increased risk for false-positive results. validation of the clinical specificity of a serologic assay requires sera from different types of sources. in the case of covid- , sera collected before the end of are presumed to be seronegative for sars-cov- ( ). the samples chosen should be representative of the population of interest. in addition, individuals known to have been infected with various common pathogens, including other human coronaviruses, but who could not have been infected with sars-cov- , should be evaluated to demonstrate the absence of cross-reactivity. finally, patients with illnesses known to stimulate high levels of polyclonal antibodies, such as epstein-barr virus infection, malaria, or conditions associated with production of rheumatoid factor, can be evaluated for cross-reactivity ( ) ( ) ( ) . without these validations, assay specificity will be difficult to establish. once a particular assay is shown to have high sensitivity and high specificity, this assay can serve as a surrogate "gold standard" for the validation of other assays, as well as a standard for quantitative assays. to date, most published sars-cov- serologic assay validations have classified patient sera according to sars-cov- pcr results ( ) . polymerase chain reaction assay is an imperfect comparator for sars-cov- diagnosis because of variable analytic performance across assays ( ) , and because pcr sensitivity depends on sample type, quality of sampling, and timing relative to illness onset ( , ) . this can lead to unpredictable directions of bias for seroassay accuracy esti- molecular testing on respiratory specimens, the current gold standard for diagnosis of sars-cov- infection, is hampered by imperfect sensitivity and limited testing capacity. antibody testing has potential to aid in particular diagnostic scenarios, such as in rt-pcr negative patients who present later during disease course. antibody testing should not be used as the sole basis for diagnosis of acute covid- . appropriately designed seroepidemiologic studies will play an essential part in the public health response to the covid- pandemic by characterizing transmission dynamics, refining disease burden estimates, and providing insight into the kinetics of humoral immunity to sars-cov- . validation of novel antibody detection tests for sars-cov- must pay careful attention to the choice of source populations and reference standards, and to possible cross-reactivity with antibodies to other human coronavirus infections. plaque reduction neutralization assays are currently the reference standard for determination of host antibodies capable of inhibiting viral replication, but must be performed in a biosafety level laboratory. urgent research is needed to determine the serologic correlates of immunity against sars-cov- . mates. there is an urgent need for validation studies to provide more detail on pcr comparators and on study populations, especially regarding disease severity and timing in the illness course. furthermore, to enable a better understanding of the diagnostic accuracy of various sars-cov- serologic tests, the development of reference panels, including seroconversion panels, by using well-characterized sera is necessary. coronavirus spike (s) and nucleocapsid (n) envelope proteins are highly immunogenic and constitute important antigenic targets for the development of serologic assays ( , ) . as with sars-cov- , the s protein of sars-cov- binds to the cell surface angiotensin-converting enzyme (ace ) receptor ( ) ( ) ( ) . host neutralizing antibodies (nabs) appear to be predominantly directed at the s protein ( ) . the n protein plays crucial roles in viral replication and assembly, is highly conserved, and induces antibodies sooner than s during infection ( , , ) . commercial sars-cov- serologic assay development has focused on enzyme immunoassays, such as laboratory-based enzyme-linked immunosorbent assays (elisas) and rapid lateral flow assays (lfas). more complex serum neutralization assays are important as a reference standard and to assess immunity. only a subset of antibodies raised against a specific antigen have the property of neutralizing viral replication. neutralization assays, such as plaque reduction and microneutralization methods, provide essential data for the validation of candidate diagnostic tests and to define correlates of protective immunity. the primary drawback of functional assays of sars-cov- neutralization is that they can only be performed by experienced staff in a biosafety level (bsl) laboratory owing to the need to culture live virus, which increases complexity and cost. thus, efforts to circumvent these obstacles have converged on finding surrogates of traditional neutralization titers. live pseudotyped viruses have been developed that incorporate the s protein of sars-cov- , can be cultivated in bsl- conditions, and express a reporter enzyme when infecting cells through binding to the ace receptor, thereby allowing for automated quantification ( ) . such reporter virus sys- the figure shows a decision tree for interpreting antibody test results by symptomatology (symptomatic, postsymptomatic, asymptomatic or subclinical) and whether the patient is a suspected case. it is presumed herein that antibody tests with the highest possible sensitivity and specificity are used, and that the symptomatology is occurring early in the pandemic, when seroprevalence is low and before the availability of a vaccine. for sars-cov- , the accuracy of antibody test results and the appropriate test interpretation both depend on clinical context. in some situations, the clinical context does not enable a single interpretation of the antibody test result. for example, a positive antibody test in a low-risk population could be the result of prior infection, or it could be a false-positive result. similarly, a negative antibody test in a high-risk population cannot a priori differentiate among preseroconversion, undetectable seroconversion, a false-negative result, or the absence of infection. sars-cov- = severe acute respiratory syndrome-related coronavirus- . * the relationship between positive antibody results and protective immunity will vary among assays and must be validated individually. † includes high exposure, high risk, hot spots, and contact tracing. serodiagnostics for sars-cov- : a narrative review tems would offer substantial advantages in terms of speed, cost, and scalability while providing a quasifunctional assessment of the host neutralizing antibody response ( ) . other groups are striving to create surrogates of neutralization that bypass the need for viral culture through the use of blocking elisa formats ( ) . for high-throughput and inexpensive (after initial capital outlay) screening in clinical laboratories, relevant antigenic targets can be purified or synthesized, and or more can be incorporated into an elisa test platform. specific antibody-antigen reactivity is detected by using enzyme conjugates that produce color changes or other detector labels that can be objectively measured ( ) . the elisas detect antibodies directed at the chosen antigen without regard for their ability to elicit viral neutralization. thus, interpretation of immune status from elisa results requires rigorous characterization of the assay with respect to a reference standard. for the moment, this work has not been done for sars-cov- . furthermore, universal standards for reporting are lacking (some assays produce semiquantitative results, others are qualitative), and assays have variable test detection limits and reproducibility and use different analytes (igg, igm, iga) or combinations thereof, with unclear effect on performance ( ) . it is thus not surprising that estimates of elisa test sensitivity and specificity vary widely across assays and even within assays evaluated by different investigators ( table ) ( - ). the lfas leverage the same capture agents as an elisa in a lateral flow strip format ( ) . the lateral flow format enables a simple and fast time to result ( to minutes), but with tradeoffs in detection that is severalfold less sensitive than their elisa counterpart, a higher cost per test, and lower throughput ( ) . for lfas, follow-up confirmatory testing is typically recommended. most provide qualitative, visual results subjectively interpreted by the operator. the use of a small instrument reader can increase test sensitivity and may permit quantitative and more reproducible results ( , ). to enable community-based and home testing, lfas should be paired with minimally invasive samples, such as finger-prick or oral fluid or swabs, and minimal sample processing. these tests are ideal for near-patient testing and low infrastructure settings, such as the lower levels of the public health system in low-and middle-income countries ( ), where they have been used to effectively screen and triage cases of epidemic and nonepidemic diseases. particularly where resources are constrained, inexpensive lfas may be useful to expand diagnostic test capacity. many sars-cov- lfa antibody tests are available; however, the performance of these tests is still under evaluation, and their value needs to be carefully weighed depending on the use case. a recent large study found heterogeneous and inconsistent results among lfas and identified signal interpretation as a major obstacle ( ). population-based seroepidemiologic studies are an important source of evidence about sars-cov- transmission dynamics and will be critical for informing interventions to mitigate the effects of the covid- pandemic ( ) . whereas reports of clinical cases identify persons with acute disease, seroepidemiologic studies identify those who were infected previously, including those who experienced mild disease or subclinical infections and thus may not be subject to biases due to health care-seeking behavior and limitations on eligibility for testing during acute disease. these assessments of seroprevalence overall and in specific groups can be used to estimate important characteristics of the pandemic ( - ) . serologic surveillance studies can also assess the accumulation of persons with antibody responses over time to estimate incidence of sars-cov- infection ( , ) and can track age-and jurisdiction-specific disease susceptibility and identify at-risk populations ( ) . utilizing standard protocols for the design and implementation of serologic studies ( ) and making protocols publicly available can improve scientific rigor and ensure comparability across studies undertaken in different populations. of note, the who unity studies aim to combine worldwide seroepidemiologic study data ( ) . cross-sectional serologic surveillance studies are a key first step toward determining the proportion of a population that has been infected with sars-cov- . when estimating age-specific seroprevalence is the primary aim, the gold-standard study design is the conduct of appropriately powered, cross-sectional, age-stratified, population-representative, randomly sampled, serologic studies in each population of interest. this study design, when implemented appropriately, ensures that the estimates obtained are representative of the population of interest and minimizes the potential that the results may have common sources of bias ( ) . in addition, many variations of this design are also valuable for estimating agespecific seroprevalence, especially when statistical methods are used that can account for alternative design elements and sources of uncertainty ( ) . layering seroprevalence surveys onto other existing observational or interventional studies or utilizing residual sera from blood donors or from routine lab tests can increase feasibility and timeliness of estimating seroprevalence at some risk to generalizability. to determine sars-cov- seroincidence, or the proportion of the population seroconverting over a certain time frame, longitudinal studies can be conducted among cohorts of individuals who are at high risk for exposure (such as health care workers) or among those for whom little is known about the risk for infection (such as children). furthermore, longitudinal serologic surveillance can be implemented to provide insight in situations where prevention and control measures are for instance, household-or workplace-based serologic studies can aid in the determination of secondary attack rates, especially when the proportion of asymptomatic infections may be high. in addition, well-designed seroepidemiologic studies are critical for informing mathematical models and forecasting tools to guide prevention and control strategies. a critical aspect in the interpretation of serologic tests is an understanding of the dynamic nature of the humoral response to sars-cov- infection. a few studies have defined the kinetics of antibody formation in patients with disease ranging from mildly symptomatic to critically ill. these studies have consistently shown that most patients seroconvert by weeks after the onset of symptoms, and almost all patients have detectable antibodies by day ( , , , , ) . antibodies can be detected as early as day after illness onset, with peak igm and iga titers occurring in the ensuing to days and waning thereafter. the igg response appears to peak simultaneously in some cases, or slightly later in others ( ) , and plateaus between and days ( ). in some cases, igg titer declines significantly within weeks ( ) . some patients appear to have weak or undetectable seroconversion ( , ) . illness severity probably affects antibody responses. critically ill patients had a delayed but more robust formation of igm and igg in one study ( ) . anti-sars-cov- responses in subclinical infections have yet to be characterized. finally, the suitability of alternative specimen types to serum, such as saliva or dried blood spots ( , ), must be established for sars-cov- serodiagnostics. correlates of protection are empirically derived, specific immune markers associated with protection against infection or disease ( ) . seropositivity is often a useful correlate for clinical immunity, though cell-mediated immunity is known to be essential and antibody production is not the sole mechanistic contribution to protection ( ) . the relationship between seropositivity and immune protection has not yet been established for coronaviruses. a recent report on patients who recovered from covid- showed that nab titers were moderately correlated with antibodies binding to s protein domains ( ) . surprisingly, % of patients developed only low titers of nabs after recovery, with younger patients ( to years of age) having significantly lower anti-sars-cov- and nab titers. this suggest that innate and adaptive cellular immunity are also likely to play a significant role in viral clearance and immunity to coronaviruses ( ) . little is known regarding seropositivity and risk for reinfection to coronaviruses. in a challenge study with hcov- e, healthy volunteers who had lower specific igg titers at baseline were more likely to develop clinically overt infection ( ) . after the challenge, specific igg and nab peaked at weeks and fell considerably at weeks. one year later, out of previously infected participants became infected after a rechallenge, though they were asymptomatic and the duration of viral shedding was shorter than during the first challenge-suggesting at least partial protection induced by the first infection. of note, the immune response dynamics after sars-cov- and middle east respiratory syndrome-coronavirus (mers-cov) infection differ substantially from what was seen with hcov- e challenge. values for igg and nab peaked months after sars-cov- and decreased after months. after mers-cov infection, % of patients had detectable igg and nabs for at least months ( ). evaluations of sars-cov- serologic assays must account for potential cross-reactivity with other coronaviruses, including the endemic human coronaviruses: hku , oc , nl , and e. a systematic review of antibody-mediated immunity to coronaviruses found that studies of serologic responses to human coronavirus n proteins suggest cross-reactivity within human alphacoronaviruses ( e and nl ) and human betacoronaviruses (oc and hku ), but not between human alpha-and betacoronaviruses ( ) . the available evidence suggests that natural infections with endemic coronaviruses produce little cross-reactivity to emerging coronaviruses sars-cov- and mers-cov. regarding sars-cov- elisa using s protein epitopes, several pilot studies report positive results with sera from patients with sars-cov- , and a lack of significant cross-reactivity when using sera from small numbers of patients seropositive for the endemic human coronaviruses ( , ) . data regarding sars-cov- elisa based on the n protein are more limited. the specificity of elisa and lateral flow assays has also been assessed against pre-covid- sera from u.s. patients collected in july , and ranged from . to % ( ). finally, in keeping with these results, a surrogate assay of sars-cov- viral neutralization tests was found to be highly specific among sera positive for endemic human coronaviruses antibodies but showed some degree of cross reactivity with sars-cov- positive sera ( ) . thus, cross reactivity of sars-cov- serologic assays may be a concern in areas where sars-cov- and mers-cov circulated widely. overall, serologic tests based on s protein appear to distinguish between emerging and endemic coronaviruses. assays based on the n protein can serve as a marker of recent infection but might be expected to cross react more with endemic coronaviruses. convalescent plasma therapy, as a means of providing "passive" immunity to susceptible individuals and as early therapy after infection, has been used for many viral infections ( ) . this approach was used in a small number of patients with sars-cov- and mers-cov and has shown promise in a few case series of sars-cov- infection ( - ) . use of covid- convalescent plasma has been approved in several jurisdictions under the category of an emergency investigational new drug ( ) . as a general principle, the efficacy of plasma therapy is a function of several factors, including timing of plasma donation (plasma obtained a few weeks after recovery during convalescence is considered more immunogenic, with higher titers of polyclonal neutralizing antibodies), dosage, and timing of administration in relation to onset of disease in the recipient. for covid- , identifying "optimal" donors will prove to be an additional challenge, given the heterogeneity in antibody titers during convalescence and the lack of an established correlation between specific antibody titers and clinical efficacy ( ) . as an example, in the treatment of influenza, plasma with high nab titers collected from a nonconvalescent general population did not show efficacy ( , ) , suggesting that donor selection should not be based solely on serologic titers. eventually, antibody derived from vaccinated donors may deserve further study. serologic tests are essential to better understand the determinants of sars-cov- immunity and to guide vaccine development. for sars-cov- and mers-cov, the s protein was shown to be the most important antigen leading to production of nabs and inhibition of viral entry into the host cells ( ) . since then, s protein has been the major target for vaccine candidates. previous experience using sars-cov- subunit vaccine based on the full-length s protein showed potent nab responses and protective immunity in animal models. however, some of these vaccines were also associated with a harmful immune enhance-review serodiagnostics for sars-cov- : a narrative review ment, as seen in vaccine candidates for dengue or respiratory syncytial virus, leading to a potentially more severe disease in vaccinated individuals ( ) . antibodydependent enhancement has also been seen among sars-cov- -infected macaques injected with antispike igg ( ) . for sars-cov- and mers-cov, the receptor-binding domain (rbd) of the s protein was shown to be the major immunodominant region. subunit vaccines targeting rbd specifically elicited high nab titers but were not associated with immune enhancement ( , ) . in sars-cov- -infected patients, among the binding antibodies to the different regions of the s protein (s , s , rbd), rbd-specific igg correlated best with nabs, suggesting that rbd is be a promising target for sars-cov- vaccine candidates ( ) . however, because rbd is the most variable region of the genome ( ) , there is still a theoretical risk for immunologic "escape," as well as immune enhancement development ( ) . the n protein, a more conserved region of the genome, has been of interest for sars-cov- and mers-cov vaccine candidates and was thought to be at lower risk for immune enhancement; however, it was not shown to elicit nabs ( ) . the role of the n protein in sars-cov- immune response is still unknown. in conclusion, the covid- pandemic has revealed several gaps in our diagnostic arsenal and is highlighting the essential role of serodiagnostics as part of our public health response. with the use of carefully validated assays, appropriately designed serologic studies will help characterize transmission dynamics and refine disease burden estimates. urgent scientific research 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review date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: wuttqw egg yolk constitutes a relevant alternative source of antibodies. it presents some advantages over mammalian serum immunoglobulins regarding productivity, animal welfare and specificity. the main immunoglobulin present in avian blood (igy) is transmitted to their offspring and accumulates in egg yolks, which enables the non-invasive harvesting of high amounts of antibodies. moreover, due to structural differences and phylogenetic distance, igy is more suitable for diagnostic purposes than mammalian antibodies, since it does not react with certain components of the human immune system and displays greater avidity for mammalian conserved proteins. igy has been extensively used in health researches, as both therapeutic and diagnostic tool. this article aims to review its applications in both human and veterinary health. antibodies are protein molecules produced in response to an antigen. due to their ability to bind to specific targets, they are widely used in research, diagnosis and therapy. most of the currently available antibodies are produced in mammals, especially in small rodents [ ] . however, the production of antibodies in mammals can be challenging due to the fact that some antigens elicit weak immune responses or are even completely non-immunogenic. moreover, the production of antibodies in mammals involves procedures that cause pain and distress to animals; such as immunization, blood sample collection and sacrifice [ ] . the search for more efficient and economical techniques, as well as for the reduction and refinement of the use of animals, has led to growing interest for egg yolk antibodies (igy). obtaining antibodies from egg yolk is a non-invasive method that eliminates the need for blood collection. hens produce a greater amount of antibodies compared to other animals, like rodents for example, considerably reducing the number of animals required for the production of antibodies [ , ] . this method presents several economical advantages over the use of other animals. for example, the cost of maintaining a hen is less expensive than maintaining animals like mice and rabbits; the amount of antibodies produced by chickens also corresponds to that of larger animals, such as goats and sheep. for instance, in one year, a hen lays about eggs and produces an average of , g of igy [ ] . moreover, the specific igy produced in chickens are about - % of the total amount of antibodies [ ] . nonetheless, the amount of antibodies produced is correlated to the quantity of antigen applied to the hen, its immunogenicity and molecular weight [ , ] . being a productive technique, as well as more refined from the point of view of animal welfare, igy technology has been used for several purposes in human and veterinary health, such as in immunodiagnostics [ ] , immunotherapy [ ] , neutralization of toxins from venomous animals [ ] and bacteria [ ] , and as functional food [ ] . considering the fast development of igy technology, this work aims to review its applications in human and animal health, in addition to increasing the potential use of these antibodies among researchers and consequently promoting the reduced use of non-invasive procedures on animals. the specific protective effect of egg yolk extracts from immunized hens, attributed to the transfer of serum chicken antibodies to eggs, was first described in [ ] . however, this knowledge remained without applications until it attracted the interest of the scientific community due to the search for animal welfare, which was driven by the works of russel & burch and the publication of the "principles of humane experimental technique" in . the use of igy increased in the s, possibly due to the development of commercially available secondary reagents, such as igy purification kits and anti-igy specific antibodies conjugated to alkaline phosphatase, fluorescein isothiocyanate and peroxidase markers [ ] . in , the european center for the validation of alternative methods (ecvam) workshop recommended the use of igy rather than mammalian igg, with the purpose of minimizing the pain generated by invasive collection of serum antibodies [ ] . igy is present in birds, reptiles, amphibians and lungfish and is the evolutionary precursor of igg and ige, present only in mammals [ ] . over time, igy was called igg due to the supposed similarity between the two. however, leslie & clem emphasized the distinct differences between igy and igg, such as antigenic differences and the major size of igy heavy chain, suggesting the use of this term instead of igg [ ] . the igy molecule has a structure similar to that of igg, with two heavy chains (h), each one with a molecular weight of to kda, and two light chains (l), with kda. the light chains have one constant region (cl) and one variable region (vl), similar to igg. the major difference between igy and igg is found at the heavy chains. igg has three constant regions at the heavy chains (ch -ch ), while igy has four constant regions (ch -ch ) (fig. ) . a further constant domain, with the corresponding carbohydrate chains, gives igy a higher molecular weight ( kda) in comparison to igg ( kda) [ ] . igy is less flexible than igg due to the absence of the hinge region between ch and ch [ ] , similar, in this aspect, to ige [ , ] . the presence of glycine and proline residues at ch -ch and ch -ch regions also limits igy flexibility [ ] . one advantage of this higher chain stiffness is that it may be associated with increased igy resistance to proteolytic degradation and fragmentation [ ] . also, igy is more hydrophobic than igg and has an isoelectric point between . and . . the half-life of purified igy is of months, retaining activity for up to six months at room temperature and one month at °c. moreover, affinity-purified and biotinylated igy retained high activity after five years of storage at °c [ ] . the structural and phylogenetic differences between igy and igg are manifested in different molecular and biochemical interactions. the fc portion of igy is unable to activate the human complement [ ] and to bind to rheumatoid factor [ ] and to protein g [ ] , it also does not react with human anti-mouse antibodies (hama) [ ] nor with the erythrocyte agglutinogens a and b [ ] . due to phylogenetic distance, chickens produce a stronger immune response against conserved mammalian proteins [ ] . although igy contains the variable (v), junction (j) and diversity (d) regions, the contribution of the v (d) j rearrangement to its immunogenetic diversity is small, due to the fact that only a single locus undergoes rearrangement [ ] . thus, the genetic diversity of igy is guaranteed by gene hyperconversion, a process in which pseudogenes donate homologous sequences to the functional gene (v) after v (d) j rearrangement [ ] . even though it is a protein molecule, igy is resistant to heat and ph, being stable between °and °c and active between ph . and . however, the affinity of igy to its antigen decreases with increasing temperature. nonetheless, the addition of sucrose, maltose and glycine protects igy from heat denaturation [ ] , while sorbitol stabilizes it at acid ph [ ] . igy is resistant to inactivation by the proteolytic enzymes trypsin and chymotrypsin, but is degraded by pepsin [ ] . the digestion of igy with papain leads to a fc fragment plus two monovalent fab fragments, like igg. nonetheless, igy digestion with pepsin leads to a pfc′ fragment and two monovalent fab fragments, unlike mammalian antibodies, which forms one bivalent fab fragment when cleaved by pepsin [ ] . the characteristics of igy, in comparison to igg, are summarized in table . several methods have been developed to protect igy from degradation by ph and pepsin present in the stomach, allowing it to arrive unscathed at the small intestine, which is especially important for the use of igy against enteric pathogens. among these treatments, chitosan and alginate microcapsules [ ] , β-cyclodextrin microcapsules [ , ] , copolymers of methacrylic acid [ ] , liposomes [ , ] , multiple emulsification [ ] , hydrogel [ ] and carbon nanotubes with hydrogel [ ] have been tested with different degrees of success. nonetheless, unprotected igy consumed as egg yolk in liquid [ ] and powder forms [ ] showed resistance to the gastrointestinal tract conditions in calves. the antibody titers are influenced by several factors, such as the antigen type and dose, the used adjuvant, the route of application, the inoculation frequency, age and stage of development in birds [ , ] . several protocols for obtaining igy, observing these different variables, have been applied [ , ] . basically, multiple immunization protocols were tested for different antigens and animals, in order to obtain the highest antibody titer for each case [ ] . several antigen types have been used to produce specific igy in birds, such as complex antigens (virus, bacteria and parasites) and single antigens (proteins, polysaccharides, peptides and nucleic acids) [ ] . different antigen concentrations can also be combined with adjuvant. however, it is common to use to μg of antigen per ml injected in two or three sites, with the age of hen ranging between seven and eight weeks [ ] . the induction of high antibodies titers depends on the use of adjuvants. though freund's complete adjuvant (fca) is the most potent to induce antibodies in laboratory animals, it can lead to severe inflammation at the injection site [ ] . although some studies have shown that the immunization with fca is more tolerated by birds than by mammals and seems to not generate tissue damage in chickens [ , ] , other studies using fca for bird immunization contradict these results [ , ] . since there is no consensus, one of the best substitutes for fca is freund's incomplete adjuvant (fia), which, unlike fca, does not contain mycobacteria. shade et al. recommended the use of fia, which can be used since the first immunization without any prejudice to the antibody titer [ ] . in spite of this, the first inoculation is generally performed using fca, while the subsequent inoculations are performed using fia, without the occurrence of inflammation at the injection site [ , ] . the most common route of antigen administration for igy production in chickens is the intramuscular route, where the inoculation is usually into the breast muscle. this technique is most suited for young hens, because subcutaneous injection into the neck is more difficult to perform and may cause more distress to the animal. the intramuscular injection into the leg must be avoided as it can lead to lameness [ ] . nonetheless, for a non-invasive method, the administration of antigen can also be performed orally [ ] . the number of inoculations required depends on the type and dose of the antigen, as well as the adjuvant used. at least two inoculations must be performed before the laying period, with an interval of four to six weeks. the titer of igy must be assessed days after the last immunization. if the antibody titer decreases, further immunizations must be done during the entire laying period to increase antibody titers year round [ ] . an increase in titer of egg yolk antibodies can be observed from the second week [ , ] or sometimes from the fifth week post antigen inoculation [ ] . after a steady increase, stabilization of the antibody titer occurs, reaching a plateau, and from there on it decreases progressively [ ] . through booster inoculations, it is possible to keep high titers of egg yolk antibodies for more than days [ ] . the igy extraction consists of the removal of lipids to form a water soluble fraction (wsf), followed by the precipitation of the antibodies contained therein. several extraction methods are available for obtaining igy from egg yolk and the choice of the suitable method depends on the purpose, which can require different purification degrees, as well as the extraction scale, cost and available technology [ ] . a frequently used method for the extraction of igy is the precipitation with polyethylene glycol (peg) . . the method consists on the delipidation by centrifuging egg yolk diluted in phosphate buffered saline (pbs) with . % of peg . next, the supernatant is twice centrifuged with % of peg to precipitate the antibodies [ ] . akita & nakai obtained highly purified igy by removing the lipids from the yolks by means of six-fold dilution in water at ph . , with an incubation time of h at °c, followed by igy precipitation with % of ammonium sulfate (nh ) so , which was supplemented by the addition of sodium chloride (nacl) or by ultrafiltration prior to gel filtration or ion exchange chromatography. the authors also achieved highly purified igy by means of ethanol precipitation at lower temperatures [ ] . nonetheless, a study conducted by araújo et al. showed that the most suited concentration of (nh ) so for igy's precipitation is %. in this study, higher concentrations of (nh ) so also caused the precipitation of non-specific proteins, while a lower concentration led to a greater loss of antibodies in the supernatant [ ] . since the fc region of igy does not bind to proteins a and g, which are commonly used in the affinity purification of mammalian igg, affinity chromatographic methods for igy purification requires other types of ligands. igy can be purified through the adsorption of the immunizing antigen to the solid phase of the affinity column, which generates monospecific antibodies that can be eluted under acid [ ] or alkaline conditions [ ] . the purification of igy can also be performed through thiophilic adsorption chromatography [ ] . in contrast to other affinity purification methods, it can be performed at mild conditions [ ] . hens are able to produce between and mg of igy antibodies by yolk, of which between and % are specific [ ] . in one year, a single laying hen produces between and g of total igy [ ] . although the production of igy for research is mainly done in chicken, other birds are also used for the same purpose, such as goose [ ] and quail [ ] , using an immunization protocol similar to that used for chickens. the production of igy and its possible applications in both human and veterinary health are summarized in fig. . the use of polyclonal igy against infectious diseases minimizes the risk of microbial resistance, since the antibody is directed to various antigens of the same microorganism, which requires multiple genes for its synthesis [ ] . therefore, specific igy antibodies are a relevant alternative to use as antimicrobials in human and veterinary health in the face of the emergence of resistant bacteria. igy antibacterial activity can be assessed by the inhibition of bacterial growth and biofilm formation in vitro, as well as adhesion ability, since they are prior conditions for successful colonization of a higher animal by bacteria and viruses [ ] . igy antibacterial activity against gastrointestinal pathogens has been widely investigated. nasiri et al. extracted igy from hens immunized with the recombinant protein fanc, from enterotoxigenic escherichia coli (etec) and these antibodies bound specifically to fanc in elisa, western blot and dot-blotting [ ] , demanding, thus, more investigations to evaluate its viability as a potential immunotherapeutic compound. li et al. evaluated the effect of specific and non-specific igy against salmonella typhimurium on the immune response of mice infected by the bacterium [ ] . specific igy reduced the expression of the pro-inflammatory cytokines tnf-α and inf-γ and elevated that of the antiinflammatory cytokine il- ; while non-specific igy also reduced the level of tnf-α, but did not alter the expression level of inf-γ and il- . although both specific and non-specific antibodies reduced the number of t lymphocytes in the lamina propria and of cd + , cd + and γδ t lymphocytes in the jejunum of mice, the animals treated with anti-s. typhimurium igy had a longer survival rate than those treated with nonspecific igy. igy activity against bacterial spores was also investigated. oral administration of igy against clostridium difficile spores both delayed the onset of diarrhea in rats treated prior to the infection and reduced its recurrence in infected rats. thus, specific igy could be used in the prophylaxis of c. difficile infection, as well as an immunotherapeutic to eliminate the remaining spores on the intestines of people who have already had the infection, which would reduce the recurrence of diarrhea [ ] . the therapeutic action of igy against helicobacter pylori has been largely studied as well, through antibodies directed to both the whole cell and to specific proteins. oral administration of anti-hpuc (h. pylori urease) igy inhibited the growth of h. pylori in vitro and reduced the inflammation of stomach in mice, with a significant reduction of neutrophils and lymphocytes infiltration on the tissue. a decrease in the serum level of antibodies against h. pylori was also observed in treated animals [ ] . similar results, as the reduction of serum levels of anti-h. pylori igg and the absence of inflammation signs on the stomach were observed in mice that received oral igy against the h. pylori vacuolating cytokine a (vaca) [ ] . specific igy against hp-nap protein, the main virulence factor of h. pylori, significantly inhibited the adhesion of the bacterium to an ags cells culture [ ] . solhi et al. evaluated the occurrence of cross reaction between strain-specific igy and four different h. pylori strains. the antibodies inhibited the cell growth and the urease enzyme not only of the strains for which they were specific, but also of other strains [ ] . the therapeutic potential of igy against bacterial respiratory tract infections has also been considered. igy against mycobacterium tuberculosis increased the proliferation of pbmc (peripheral blood mononuclear cells) in rats in a dose-dependent manner. an increased expression of cytokines that stimulate the cellular immune response was observed and an increase of mrna level of il- and ifn-γ was detected by rt-pcr, which indicates that anti-m. tuberculosis igy is a potential immunotherapeutic that stimulates t-helper immune response [ ] . shi et al. produced specific igy against multi-drug resistant strains of acinetobacter baumannii. the antibodies inhibited bacterial growth in vitro, significantly reduced the mortality of mice infected with the bacterium and attenuated the lung inflammation [ ] . igy has also been tested as a potential therapeutic resource in oral infections against prevotella intermedia. specific igy inhibited bacterial growth in liquid medium and showed therapeutic activity in rats with p. intermedia induced gingivitis. the reduction of gingival inflammation, bacterial plaque and bleeding, as well as the normalization of wbc (white blood cells) levels in the blood, were observed in treated animals. histopathological examination showed no signs of inflammation in the gums of rats treated with anti-p. intermedia igy [ ] . additionally, studies have shown inhibitory growth effect of therapeutic igy produced against fusobacterium nucleatum in liquid medium and in the formation of biofilm in polyestyrene plates. anti-f. nucleatum igy also reduced bone loss in mice with periodontal disease when applied after the infection, thus presenting therapeutic function [ ] . the therapeutic effect of igy against acne was also evaluated. revathy et al. produced and assessed the efficiency of specific igy against the bacterium propionibacterium acnes. the antibody inhibited bacterial growth and biofilm formation in vitro, proving to be a possible alternative to antibiotics in the treatment of acne [ ] . more recently, igy was tested for antibacterial effect in aquatic animals. gao et al. produced igy against vibrio spp., the major cause of death in white shrimp (litopenaeus vannamei), and demonstrated that powder egg yolk containing anti-vibrio igy significantly reduced the mortality of white shrimp infected with v. harveyi and v. parahaemolyticus [ ] , which indicates that therapeutic igy can also be applied in aquafarming. the prophylactic potential of igy against dental caries has been widely studied. an interesting study carried by bachtiar et al. evaluated the effect of soybean milk containing igy against the cariogenic bacterium streptococcus mutans in rats. the animals fed with this milk presented a decrease in the number of s. mutans in dental biofilm and fifteen days after milk intake igy could still be detected in the saliva [ ] . in other research, the effect of specific igy against the cell-associated glucosyltransferase enzyme (ca-gtf), of s. mutans, was evaluated. as a result, anti-ca-gtf igy inhibited bacterial adhesion in vitro and suppressed the oral cavity colonization [ ] . in addition, specific igy action against the comd protein, a quorumsensing signals receptor of s. mutans, was also explored. it inhibited, in vitro, the development of biofilm of s. mutans from the oral cavity of people with and without caries, and changes in the protein expression pattern were found in the bacteria treated with anti-comd igy [ ] . the protective effect of igy against opportunistic infections was also investigated by thomsen et al., where, by means of chemiluminescence, the production of reactive oxygen species (ros) in polymorphonuclear neutrophils (pmn) was evaluated as an indicator of the intensity of pmn mediated immune response against pseudomonas aeruginosas in vitro, as well as the influence of anti-p. aeruginosas igy in this process. the specific igy opsonized the bacterium and intensified the cellular immune response, probably due to the recognition of igy by receptors resembling avian igy receptors or physical-chemical changes in the bacterium, since igy does not activate mammalian fc receptors. anti-p. aeruginosas igy could be used in the prophylaxis of p. aeruginosas infection in patients with cystic fibrosis by increasing the cellular immune response [ ] . prophylaxis of bacterial infections in animals for consumption, using igy, was also considered. specific igy incorporated into hydrogel added to carbon nanotubes and chitosan (h-cnt) showed protective activity against enterotoxigenic escherichia coli (etec) in piglets [ ] . thus, anti-etec igy incorporated into h-cnt and orally applied could be used to prevent the intestinal infection in these animals. in order to generate antibodies against the bacterium campylobacter jejuni to be used as food additive aiming to control c. jejuni colonization in chickens, thibodeau et al. produced anti-c. jejuni igy in three different ways: oral administration of a pool of living bacteria from four c. jejuni strains, subcutaneous injection of extracts from the outer membrane of the bacterium or of formalin inactivated bacterium. the hens submitted to subcutaneous inoculation produced igy more intensely, although the antibodies obtained through oral inoculation also showed good activity. anti-c. jejuni igy, obtained from the three ways, presented bactericidal activity in vitro [ ] . igy against the bacterium aeromonas salmonicida, the causative agent of ulcers on fish skin, was added, in powder form, to the water in which koi carp (cyprinus carpio koi) ornamental fish were raised. anti-a. salmonicida antibodies showed good prophylactic activity against the infection, and a small amount of specific igy was enough to completely prevent the appearance of skin ulcers in healthy fish that cohabited with sick fish previously infected with a. salmonicida [ ] . the potential of igy technology to prevent bacterial infections in animals could be useful in the needful strategies to reduce the use of antibiotics in food animal husbandry, which has been linked to the selection and spread of resistant bacterial strains that threaten antibiotics as a therapeutic resource [ , ] . the therapeutic potential of igy against viral infections of the gastrointestinal tract in animals has been extensively investigated. oral administration of egg yolk powder rich in igy against bovine group a rotavirus (rva) was performed on calves infected by the virus. as a result, disease attenuation occurred in treated animals, with a reduction of the period of diarrhea and hyperthermia and the absence of other symptoms observed in untreated calves, such as anorexia, dehydration and depression. the egg yolk powder enriched with anti-rva igy retained the activity after two years, when kept at °c [ ] . in another study, igy against the s protein of porcine epidemical diarrhea virus (pedv) was produced and orally administered in piglets previously infected with pedv. anti-s igy reduced the severity of diarrhea and intestinal lesions caused by pedv infection and nullified the mortality of piglets due to the disease [ ] . another possible therapeutic use of specific igy is against dengue fever, as demonstrated by fink et al. anti-denv igy produced in goose was able to neutralize the virus in vitro and in vivo without binding to fcγ receptors on myeloid cells and generating ade (antibody dependent enhancement) in mice [ ] . the protective effect of igy against influenza has been widely studied. igy against the avian influenza a virus (h n ) were extracted from eggs available in supermarkets of vietnam, where the vaccination of chickens against h n virus is required. anti-h n igy was administrated intranasally in mice before and after their infection with h n and h n and completely prevented the disease onset [ ] . these results reveal that commercially available eggs that are produced in countries where anti-h n vaccination is mandatory constitute a considerable source of igy that could be used to prevent a potential pandemic of h n virus. following this rationale, wallach et al. produced igy against h n , h n and h n strains of influenza virus, which were tested for their ability to neutralize homologous and heterologous strains in vitro and to prevent the infection in vivo. the antibodies inhibited only homologous strains in the seroneutralization and in the haemagglutination inhibition assay, except anti-h n igy, which also inhibited h n . anti-h n and anti-h n igy were applied intranasally in mice h before the infection by influenza virus and showed prophylactic activity. thus, igy against influenza virus strains could be used in nasal, oral or spray applications to protect individuals and environments [ ] . igy against influenza b virus (ibv) was also tested and neutralized the activity of haemagglutinins and neuraminidase present in the virus and inhibited viral replication in vitro. when applied intranasally, anti-ibv igy prevented influenza development in mice treated prior to virus exposure and attenuated the disease in those treated post-infection [ ] . the prophylactic effect of igy against other viral infections of the respiratory tract was also evaluated. igy against andes virus (andv), the causative agent of the hantavirus pulmonary syndrome (hps), was obtained from geese eggs by inoculating these animals with the dna encoding the virus envelope glycoprotein. anti-andv igy was able to prevent hps development in recently infected mice but failed when applied after the onset of viremia, thus presenting prophylactic and non-therapeutic activity [ ] . the protective effects of igy were also considered for viral infections in poultry. aizenshtein et al. produced, in the same eggs, efficient igy against the newcastle disease virus (ndv), infectious bursal disease virus (ibdv), influenza and reovirus, which are pathogenic for birds [ ] , demonstrating that passive immunization with igy against several viruses is possible; nonetheless, it is a technology that must be further explored. a gel preparation for oral use containing igy against candida albicans was tested by tekeuchi et al. and caused a reduction in the number of colony-forming units (cfu) on the oral cavity of elderly people, showing promise for prophylactic use against c. albicans oral infection [ ] . in other research, specific igy inhibited the adhesion of candida albicans and candida glabrata to denture base material. anti-c. albicans igy was more effective against c. albicans than anti-c. glabrata igy, while both antibodies were equally effective in preventing the adhesion of c. glabrata [ ] . sampaio et al. produced a high avidity igy against the protozoan trypanosoma evansi. there was neither prophylactic action nor infection control in the treated rats, but anti-t. evansi igy increased their survival rate when used concomitantly to anti-hematozoa drugs [ ] . in another study, grando et al. inoculated trypanosoma cruzi trypomastigots in hens and extracted a high amount of anti-t. cruzi igy that cross-reacted with t. evansi antigens in western blot, showing that the anti-t. cruzi igy is not reliable as a diagnostic tool, but deserves more investigations as a possible therapeutic resource for trypanosomes infection [ ] . the phylogenetic distance between birds and mammals ensures a stronger immune response against mammalian antigens by birds [ ] . such a feature may be advantageous to produce igy against human tumor antigens. following this rationale, amirijavidv et al. produced highly specific igy against a sequence of amino acids present on the ectodomain of the trail (tnf-related apoptosis-inducing ligand) receptor trail-r (dr ). the antibodies bound to the amino acid sequence and activated the dr receptors in human breast cancer cells mcf , acting as a trail agonist and inducing apoptosis [ ] . igy against other receptors, such as the her receptor, was tested coupled to single walled carbon nanotubes (swnts) and specifically detected the her receptors on the surface of sk-br- cells. the binding of the complex to the receptors was measured by raman signals emitted by the nanotubes. swnt has a near infrared absorption (nir), which can be used for tumor ablation, and, coupled to anti-her igy, was able to kill sk-br- cells without needing internalization of the complex by the cell [ ] . these findings show that igy produced against tumor antigens is an attractive alternative for a more selective treatment of cancers and its use could, therefore, minimize the side effects of traditional chemotherapy. igy raised against porcine pancreatic lipase was used against the enzyme in vitro and in vivo. later, mice with obesity induced by high fat diet were orally treated with the antibody, which was given concomitantly with food, and a reduction of adipose tissue and liver fat level was observed, as well as an increase of fecal excretion of triglycerides and their decrease in blood plasma. anti-lipase igy inhibited the hydrolysis of diet fat and reduced its intestinal absorption, showing anti-obesity activity [ ] . wei-xu et al. evaluated the antiallergic effect of specific igy against the pro-inflammatory cytokines il-β and tnf-α in guinea pigs with induced allergic rhinitis. a reduction of the eosinophils number in the blood and in the nasal and bronchial lavages was found, as well as a decrease of eosinophils, neutrophils and lymphocytes infiltration into the nasal mucosa and the lungs of animals treated with anti-il-β and anti-tnf-α igy, alone or jointly [ ] . one of the side effects that occur in individuals receiving antivenom serum produced in goats, sheep and horses is due to the presence of serum proteins on the anti-venom serum derived from these animals, in which igg is not sufficiently purified [ , ] . one advantage of using igy in anti-venom serotherapy is that it is easily purified, which would minimize the occurrence of side effects due to nonspecific proteins. araujo et al. demonstrated this property when specific igy was produced as anti-venom of the snake genus bothrops sp. these antibodies neutralized a pool of venoms from five bothrops species, with an ed of μl/ ld , showing little to no side effects in mice [ ] . mendonza et al. also produced igy capable of neutralizing the venom of the peruvian snake bothrops atox. the anti-venom igy showed considerable cross reaction with the venom of bothrops brazili and could be used not only as b. atox anti-venom, but also as a tool for the research of cross reaction with venoms from different species [ ] . in another elegant work, andrade et al. produced igy against a pool of venoms from snakes of the genus bothrops and against the venom from the species crotalus durissus terrificus. anti-venom igy extracted from eggs was compared to the horse anti-venom igg in western blot. the results showed that specific egg's igy recognized the same antigens as the equine anti-venom [ ] . igy against coral snake venom was first produced in response to a pool of venoms from different species of micrurus. these antibodies recognized, by western blot, venom proteins from several snakes: m. isozonus, m. surinamensis, m. f. fulvius, naja kaouthia, n. pallida, bothrops colombiensis, crotalus durissus cumanensis and c. vegrandis and could, therefore, be used as a broad-spectrum snake anti-venom [ ] . zolfagharian and dounighi produced igy by inoculating the vipera lebetina snake venom, inactivated by γ radiation, in hens [ ] . these antibodies were effective in neutralizing the crude venom of vipera lebetina in mice. anti-venom igy were also obtained from eggs of hens immunized with the venom of the snake trimeresurus albolabris. these igy recognized, by western blot, most of the proteins present in the t. albolabris venom and neutralized it in mice in a dose-dependent manner [ ] . more recently, liu et al. extracted and purified igy from eggs of hens inoculated with the venom of the deinagkistrodon actus snake. these antibodies were able to neutralize the lethal effects of the venom, such as bleeding, edema formation and myotoxicity in a dose-dependent manner [ ] . da rocha et al. produced igy against ophidian toxins of crotalus durissus terrificus, bothrops jararaca and bitis arietans. the antibodies were able to bind to specific components of the venoms in western blot and protected % of the intoxicated mice when obtained after the ninth inoculation. the authors recommended the use of a small antigen dose ( μl) applied in successive inoculations for igy production, since this dose was enough to genetically alter the v(d)j segments on the naïve cells and to generate immunological memory [ ] . however, igy raised against the venom of the snake oxyuranus scutellatus was less effective than equine igg, being unable to neutralize the neurotoxic and coagulant effects of the venom [ ] . nonetheless, this result cannot be extended to igy produced against other venoms. igy against the tityus caripitensis scorpion venom, produced by alvarez et al., neutralized not only the venom of t. caripitensis, but also that of other tityus species (t. quirogae, t. discrepans and t. gonzalespongai), and inactivated the hyaluronidase, an enzyme that facilitates the toxin spread in the tissues, present in the t. serrulatus venom [ ] . thus, igy raised against t. caripitensis venom could be used as a broadspectrum anti-scorpionic serum. another application of igy technology, the prophylaxis of celiac disease, was demonstrated by gujral et al., who developed powdered egg yolk formula with protective sugars containing anti-gliadin igy, among which, the formula with mannitol (eyp-m) retained its activity after being submitted, in vitro, to chemical conditions analogous to those of the stomach and small intestine. the formula igy-epy-m neutralized in vitro both the isolated gliadin and that present in food matrix and inhibited its intestinal absorption in mice, showing promising for the prevention of celiac disease [ ] . bobeck et al. used igy against the human intestinal alkaline phosphatase (hiap) to assess the influence of iap on increased bioavailability of phytate phosphate in the presence of α-dihydroxycholecalciferol (vitamin d ) in chickens. anti-hiap igy was ingested by chickens and reduced the absorption of phytate phosphate, which suggests that although it performed less adequately than sevelamer chorhydrate, already used for the same purpose, anti-hiap igy can be optimized for the prevention of phytate phosphate toxicity induced by the consumption of the active form of vitamin d [ ] . the ability of igy to detect viral pathogens of the gastrointestinal tract in humans and animals has been widely studied. igy raised against canine parvovirus viral like particles (cpv-vlps) were used in elisa and immunochromatography, showing sensitivity and specificity in the detection of canine parvovirus in dog fecal samples [ ] . specific igy against the e protein of bovine viral diarrhea virus (bvdv) were used in elisa to detect pathogens that cause diarrhea in cattle. anti-e igy showed a high specificity, recognizing only bvdv. elisa and immunochromatography tests using these antibodies were efficient in detecting bvdv in serum samples of cows with diarrhea, showing a concordance of , % and % with rt-pcr, respectively [ ] . da silva et al. used igy against hepatitis a virus (hav) in a competitive immunoenzymatic assay to detect anti-hav igg in serum samples, showing satisfactory sensitivity and specificity [ ] . more recently, igy against hav was used to detect the virus in hepatic sections of infected rhesus monkeys by means of indirect immunofluorescence (iif). anti-hva igy was more efficient than the commercially available igg for the detection of the same antigen [ ] . igy was also used to detect viral pathogens in aquatic animals. specific igy against the soft-shelled turtle systemic septicemia spherical virus (stsssv) was used to compose a lateral flow assay to detect the virus in turtles. this assay was sensitive and specific, detecting stsssv in all infected turtles in serum and feces samples [ ] . a potential for diagnosis was presented by igy raised against the nucleocapsid protein (np) of coronavirus (cov). anti-np igy, used as capture antibody in elisa for detecting np, lowered its detection limit to a picogram level, which indicates that the antibody is promising for use in the diagnosis of acute respiratory syndrome associated to coronavirus (sars-cov) and is sensitive enough to detect small amounts of np [ ] . moreover, the ability of igy in diagnosing dengue fever was also evaluated. figueiredo et al. produced igy against the non-structural protein (ns ) of dengue virus (denv ). these antibodies were used to compose an immunosensor that was effective in electrically detecting the ns protein of denv in standard samples and could be used for dengue diagnosis in biological samples [ ] . one of the most studied diagnostic potential of igy is against staphylococcus aureus. the fact that the fc portion of igg reacts with the staphylococcal protein a makes igy a relevant resource for more specific detection of different s. aureus strains and their toxins, since, due to structural differences, the fc portion of igy does not react with protein a [ ] . following this rationale, walczak et al. produced igy against the fibrinogen binding protein (efb) of staphylococcus aureus and against a peptide epitope that encompasses the residues - of efb protein. anti-efb and anti-efb - antibodies presented high titers in elisa and strong avidity in western blot, showing promising use in the diagnosis of s. aureus infection [ ] . another elisa test using igy against the staphylococcal enterotoxin b (seb) of s. aureus as capture antibody and specific ssdna aptamers coupled to biotin as revealing was developed by mulidi et al. this assay was efficient in detecting seb, but also reacted with others staphylococcal toxins, such as staphylococcal endotoxins a (sea) and c (sec), toxic shock syndrome toxin (tsst) and α-hemolysin [ ] . igy raised against α-hemolysin was applied as capture antibody in elisa for detecting the toxin in the supernatant of s. aureus cultures. anti-αha igy showed high specificity against the toxin, without reacting with protein a, which is present in all s. aureus strains [ ] . the ability of igy to diagnose resistant s. aureus was also investigated. yamada et al. produced igy against the penicillin binding protein (pbp) a, which is present in methicillin resistant s. aureus (mrsa) strains. anti-pbp a igy was used in elisa, lateral flow and latex to detect mrsa and other s. aureus strains sensitive to methicillin and beta-lactams. the antibody was mrsa specific and did not react with the sensitive strains that express significant amounts of protein a [ ] . among the parasitic infections tested, cakir-koc evaluated the potential of igy in detecting the protozoan toxoplasma gondii by producing igy against its surface protein sag- , which reacted with the target antigen in elisa and western blot [ ] . therefore, this study revealed a potential diagnostic test for toxoplasmosis. more recently, anti-sag igy conjugated to fluorescein isothiocyanate detected t. gondii tachyzoites in a colony, by means of immunofluorescence, and may be used to detect the protozoan in other types of samples [ ] . in other interesting research, an indirect elisa using igy to detect cathepsin f (cf), a cysteine protease present in the helminth opisthorchirs viverrini, was developed. this assay showed good sensitivity in the detection of o. viverrini in fecal samples from humans living in endemics places; however, a cross reaction with taenia spp., echinostoma spp. and minute intestinal fluke (mif) was observed [ ] . therefore, improvements still have to be made before this technology is available. miura et al. produced igy against the gp protein, from the cryptosporidium hominis protozoan. these igy bound to gp in western blot and to c. parvum sporozoites in fecal samples by indirect immunofluorescence, suggesting that anti-gp igy could be used in the diagnosis of cryptosporidiosis caused by both c. hominis and c. parvum [ ] . several authors have been investigating the potential of igy in the detection of tumor markers. igy against the peptide antigen ca - , a commonly used breast cancer marker, was used as secondary antibody in a sandwich elisa aiming to detect ca - , showing potential for clinical use [ ] . sun et al. produced igy against two portions of the transmembrane glycoprotein her : her -a, proximal region, and her -b, distal region. anti-her -a and anti-her -b igy effectively detected the her glycoprotein in cultured breast cancer sk-br- cells by immunofluorescence and in sections of breast tumors over expressing her -b, using immunohistochemistry. in addition, the antibodies bound to the glycoprotein in elisa and western blot, which indicates that igy against her is promising for use in breast cancer diagnosis [ ] . in another study, Łupicka-słowik et al. developed a direct elisa test using a lysate of human malignant tumor cells and igy against bovine adenosine deaminase (c-ada). the assay was efficient in detecting human adenosine deaminase (h-ada) present in the tumor cells lysate, which was possible due to the high homology between c-ada and h-ada. the elisa test using anti-c-ada igy could be used to diagnose several types of malignant tumors in humans, as well as be an alternative to currently employed enzymatic methods for the detection and quantification of ada for pleural tuberculosis diagnosis [ ] . another igy developed to detect prostatic specific antigen (psa) and two peptide fragments of this protein demonstrated specificity and marked the antigen more strongly than the igg counterpart using western blot analysis. however, anti-psa igy had an unsatisfactory sensitivity when applied as secondary antibody in indirect elisa [ ] , thus deserving more investigation. in general, these findings suggest that the phylogenetic distance between birds and mammals, that ensures a stronger immune response against mammalian antigens by birds than by other mammals [ ] , makes the production of igy against tumor antigens advantageous not only for therapeutic purposes, as described above, but also for usage in several types of immunoassays for tumor detection in humans. hens immunized with umbilical cord sera produced specific igy against igg and the complement fractions c b and c d. these antibodies did not react with the c b fraction -which configures a higher specificity, since anti-c b antibodies often cross-reacts with the antigens of chido/rodgers rbc group -nor with erythrocyte antigens from abo group. these antibodies are, therefore, promising as a reagent for coombs test [ ] . igy immunoassays were used to detect the hepatic expression of cytochrome p e (cyp e ) in mice treated with medicinal herbs and products derived from plants rich in flavonoids. cyp e metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. anti-cyp e igy was specific, without reacting with other cytochromes, and was able to detect the reduction of hepatic cype e expression due to the ingestion of natural products with hepatoprotective effects [ ] . the ability of igy in identifying harmful substances in consumer products has been evaluated. an elisa test was developed to detect the staphylococcal enterotoxin g (seg), using specific igy, and showed satisfactory sensitivity and specificity, reducing the interference of protein a that occurs in igg tests. this test was successfully used to detect seg in milk and dairy products samples and could therefore be used to identify the toxin in food [ ] . bittner et al. used igy in elisa to detect potentially allergenic proteins in commercially available latex gloves. this assay presented similar results to that of the gold standard test, which uses mammalian igg [ ] . igy can also be used in assays to identify antibiotic residues in food products of animal origin, as demonstrated in a study by he et al., in which produced anti-gentamycin igy specifically detected the target antibiotic present in animal origin products [ ] . following this rationale, li et al. used specific igy to detect kanamycin and gentamycin residues in milk and meat samples by means of competition elisa and fpia (fluorescence polarization immunoassay) [ ] . the potential of igy in identifying substances has also been used to evaluate the toxicity of a natural product employed in the alternative medicine. igy labeled with quantum dot were successfully used in a lateral flow assay for the detection of rhein; a toxic substance found in the plant rheum officinale, widely used in chinese traditional medicine; in plant samples and serum from users [ ] . igy raised against the bacterium listeria monocytogenes showed a significant inhibitory effect of bacterial growth in liquid medium and in fish samples stored between and °c in a dose-dependent manner, which indicates that anti-l. monocytogenes igy is a potential antimicrobial for use in the food industry [ ] . taking into consideration the versatility and the range of igy already tested against several bacteria, this result could be easily apply to other food poisoning bacteria and viruses. among the importance of this technology, leclair et al. demonstrated that igy produced against the staphylococcal enterotoxin b (seb), a potential biological weapon, can save individuals exposed to this material. the results with rhesus monkeys showed that animals that received anti-seb igy min before or h after exposure to a lethal seb aerosol survived [ ] , which indicates that anti-seb igy could be used to protect populations in a hypothetical context of bioterrorism involving seb [ ] . the latest findings using igy have clearly demonstrated the versatility of this technology. obtaining igy from birds presents several technical and economical advantages over mammalian igg, and as described in this review, igy technology has a broad spectrum of applications in human and veterinary health. it can be used in multiple types of therapies; it is useful in the prevention of various types of diseases and detects, by means of different techniques, several classes of antigens, such as microorganisms, tumor markers and substances. among the advantages of this technology, the replacement of invasive antibody collection by its extraction from eggs is one of the most interesting, considering the animal welfare benefits, with this technology it is possible to achieve great quantities of antibodies with a lower cost of production and less damage to animal welfare. due to its structural differences and phylogenetic distance, igy is more specific for diagnostic use and displays greater avidity for mammalian conserved proteins than igg, being, thus, an important alternative in the search for more effective diagnostics and therapies. in addition, in view of its proven ability to neutralize microorganisms, igy represents an important therapeutic resource in times of increasing resistance to antibiotics and emergence of viral diseases for which there is no treatment. there are no funding or competing interests to report. chicken egg yolk antibodies (igy) as an alternative to mammalian antibodies production of antibodies in chickens the production of avian (egg yolk) antibodies: igy. the report and recommendations of ecvam workshop igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation avian antibodies (igy) against trypanosoma cruzi: purification and characterization studies antibodies to proteins from yolk of immunized hens chicken egg yolk antibodies (igy) for detecting circulating antigens of schistosoma japonicum oral passive igybased immunotherapeutics: a novel solution for 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antibody against h. pylori, microbial pathog koerniasari, the activity of immunoglobulin y anti-mycobacterium tuberculosis on proliferation and cytokine expression of rat peripheral blood mononuclear cells effects of specific egg yolk immunoglobulin on pan-drug-resistant acinetobacter baumannii protective effect of an egg yolk-derived immunoglobulin (igy) against prevotella intermedia-mediated gingivitis effectiveness of egg yolk immunoglobulin (igy) against periodontal diseasecausing fusobacterium nucleatum in vitro evaluation of the efficacy of chicken egg yolk antibodies (igy) generated against propionibacterium acnes effects of soybean milk, chitosan, and anti-streptococcus mutans igy in malnourished rats' dental biofilm and the igy persistencyin saliva anticell-associated glucosyltransferase immunoglobulin y suppression of salivary mutans streptococci in healthy young adults anti-pseudomonas aeruginosa igy antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils igy against enterotoxigenic escherichia coli administered by hidrogel-carbon nanotubes composites to prevent neonatal diarrhoea in experimentally challenged piglets ulcer disease prophylaxis in koi carp by bath immersion with chicken egg yolk containing anti-aeromonas salmonicida igy medical consequences of antibiotic use in agriculture industrial food animal production, antimicrobial resistance, and human health immunoprophylactic effect of chicken egg yolk antibody (igy) against a recombinant s domain of the porcine epidemic diarrhea vírus spike protein in piglets prophylactic and therapeutic efficacy of avian antibodies against influenza virus h n and h n in mice cross-protection of chicken immunoglobulin y antibodies against h n and h n viruses passively administered in mice antiviral biologic produced in dna vaccine/goose platform protects hamsters against hantavirus pulmonary syndrome when administered post-exposure practical aspects in the use of passive immunization as an alternative to attenuated viral vacines effects of oral moisturising gel containing egg yolk antibodies against candida albicans in older people use of candida-specific chicken egg yolk antibodies to inhibit the adhering of candida to denture base materials: prevention of denture stomatitis production, purification and therapeutic potential of egg yolk antibodies for treating trypanosoma evansi infection apoptotic killing of breast cancer cells by igys produced against a small aminoacid epitope of the human trail- receptor, asian pac anti-her igy antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells antiobesity activity of hen egg anti-lipase immunoglobulin yolk, a novel pancreatic lipase inhibitor anti-interleukin- beta/tumor necrosis factor-alpha igy antibodies reduce pathological allergic responses in guinea pigs with allergic rhinitis a comparison of ovine and equine antivenoms the production and characterization of antibothropic and anti-crotalic igy antibodies in laying hens: a long term experiment coral snake antivenom produced in chickens (gallus domesticus) study on development of vipera lebetina snake anti-venom in chicken egg yolk for passive immunization anti-trimeresurus albolabris venom igy antibodies: preparation, purification and neutralization efficacy preparation and neutralization efficacy of igy antibodies raised against deinagkistrodon acutus venom development of igy antibodies against anti-snake toxins endowed with highly lethal neutralizing activity development of a chicken-derived antivenom against the taipan snake (oxyuranus scutellatus) venom and comparison with an equine antivenom antibodies anti-tityus caripitensis venom: purification and neutralization efficacy in-vitro and in-vivo binding activity of chicken egg yolk immunoglobulin y (igy) against gliadin in food matrix oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary α-hydroxycholecalciferol evaluation of chicken igy generated against canine parvovirus viral-like particles and development of enzyme-linked immunosorbent assay and immunochromatographic assay for canine parvovirus detection preparation of chicken igy against recombinant e protein of bovine viral diarrhea virus (bvdv) and development of elisa and ica for bvdv detection an immunoenzymatic assay for the diagnosis of hepatitis a utilising immunoglobulin y using immunoglobulin y as an alternative antibody for the detection of hepatitis a virus in frozen liver sections development of a colloidal gold immunochromatographic strip for the rapid detection of soft-shelled turtle systemic septicemia spherical vírus diagnostics of severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid antigen using chicken immunoglobulin y electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element the binding of staphylococcal protein a by the sera of different animal species method for generation of peptide-specific igy antibodies directed to staphylococcus aureus extracellular fibrinogen binding protein epitope a novel igy-aptamer hybrid system for cost-effective detection of seb and its evaluation on food and clinical samples detection of methicillin-resistant staphylococcus aureus using a specific anti-pbp a chicken igy antibody production of anti-sag- igy antibody against toxoplasma gondii parasites and evaluation of antibody activity by elisa method novel fitc-labeled igy antibody: fluorescence imaging toxoplasma gondii in vitro chicken igy-based coproantigen capture elisa for diagnosis of human opisthorchiasis evaluation of recombinant cryptosporidium hominis gp protein and anti-gp chicken polyclonal igy for research and diagnostic purposes highly sensitive detection of cancer antigen human epidermal growth factor receptor using novel chicken egg yolk immunoglobulin development of adenosine deaminase-specific igy antibodies: diagnostic and inhibitory application evaluation of igy antibody as a polyspecific coombs-reagent development of an igy antibody-based immunoassay for the screening of the cyp e inhibitor/enhancer from herbal medicines development of igy based sandwich elisa for the detection of staphylococcal enterotoxin g (seg), an egc toxin content of asthmagen natural rubber latex allergens in commercial disposable gloves development of indirect competitive elisa using egg yolk-derived immunoglobulin (igy) for the detection of gentamicin residues detection of kanamycin and gentamicin residues in animal-derived food using igy antibody based ic-elisa and fpia quantum dot-based lateral-flow immunoassay for rapid detection of rhein using specific egg yolk antibodies key: cord- -oluq v h authors: oliphant, theodore; engle, michael; nybakken, grant e; doane, chris; johnson, syd; huang, ling; gorlatov, sergey; mehlhop, erin; marri, anantha; chung, kyung min; ebel, gregory d; kramer, laura d; fremont, daved h; diamond, michael s title: development of a humanized monoclonal antibody with therapeutic potential against west nile virus date: - - journal: nat med doi: . /nm sha: doc_id: cord_uid: oluq v h neutralization of west nile virus (wnv) in vivo correlates with the development of an antibody response against the viral envelope (e) protein. using random mutagenesis and yeast surface display, we defined individual contact residues of newly generated monoclonal antibodies against domain iii of the wnv e protein. monoclonal antibodies that strongly neutralized wnv localized to a surface patch on the lateral face of domain iii. convalescent antibodies from individuals who had recovered from wnv infection also detected this epitope. one monoclonal antibody, e , neutralized different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose d after infection. a humanized version of e was generated that retained antigen specificity, avidity and neutralizing activity. in postexposure therapeutic trials in mice, a single dose of humanized e protected mice against wnv-induced mortality, and may therefore be a viable treatment option against wnv infection in humans. supplementary information: the online version of this article (doi: . /nm ) contains supplementary material, which is available to authorized users. development of a humanized monoclonal antibody with therapeutic potential against west nile virus e protein by nickel-affinity chromatography (data not shown). after immunization and screening , hybridomas, we isolated new monoclonal antibodies that recognized wnv e protein (supplementary table online). we evaluated the antibodies for their ability to block wnv infection in bhk cells using a standard plaque-reduction assay . twelve had strong neutralizing activity that greatly exceeded the potency of immune human γ-globulin, with % plaque reduction neutralization titers (prnt ) below µg, whereas immune human γ-globulin had a prnt value of µg . the inhibitory activity of two neutralizing antibodies, e and e , was reproduced in j . mouse macrophages and sw human adrenal carcinoma cells ( supplementary fig. online) and thus was not specific to fibroblasts. one of the potent neutralizing monoclonal antibodies, e , inhibited infection of genetically diverse wnv lineage i strains that were isolated from mosquitoes, birds and horses in new york. e neutralized all wnv strains with prnt values of - ng and prnt values of - ng (supplementary table online) . notably, fab fragments of e inhibited wnv (prnt , ng), suggesting that neutralization does not require bivalent e protein binding. e potently blocked infection with strain , the original lineage ii strain isolated in (ref. ), yet was virus specific, as it neither recognized nor neutralized other flaviviruses including distantly related dengue and yellow fever viruses (data not shown) and closely related japanese and st. louis encephalitis viruses (supplementary table figure mapping of monoclonal antibodies to diii with yeast. (a) surface display of wnv e protein on yeast. a fusion protein is composed of the ectodomain or diii of wnv e protein and the yeast aga protein, which becomes attached to the aga protein on the yeast cell wall. yeast were transformed with the vector alone (pyd ; top row), the entire wnv e ectodomain (amino acids - ; middle row), or diii alone (amino acids - ; bottom row). h after induction, yeast were stained with the indicated monoclonal antibodies (negative control, anti-sars orf a) and processed by flow cytometry. data for a representative neutralizing (e ) and non-neutralizing (e ) antibody are shown. (b) flow cytometric enrichment for diii-expressing yeast variants that lose binding of e . after each round, an increased percentage of diii expressing yeast are recognized by the polyclonal wnv e-specific antibody but not by e . after the final round, diii-expressing variants (boxed region) were harvested. a b figure fine epitope mapping of diii neutralizing and non-neutralizing monoclonal antibodies. (a) flow cytometry profiles for immunoreactivity by nonneutralizing (e ), weakly neutralizing (e ) and strongly neutralizing (e ) monoclonal antibodies with yeast expressing wild-type and variant diii. the red, yellow and green arrows point to mutations that abolish yeast surface binding of individual monoclonal antibodies and correspond to distinct regions of diii (see b). (b) mapping of neutralizing and non-neutralizing monoclonal antibodies on the surface of wnv diii. a molecular surface representation is depicted based on the nuclear magnetic resonance structure of wnv diii . the indicated amino acid residues associated with binding of neutralizing, weakly neutralizing and non-neutralizing monoclonal antibodies are shown in red, yellow and green, respectively. to map our strongly neutralizing monoclonal antibodies, we developed a strategy using yeast surface display . the ectodomain (amino acids - ) or diii (amino acids - ) of wnv e protein were expressed as fusion proteins on the yeast cell surface (fig. a) . monoclonal antibodies that recognize diii alone are considered diii specific. monoclonal antibodies that recognized the e ectodomain but not diii alone may contact residues that map to domain i or ii or both, although cooperative contacts with diii cannot be ruled out. most of our monoclonal antibodies recognized yeast that displayed the entire ectodomain of e ( fig. a and supplementary table online) . sixteen antibodies recognized yeast that displayed diii alone, and of strongly neutralizing antibodies localized to diii. only two neutralizing antibodies (e and e ) recognized the e ectodomain but not diii alone. we used error-prone pcr mutagenesis of diii of wnv e protein and yeast surface expression to map antibody contact residues in a highthroughput manner. we performed individual screens to identify diii mutants that lost binding selectively to strongly neutralizing (e , e and e ), weakly neutralizing (e ), and non-neutralizing (e and e ) monoclonal antibodies. to eliminate mutants that abolished surface expression of diii, yeast were stained sequentially with an alexa fluor -conjugated individual monoclonal antibodies and an alexa fluor -conjugated oligoclonal antibody derived from a pool of individual monoclonal antibodies. after cell sorting, we identified yeast that selectively lost expression of an individual monoclonal antibody epitope but retained surface expression of diii (fig. b) . multiple independent yeast that lost binding of individual monoclonal antibodies were subjected to plasmid recovery and sequencing. monoclonal antibodies that localized to diii and strongly neutralized wnv had reduced binding when residues s , k , t or t were altered ( fig. a and table ). these are located on adjacent loops and form a contiguous patch on the solvent-exposed surface at the lateral tip of the diii (fig. b) . only two other mutations caused considerable loss of binding of any of the ten neutralizing monoclonal antibodies tested: k e or p r reduced binding only of e . no two neutralizing monoclonal antibodies had identical loss-of-binding patterns. for example, s l reduced binding of e , e , e , e and e but not e , e , e , e and e . k r abolished binding of e , e , e , e , e and e but affected e , e , e and e less strongly. in contrast, k e decreased binding of all neutralizing monoclonal antibodies yet did not affect non-neutralizing or poorly neutralizing monoclonal antibodies. changes in residues t and t also abolished binding of neutralizing but not non-neutralizing monoclonal antibodies. t i or t m strongly reduced binding of all neutralizing monoclonal antibodies with the exception of e , and t v or t a weakened binding of only e , e , e and e . six monoclonal antibodies that recognized diii were either poorly neutralizing or non-neutralizing, and none engaged the dominant neutralizing epitope defined by s , k , t or t . e and e were abolished or reduced by mutation of d and h , and binding of e was weakened by a change in p . d and h are proximal to one another but physically distinct from the four residues that affect binding of neutralizing monoclonal antibodies (fig. b) . none of the mutations identified by loss-of-binding sorts for e or e had any effect on two other non-neutralizing monoclonal individual wnv-specific monoclonal antibodies ( µg/ml) were mixed with yeast that displayed wild-type or mutant diii on their surface. after washing, and incubation with an alexa- goat-anti mouse igg secondary antibody, yeast were analyzed for antibody binding by flow cytometry. the two values represent the percentage of yeast that were positive for diii expression with a given monoclonal antibody, and in parentheses, the mean linear fluorescence intensity of the positive cells. yeast were analyzed at h after induction with galactose, which gives a baseline surface expression of diii of - % positive cells (fig. a) . bold values indicate an almost complete (> %) loss of binding, whereas underlined values show a marked ( - %) reduction in either the percentage or the mean fluorescence intensity of the positive cells. results are representative of at least two independent experiments for each antibody and diii variant. antibodies, e and e . e , a monoclonal antibody with weak neutralizing activity, mapped to a site between the non-neutralizing and neutralizing monoclonal antibodies, as mutation of k and n strongly inhibited binding. to determine whether human antibodies specific for wnv recognize the neutralizing epitope on diii during infection, plasma was obtained from wnv-positive individuals. samples from convalescent individuals were negative for wnv rna but positive for neutralizing wnv-specific antibodies. the individuals reported mild systemic illness, though none progressed to severe disease. to determine whether these samples contained antibodies that localized to the neutralizing epitope on diii, we tested whether e fab or igg could compete binding to recombinant, wildtype and mutant n k and k n forms of diii ( supplementary fig. online); n k retains wild-type binding to e , whereas k n has markedly reduced binding. e equivalently inhibited binding of patient wnv-specific antibodies to wild-type (fab, % ± ; igg, % ± ) or n k diii (fab, % ± ; igg, % ± ), whereas e igg, a wnv-specific monoclonal antibody that recognizes an epitope outside diii, did not compete binding to wild-type ( % ± ) or n k (- % ± ) diii. as expected, e , which only weakly recognizes k n, poorly competed (fab % ± ; igg % ± ) binding to k n diii. these data suggest that humans, who clear wnv infection, develop antibodies that recognize an epitope in close proximity to that defined by e . to evaluate the correlation between neutralization, epitope localization and in vivo protection, we assessed the therapeutic activity of different neutralizing monoclonal antibodies in an established mouse model . studies were performed with -week-old wild-type c bl/ mice, which have a ∼ % survival rate . mice were inoculated subcutaneously with plaque-forming units (pfu) of wnv and administered a single dose of monoclonal antibody at day after infection. notably, µg of the non-neutralizing monoclonal antibody e provided no protection (data not shown). in contrast, µg of any of three different neutralizing monoclonal antibodies that map to k (e , e or e ) protected greater than % of mice from lethal infection ( fig. a-c) . even a single µg treatment of e or e on day after infection prevented mortality. given that humans can present with wnv infection of the cns, we evaluated the therapeutic efficacy of monoclonal antibodies at later , or (c) e monoclonal antibodies. as controls, mice were independently administered saline (pbs) or a negative control monoclonal antibody (anti-sars orf a, µg). the survival curves were constructed using data from two independent experiments. the number of animals for each antibody dose ranged from to . the difference in survival curves was statistically significant for all wnv-specific monoclonal antibody doses shown (p < . ). (d) wnv burden in the brain of -week-old wild-type mice. at days , and after wnv infection, brains were harvested and viral burdens were determined by plaque assay. the following percentage of mice had viral burdens below detection (< pfu/g): day , %; day , %; day , %. (e,f) efficacy of wnv-specific monoclonal antibody therapy at days (e) and (f) after infection. a single dose ( . mg at day or mg at day ) of monoclonal antibody (e , e , e or anti-sars orf a) was administered either or d after wnv infection. data reflect approximately mice per condition. the difference in survival curves was statistically significant for all wnv-specific monoclonal antibody doses shown at day (p < . ) and day (e , p = . ; e , p = . ). (g) effect of e therapy on viral burden. mice were treated with mg of e or pbs on day after wnv infection. on day , brains were recovered, homogenized and subjected to plaque assay. for a subset ( ) that received pbs treatment, brains were harvested at days and from moribund mice. the data is expressed as pfu/g. of mice treated with e % ( ) had undetectable viral loads in the brain at day whereas all mice ( of ) treated with pbs had detectable virus at the time of harvest. the dotted line represents the limit of sensitivity of the assay and the dark bars represent the mean of the log values. the differences were statistically different (p < . ). time points. at days , and after wnv infection, we detected virus in the brains of %, % and % of mice, respectively (fig. d) . a single µg dose of e or e at day resulted in an - % survival rate (fig. e) . a single mg dose of e at day resulted in % survival (fig. f) and complete clearance of wnv from the brain in % of mice by day (fig. g) . thus, administration of neutralizing monoclonal antibodies to mice with active cns infection improved survival and induced a virologic cure. as expected, lengthening the interval before treatment was associated with decreased benefit. administration of e at day did not enhance survival (data not shown), although average survival time was increased ( . d ± . to . d ± . ; p = . ). we considered humanizing e or e as a possible therapeutic measure. humanized monoclonal antibodies have substantially longer half-lives in humans than their mouse counterparts , . sequencing studies indicated that e had greater homology to human framework regions, making it simpler to construct a humanized version of e . we amplified the cdna encoding the heavy (vh) and light (vl) variable domains from the hybridoma cellular rna by a ′ rapid amplification of cdna ends (race) procedure. the vh belongs to mouse heavy chain subgroup ii (j family) and the vl belongs to mouse κ chain subgroup v. the complementarity-determining regions of e were grafted onto the human vh - backbone (fig. a) and human vκ-b backbone (fig. b) to create hm-e . . one (vl-y s) and two framework back-mutations (vh-t a and vl-y s) were introduced to create two variants, hm-e . and hm-e . , respectively. the resulting humanized vh and vl were combined with human γ and κ constant regions, fused to an igg signal sequence and inserted into expression plasmids. to construct the chimerized antibodies, the mouse vh and vl sequences were combined with the human γ and κ constant regions. we expressed humanized (hm-e ) and chimerized (ch-e ) e in hek- cells, and purified them from supernatants by affinity and size-exclusion chromatography (data not shown). the affinity was analyzed by surface plasmon resonance using purified antibody in the solid phase. mouse e binds diii with an affinity of . nm and a half-life of . min. the affinity of the ch-e and hm-e was similar with k d ranging from . to nm (fig. c) . hm-e , ch-e , and the parent e all had similar prnt values (fig. d) . we hypothesized that e could also control infection in mice through effector functions including antibody-dependent complement fixation and cytotoxicity. to test this, we generated a ch-e n q aglycosyl variant that neutralizes wnv (fig. d) but does not efficiently fix complement or bind fc γ receptors . mice were administered ch-e or ch-e n q at day after wnv infection. although high doses of ch-e and ch-e n q provided virtually complete protection, lower doses of the aglycosyl variant afforded less protection (fig. e) . administration of µg of ch-e resulted in % survival, whereas µg of ch-e n q provided only % protection. to test which effector function enhanced the activity of e , we performed studies with -week-old c qa, c , or fcgr and fcgr -deficient c bl/ mice (fig. ) . these mice all show increased susceptibility to lethal wnv infection compared to wild-type controls. in c qa or c -deficient mice, which cannot activate complement by the antibody-dependent classical pathway, e had a similar potency compared to wild-type mice. in contrast, in fcgr -and fcgr -deficient mice, although high doses afforded complete protection, lower doses resulted in higher mortality rates. a dose of µg at day , which strongly protected wild-type, c qa or c -deficient mice, did not improve the survival rate of fcgr -and fcgr -deficient mice (p = . ). thus, the fc region enhances the potency of e in mice, by virtue of its ability to bind to fc γ receptors. to confirm the efficacy of humanized e , wild-type mice were administered three different versions of purified hm-e at day after infection. although hm-e variants protected mice against lethal infection (fig. f) , at a dose of µg, the variant ( . ) that showed the highest affinity for diii (fig. c) was more protective ( % versus % survival; mean survival time of ± d versus ± d, p = . ). we generated a panel of monoclonal antibodies against wnv e protein, and applied a new high-throughput epitope-mapping strategy to identify a dominant epitope that was recognized by the majority of neutralizing monoclonal antibodies in diii. this epitope was also detected by convalescent antibodies from individuals who had recovered from wnv infection without clinical consequence. three neutralizing monoclonal antibodies protected against wnv mortality in a postexposure therapy model. one of these, e , was humanized and confirmed as therapeutically effective in mice. previous studies have mapped amino acid contact residues of neutralizing monoclonal antibodies by sequencing in vitro neutralization escape variants, through site-specific substitution of specific charged or polar residues, and by performing binding assays with overlapping peptide libraries. we used error-prone pcr mutagenesis and yeast surface expression to identify contact residues in a high-throughput manner. although this technique has been used previously , this is the first highthroughput epitope-mapping application. by having a large panel of diii monoclonal antibodies and selecting only variants that abolished or markedly reduced binding of a few monoclonal antibodies, we minimized the possibility that mutations altered protein folding. we have recently confirmed the recognition sites on diii for e and the validity of the yeast display strategy by solving the crystal structure of the e fab-diii complex (g.n., t.o., m.d, & d.f., unpublished data). of neutralizing monoclonal antibodies, localized to the distal lateral surface of diii, results that are consistent with prior studies that mapped three neutralizing monoclonal antibodies against wnv using in vitro escape variants , . our weakly neutralizing and non-neutralizing monoclonal antibodies did not recognize this epitope but localized to distinct regions. e recognized the dominant epitope and neutralized all strains that were tested. sequence analysis of wnv strains in public databases showed almost complete ( . - %) conservation of the contact residues s , k , t and t . only two clinically attenuated lineage ii isolates had mutations at these residues. because of the structural homology among flaviviruses , , , the analogous amino acids that map to the distal lateral surface of diii can be readily identified. although this region is highly variable among flaviviruses, most mutations that abolish binding of virus-specific neutralizing antibodies map here , , , , suggesting the existence of an analogous dominant neutralizing epitope for other flaviviruses. we speculate that successful vaccines against wnv or other flaviviruses should induce potent humoral responses against this neutralizing epitope. although passive administration of immune human γ-globulin after wnv infection improved survival in mice , , it may be limited by its low-titer neutralizing activity, variability and risk of transmission of infectious agents. only two prior studies have shown postexposure therapy of neutralizing monoclonal antibodies with flaviviruses: b a- reduced mortality - d after infection with st. louis encephalitis virus ; and reduced mortality resulting from infection with japanese encephalitis virus d . here, we show that three different neutralizing monoclonal antibodies improved survival even when administered and d after wnv infection. moreover, therapy with e at day completely cleared wnv from the brain at day in % of mice. thus, inhibitory wnv monoclonal antibodies improve clinical and virologic outcome even after viral spread through the cns, results that agree with studies showing that antibody can mediate viral clearance from infected neurons , . our experiments are consistent with a model in which the therapeutic efficacy of monoclonal antibodies is determined by properties in addition to neutralization: (i) the monoclonal antibody (e ) with the strongest neutralizing activity in vitro did not have the greatest efficacy in vivo; (ii) an aglycosyl version of e that lacked the ability to fix complement or bind to fc γ receptors had equivalent neutralizing but reduced therapeutic activity; (iii) e was less potent in mice that lacked fc γ receptors. e was humanized as a possible therapeutic measure for humans. hm-e bound diii with similar affinity and showed efficacy as postexposure therapy. moreover, it may be possible to improve hm-e by a b c introducing mutations that enhance affinity, creating forms of e that more readily cross the blood-brain barrier, and combining monoclonal antibodies that neutralize wnv infection through independent mechanisms. our results are the first successful demonstration of a humanized monoclonal antibody as postexposure therapy against a viral disease, and suggest that antibody-based therapeutics may have more broad utility than previously appreciated, especially in the treatment of cns infections in which an effective antibody response is important for limiting virus dissemination and injury to neurons. we cultured bhk- , vero and c / aedes albopictus cells as previously described , ) . we also performed neutralization experiments with prototype strains of st. louis ( (parton)) and japanese encephalitis (nakayama) viruses . for in vivo experiments, viruses were diluted and injected into mice as described . purified wnv e protein expression. wnv e protein ectodomain was generated using a baculovirus expression system according to previously described methods for related flaviviruses . the last nucleotides of prm (endogenous signal sequence) and the first , nucleotides of wnv e protein from the new york strain were fused downstream of the polyhedrin promoter and upstream of a histidine repeat in a baculovirus shuttle vector (pfastbac, invitrogen) by pcr using a high-fidelity taq polymerase (platinum taq, invitrogen). three days after baculovirus infection of hi- insect cells at a multiplicity of infection (moi) of , supernatants were harvested, filtered, buffer-exchanged and purified by nickelaffinity chromatography according to the manufacturer's instructions. the purified wnv e ectodomain lacks the c-terminal amino acids that are associated with the membrane proximal, transmembrane and cytoplasmic domains. purified wnv diii. the construction, expression, purification and refolding of diii of wnv e protein is described in greater detail elsewhere (g.n., t.o., m.d. & d.f., unpublished data). briefly, wild-type, n k and k n diii were generated from an infectious cdna clone of the new york strain of wnv (gift of r. kinney, centers for disease control and prevention, fort collins, co) using pcr and quik-change mutagenesis (stratagene). after cloning into a pet vector (novagen) and sequence confirmation of the mutations, we transformed plasmids into bl codon plus e. coli cells (stratagene). bacteria were grown in luria broth, induced with . mm isopropyl thiogalactoside (iptg) and pelleted. subsequently, we lysed bacteria after the addition of lysozyme, sonicated them and recovered diii as insoluble aggregate from the inclusion bodies. diii was denatured in the presence of guanidine hydrochloride and β-mercaptoethanol and refolded by slowly diluting out the denaturing reagents in the presence of l-arginine, edta, pmsf, reduced glutathione and oxidized glutathione. we separated refolded diii from aggregates on a superdex / size-exclusion column (amersham bioscience) and concentrated it using a centricon- spin column into mm hepes ph . , mm nacl and . % nan . after refolding, wild-type diii reacted with all domain iii-specific monoclonal antibodies including those that recognized conformationally sensitive epitopes. generation and purification of monoclonal antibodies. balb/c mice were primed and boosted at -week intervals with insect cell-generated, purified, recombinant wnv e protein ( µg) that was complexed with adjuvant (ribi immunochemical). approximately month after the last boost, we harvested serum and tested it for immunoreactivity against solid-phase purified e. mice with high titers (> / , ) were boosted intravenously with purified e protein ( µg) in pbs. we harvested splenocytes d later and fused them to p x ag . myeloma cells to generate hybridomas according to published procedures . we purified monoclonal antibodies against wnv or other control antigens by stan-dard protein a or protein g chromatography according to the manufacturer's instructions (pharmacia). for wnv infection experiments, all wild-type c bl/ j mice were purchased from a commercial source (jackson laboratories). we obtained the congenic c qa-deficient and c -deficient mice from g. stahl (beth israel deaconess medical center, boston, ma) and m. carroll (the cbr institute for biomedical research, harvard medical school, boston, ma), respectively. we obtained the congenic fcgr -and fcgr -deficient mice commercially (taconic). we used mice between and weeks of age depending on the particular experiment and inoculated them subcutaneously with wnv by footpad injection after anesthetization with xylazine and ketamine. mouse experiments were approved and performed according to the guidelines of the washington university school of medicine animal safety committee. for passive-transfer experiments, we administered to mice a single dose of purified monoclonal antibody by intraperitoneal injection at a given time point (day , or ) after infection. to analyze virus production in the brain, infected mice were killed on a given day after inoculation. after cardiac perfusion with pbs, we removed the brains, weighed and homogenized them, and performed plaque assays as previously described . expression of wnv e protein on yeast. the ectodomain or diii of wnv e protein was expressed on the surface of yeast using a modification of a previously described protocol for surface expression of t cell receptors . amino acid residues - (ectodomain) or - (diii) of wnv e protein were amplified with bamhi and xhoi sites at their ´ and ´ ends, respectively, by pcr from the new york infectious cdna clone (r. kinney, centers for disease control and prevention, fort collins, co). the resulting products were digested with bamhi and xhoi, and cloned as downstream fusions to the yeast aga and xpress epitope tag genes in the yeast surface display vector pyd (invitrogen). an upstream gal promoter controls fusion protein expression. these constructs were transfected into the s. cerevesiae yeast strain eby (refs. , ) resulting in yeast that expressed the wnv e ectodomain or diii. yeast that only expressed the xpress epitope tag linked to aga were prepared in parallel by transfecting eby cells with the parent vector pyd . individual yeast colonies were grown to log phase overnight in tryptophan-free media containing % glucose at °c and harvested in log phase. fusion protein expression was induced on the yeast surface by growing yeast for an additional h in tryptophan-free media containing % galactose at °c. we harvested yeast, washed them with pbs supplemented with mg/ml bsa and immunostained them with µl of monoclonal antibody ( µg/ml) against the xpress tag or wnv e protein. after min, we washed yeast three times and stained them with a goat anti-mouse secondary antibody conjugated to alexa fluor (molecular probes). subsequently, the yeast cells were analyzed on a becton dickinson facscaliber flow cytometer. library construction and screening. we mutated diii of the wnv e protein using an error-prone pcr protocol that included mn + and mg + at concentrations of . and . mm respectively. subsequently, the cdna library was ligated into pyd and transformed into xl -blue ultracompetent cells (strategene). the colonies were pooled and the plasmid dna was recovered using the qiagen hispeed maxi kit. for each individual antibody, we screened the yeast library of diii mutants according to the following protocol. to identify yeast that selectively lost binding to a given monoclonal antibody epitope, the library was initially stained with an alexa fluor -conjugated wnv-specific monoclonal antibody for min at °c. to control for the surface expression of diii, after washing, yeast were subsequently stained for min at °c with an alexa fluor -conjugated oligoclonal antibody that was derived from a pool of individual monoclonal antibodies (e , e , e , e , e and e ). after immunostaining, we subjected yeast to flow cytometry and identified the population that was single monoclonal antibody negative but pooled oligoclonal antibody positive. the yeast cells were sorted at an event rate of ∼ , cells/s and this population (monoclonal antibody-negative and oligoclonal antibody-positive) was enriched after three rounds of sorting. after the final enrichment sort, we plated yeast and selected individual colonies and tested them for binding to individual monoclonal antibodies. for individual clones that had lost only the desired monoclonal antibody epitope, the diii-pyd plasmid was recovered using the zymoprep yeast miniprep kit (zymo research). the plasmid was then transformed into dh α cells, purified using the qiaprep spin miniprep kit (qiagen) and sequenced. in some cases, diii variants with two independent mutations were isolated. to determine which mutation conferred the loss-of-binding phenotype, single independent mutations were engineered by site-directed mutagenesis of diii-pyd using mutant oligonucleotides and the quik change ii mutagenesis kit (strategene). all mutations were confirmed by sequencing. we determined the titer of neutralizing antibodies by a standard plaque reduction neutralization titer (prnt) assay using either bhk or sw cells . results were plotted and the titers for % (prnt ) and % inhibition (prnt ) were calculated. the inhibition assay with j . mouse macrophages was performed as follows: we mixed medium and e or e ( . µg of monoclonal antibody) with × pfu of wnv, incubated the mixture for h at °c, and then added to × j . mouse macrophages in individual wells of a -well plate. after h, cells were washed four times with pbs to remove free virus and monoclonal antibody, dmem with % fbs was added, and the cells were incubated for an additional h. we subsequently harvested supernatants for a viral plaque assay on vero cells. competition elisa with human anti-wnv antibodies. after purification and refolding, wild-type, k n and n k diii were diluted ( µg/ml) in . m na carbonate buffer (ph . ) and adsorbed to -well plates overnight at °c. after blocking with pbs, % bsa and . % tween (pbs-bt), wells were preincubated for h at °c with pbs-bt containing no antibody, e igg ( µg/ml), e fab ( µg/ml) or e igg ( µg/ml). e serves as a negative control as it recognizes an epitope in domain i and ii of wnv e protein. subsequently, human plasma ( / dilution in pbs-bt, heat-inactivated) was directly added for an additional h at °c. we obtained the human samples with informed consent from seven different wnv-infected patients (gift of m. busch and l. tobler, blood systems research institute, san francisco, ca). because the samples were sequentially numbered and not linkable back to the original subjects, they satisfied the criteria for exemption from approval from the human studies committee at washington university. after six washes with pbs-bt, plates were serially incubated with biotin-conjugated goat anti-human igg ( µg/ml), streptavidin-horseradish peroxidase ( µg/ml) and tetramethylbenzidine developing substrate (dako). we determined optical densities at nm with an automatic elisa plate reader (tecan) and adjusted them after subtraction of the value obtained from nonimmune human plasma. surface plasmon resonance. antibody affinity analysis for diii of wnv was performed by surface plasmon resonance (biacore , biacore, inc). binding curves and kinetic parameters were obtained as follows: we captured e antibodies by flowing ( nm, rate of µl/min for min) them over immobilized f(ab)' fragment specific for goat anti-human or mouse igg with fc region specificity. subsequently, diii of the new york strain of wnv e protein (amino acids - ), which was generated in e. coli, was injected ( . - nm, flow rate µl/min for . min and then allowed to dissociate over min). the f(ab)' surface was regenerated by pulse injection of mm glycine (ph . ) and mm naoh before each e injection. we analyzed curves with a global fit : binding algorithm with drifting baseline. cloning and humanization of e . e heavy-and light-chain rna was isolated from hybridoma cells after guanidinium thiocyanate and phenol-chloroform extraction, and converted to cdna by reverse transcription. the vh and vl segments were amplified by pcr using the ′ rapid amplification of cdna ends (race) system (invitrogen). gene-specific primers (gsp) for vh and vl were as follows: vh-gsp : ´-ggtcactgtcactggctcaggg- ´; vh-gsp : ´-aggcggatccaggggccagtggatagac- ´; vl-gsp : ´-gcacacg actgaggcacctccagatg- ´; and vl-gsp : ´ cggatccgatggatac agttggtgcagcatc- ´. the pcr products were inserted into the plasmid pcr . -topo using the topota kit (invitrogen). we then subjected the resulting plasmids to dna sequencing to determine the vh and vl sequences for e . the cdna sequences were translated and the predicted amino acid sequence determined. from these sequences the framework and cdr regions were identified as previously defined . we joined the mouse vh to a human c-γ constant region and an ig leader sequence, and inserted it into pci-neo for mammalian expression. we joined the mouse vl to a human cκ segment and an ig leader sequence and also cloned it into pci-neo for mammalian expression of chimeric e (ch-e ). for ch-e , site-directed mutagenesis was also performed to change residue from asparagine to glutamine of the heavy chain to eliminate the single glycosylation site on the γ fc. humanized e vh consists of the framework segments from the human germline vh - vh segment and jh segment , , and the cdr regions of the e vh, respectively. the humanized e vl consists of the framework segments of the human germline vk-b vl segment and jk , (refs. - ) segment and the cdr regions of e vl. the humanized vh segments were assembled de novo from oligonucleotides and amplified by pcr. the humanized vl segments were assembled by pcr and overlapping pcr. the resulting vh and vl segments were subsequently combined by overlapping pcr with a leader sequence and the appropriate constant region segment and cloned into the expression vector pci-neo as nhei-ecori fragments. we confirmed the dna sequence of the resulting plasmids by sequence analysis. site-directed mutagenesis was then performed to substitute mouse for human residues at key framework positions vh- (t a) and vl- (y s). the resulting plasmids were cotransfected into human cells using lipofectamine- and humanized antibody was recovered from the resulting conditioned medium and purified by protein a and size-exclusion chromatography. statistical analysis. all data were analyzed with prism software (graphpad software). for survival analysis, kaplan-meier survival curves were analyzed by the log-rank and mantel-haenszel test. for viral burden experiments, we determined statistical significance using the mann-whitney test. note: supplementary information is available on the nature medicine website. west nile virus: where are we now? innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus immunity to west nile virus prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating west nile virus infection in mice antibody prophylaxis and therapy against west nile virus infection in wild type and immunodeficient mice antibody prophylaxis and therapy for flaviviral encephalitis infections a ligand-binding pocket in the dengue virus envelope glycoprotein the envelope glycoprotein from tick-borne encephalitis virus at angstrom resolution dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry structure of the dengue virus envelope protein after membrane fusion visualization of membrane protein domains by cryo-electron microscopy of dengue virus structure of west nile virus biophysical characterization and vector-specific antagonist activity of domain iii of the tick-borne flavivirus envelope protein inhibition of west nile virus entry by using a recombinant domain iii from the envelope glycoprotein identification of neutralizing epitopes within structural domain iii of the west nile virus envelope protein solution structure and antibody binding studies of the envelope protein domain iii from the new york strain of west nile virus epitopes on the dengue virus envelope protein recognized by neutralizing igm monoclonal antibodies monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells monoclonal antibody mapping of the envelope glycoprotein of the dengue virus nucleotide changes responsible for loss of neuroinvasiveness in japanese encephalitis virus neutralization-resistant mutants japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence localization of a neutralizing epitope on the envelope protein of dengue virus type b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus a neurotropic virus isolated from the blood of a native of uganda yeast surface display for screening combinatorial polypeptide libraries mouse/human chimeric monoclonal antibody in man: kinetics and immune response pharmacokinetics, immune response, and biodistribution of iodine- -labeled chimeric mouse/human igg ,k - a monoclonal antibody studies of aglycosylated chimeric mouse-human igg. role of carbohydrate in the structure and effector functions mediated by the human igg constant region directed evolution of a stable scaffold for t-cell receptor engineering nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain iii of the japanese encephalitis virus envelope protein protection of mice against japanese encephalitis virus by passive administration with monoclonal antibodies antibody protects against lethal infection with the neurally spreading reovirus type (dearing) antibody-mediated clearance of alphavirus infection from neurons modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons genetic and phenotypic variation of west nile virus in new york international catalogue of arboviruses including certain other viruses of vertebrates rd edn analysis of c-terminally truncated dengue and dengue virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states antibodies: a laboratory manual optimal screening of surface-displayed polypeptide libraries sequences of proteins of immunological interest the complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged j and d genes human immunoglobulin kappa light chain genes of subgroups ii and iii subgroup iv of human immunoglobulin k light chains is encoded by a single germline gene evolution of human immunoglobulin kappa j region genes the authors declare competing financial interests (see the nature medicine website for details). key: cord- - eyetb i authors: mandel, benjamin title: neutralization of animal viruses date: - - journal: advances in virus research doi: . /s - ( ) - sha: doc_id: cord_uid: eyetb i publisher summary various aspects of the interaction of bacterial viruses and antibody were studied by andrewes and elford in england. similar studies, as well as studies on animal viruses, were carried out in australia by burnet and his colleagues. one result of their extensive studies, which were summarized in great detail, was the conclusion that, with respect to their interaction with antibody, bacterial and animal viruses were basically different. specifically, the difference resided in the stability of the union of virus and antibody, whereas bacterial viruses formed stable complexes, animal viruses formed complexes that tended to dissociate readily. the introduction of animal cell cultures as host systems greatly aided in the study of animal viruses, with respect to fewer and more readily controlled variables, and by the use of the plaque assay in enhanced quantitative reliability. in , dulbecco et al. described the interaction of two animal viruses with their respective antibodies. the results of these studies led these investigators to conclude, among other things, that animal viruses, at least the two they studied, reacted with antibodies to form complexes that did not dissociate spontaneously. this interpretation was challenged by fazekas de st. groth and reid. as more animal virus-antibody systems were studied by many investigators, there seemed to be a greater accord for irreversible, rather than reversible, interaction. for this reason, in this chapter it is assumed that there are no differences between bacterial viruses, as one category, and animal viruses, as a separate category, concerning their interaction with antibodies. rather, differences, when they exist, are considered to be related to the viruses per se. although this chapter is intended to survey the neutralization of animal viruses, occasional reference is made to the studies on bacterial viruses when these studies are pertinent and illuminating to the topic at hand. years later, in the late oos, the validity of jenner's approach to combatting smallpox was reaffirmed by pasteur when he succeeded in selecting an avirulent strain of rabies virus for use as a vaccine. at about the same time the studies of iwanowski and beijerinck began to reveal the nature of a then unknown form of infectious agent of plants, and somewhat later a similar revelation emerged from the studies of loeffler and frosch on infected cattle. the ubiquity of this kind of agent was revealed by the studies of twort and d'herrelle who showed that bacteria too were susceptible to what became known as filterable viruses. as knowledge of the existence and the nature of viruses accrued, so did knowledge of the existence and the nature of antibodies through the studies of von behring, kitasato, and bordet. i n sternberg (see hahon, ) inferred that the blood of an individual recently recovered from an infection such as smallpox contained an "antitoxine which would neutralize the active poison of the disease." this inference proved t o be correct when he showed that serum from a recently vaccinated calf neuralized cowpox virus. these observations presaged the start of experimental viral immunology. the relative ease and simplicity of cultivating bacterial viruses led, in the early s, to highly productive studies on their interaction with antibody. the introduction by woodruff and goodpasture of the use of embryonated chick eggs as an animal virus host greatly stimulated research on animal viruses. the usefulness of this host was extended when burnet developed a technique for infecting the cliorioallantoic membrane in a manner that resulted in discrete lesions (pocks) which could be readily recognized and enumerated. with the application of the plaque assay for bacterial viruses, and the pock-counting assay for animal viruses, the era of quantitative virology had its inception. various aspects of the interaction of bacterial viruses and antibody were studied by andrewes and elford in england. similar studies, as well as studies on animal viruses, were carried out in australia by burnet and his colleagues. one result of their extensive studies, which were summarized in great detail (burnet et al., ) , was the conclusion that, with respect to their interaction with antibody, bacterial and animal viruses were basically different. specifically, the difference resided in the stability of the union of virus and antibody. whereas bacterial viruses formed stable complexes, animal viruses formed complexes that tended to dissociate readily. the introduction of animal cell cultures as host systems greatly aided in the study of animal viruses with respect to fewer and more readily controlled variables, and, by use of the plaque assay, in enhanced quantitative reliability. in dulbecco et al. described the interaction of two animal viruses with their respective antibodies. the results of these studies led these investigators to conclude, among other things, that animal viruses, at least the two they studied, reacted with antibodies to form complexes that did not dissociate spontaneously. this interpretation was challenged by fazekas de st. groth and reid ( ) . as more animal virus-antibody systems were studied by many investigators, there seemed to be greater accord for irreversible, rather than reversible, interaction. for this reason, in this article it is assumed that there are no differences between bacterial viruses, as one category, and animal viruses, as a separate category, concerning their interaction with antibody. rather, differences, when they exist, are considered to be related to the viruses per se. although this article is intended to survey the neutralization of animal viruses, occasional reference is made to studies on bacterial viruses when these studies are pertinent and illuminating to the topic at hand. the subject was reviewed in great detail by fazekas de st. groth ( ) and, wherever feasible, relationships were reduced to mathematical terms. subsequent reviews were presented by svehag ( svehag ( , with emphasis on the biochemical aspects of virus-antibody interactions and the role of antibody diversity on these interactions. in . , notkins and, in , majer reviewed the topic of viral sensitization by antibody, i.e., the formation of a virus-antibody complex that continues to be infectious but has acquired sensitivity to such reagents as complement, or antibody specific for the antibody that is bound to the virus. more recently, the same topic was surveyed by who also reviewed the subject of immunopathology as it relates to virus-induced immunocomplexes. other aspects of the general subject of viral immunology have been reviewed by cowan ( ) who described the characteristics of the immune response to viral antigens, and by burns and allison ( ) who discussed the immune response, the mechanism of neutralization, and the nature of cell-mediated immunity. methods for studying virus-antibody complexes have been reviewed by mandel ( a) . of particular relevance are the reviews of daniels ( ) and della-porta and westaway ( ) that deal with various aspects of the mechanism underlying the neutralization reaction. studies of the in vitro interaction of viruses with antibody have included a wide array of animal and bacterial viruses and antibody derived from a variety of animal species. i n some instances antibody was obtained from animals responding to an active infection. i n other instances nonsusceptible animals were induced to synthesize antibody by inoculation of the viral immunogen. in the latter instance, rabbits were used most frequently. as indicated in section ii,c,l antibodies are a diverse group of molecular species that vary both physicochemically as well as functionally. in the various studies of the in vitro reaction of virus with antibody, knowledge of the specific type of antibody was not always available, nor were sufficient details provided of the conditions under which antibody (i.e., the serum) was obtained. it has been reported (petty and steward, ) that the use of adjuvants to enhance the immune response may affect the level and the quality of antibody according to the composition of the adjuvant. nonetheless, the general characteristics of the in vitro reaction have been found to be reasonably consistent to warrant generalizations. when the concentrations of virus and antibody are appropriately adjusted, the rate of the reaction, i.e., loss of infectivity, can be measured. the quantitative expression of the rate is based on the early part of the reaction which can be mathematically described by the equation for first-order reactions: ( ) where ct and co are the concentrations at times t and zero, respectively, and k is a constant. when adapted for use in the measurement of neutralization, the equation becomes (adams, ) : where v t and v , are the respective concentrations of virus, t most frequently is in minutes, and d is the final dilution of serum. the constant le is the rate constant (min-l). when the information is available, the d term can be replaced by the known molar concentration of antibody, in which case k will have the dimensions of min-l mole-l. the effects of several environmental conditions on the reaction of virus and antibody have been studied. the procedure is quite straightforward, involving measurement of the rate constant under specified conditions. various studies, covering a range of temperatures consistent with the stability of the reactants, have indicated that the reaction is not activated by temperature. activation energies of the order of - kcal/mole have been reported for most animal and bacterial viruses. the interpretation is that the change in rate is dependent on thermal diffusion of the reactants, but the efficiency of the interaction is unaffected. the concentration of salt has considerable influence. for example, jerne and skovsted ( ) reported that, when the salt concentration was reduccd from . m to . m, the rate increased -fold. others (svehag, ; wallis et al., ) have reported similar findings but not of the same order of magnitude. it is quite likely, as shown by svehag ( ) , that the degree of the salt effect varies with the quality (association affinity) of the antibody. wallis et al. stated that the salt effect was seen with igg but not igm antibody, and that antiserum from baboon but not from rabbit or sheep showed this effect. although not highly dependent on an optimum ph, the reaction becomes decreasingly efficient at either extreme. this undoubtedly reflects a decrease in the number of reactive complementary ionic groups due to their titration a t low and high ph. it has been shown that restoration of the ph to neutrality restores the reactivity of virus and antibody, hence the reduced rate at ph extremes is not a consequence of denaturation. the binding of an antibody molecule to the antigenic site of a virus is mediated by noncovalent interactions. these are short-range interactions such as hydrogen bonding, electrostatic interactions, van der waal's forces, and possibly nonpolar hydrophobic reactions. thermodynamic studies have indicated that the forward reaction is accompanied by an increase in entropy and a change in mean free energy of the order of - kcal/mole. the reaction therefore is "downhill," i.e., is entropy-driven. studies on the effect of temperature on the reaction rate have indicated that the system is not subject to activation and that the rate changes observed are related simply to changes in collision frequency induced by changes in thermal diffusion, reaction rates of the order of ' mole-' sec-' have been reported for several systems, indicating a very rapid process. hornick and karush ( ) have suggested that the rate-limiting variable is collision efficiency rather than collision frequency. a successful collision requires that both reactive sites be in apposition a t the time of approach. it has been indicated that, when the functional binding affinity of virus-antibody interaction is examined, the effect of valence is extremely important (karush, ) . in studies with bacteriophage x there was an increase of more than lo' for bivalent compared with monovalent binding. blank et al. ( i similarly stressed this aspect. in view of the fact that most virus-antibody interactions involve bivalent antibody and multivalent viral particles, it is likely that the majority of virus-antibody complexes are characterized by monogamous bivalent unions, hence by a very high binding affinity. although the tendency may be to focus on the interactions of virus with antibody as the heart of the reaction, it has become clear that the host cell too must be scrutinized. the reason for this is twofold: ( ) the outcome of a given reaction may vary with the host cell or host animal. ( ) since viruses are inert, antibody does not inhibit any viral function other than adsorptive behavior. therefore, in instances in which neutralized virus adsorbs, the failure to infect is due to the inability of the cell to react to the virus as it would to an unneutralized particle. the diversity of antibodies in the serum of an animal at a given time is a function of several sources of variability. ( ) different classes and subclasses of antibodies exist that vary in their physicochemical as well as their functional characteristics. ( ) the class of antibody produced may depend on the mass of immunogen. it has been shown (svehag and mandel, a ) that, with a relatively low amount of poliovirus, only the s class of antibody is induced in rabbits. with highcr amounts both and antibodies arc elicited. ( ) it has been shown in many instances that the quality, i.e., the effectiveness, of antibodies improves with increasing time during the immune response. this characteristic implies a maturation proccss reflecting a progressive (either continuous or discrete) improvement in the combining characteristics of the antibody. ( ) viral immunogcns that have more than one determinant induce antibodies to each one. interaction of such an unfractionated serum with the respective virus can be expected to involve a complex set of interactions involving each refipective antibody-antigen system as well as possible influences among the different systems due to steric complications. ( ) each of the technical aspects of the immunization procedure --dose of immunogen, route of inoculation, frequency of inoculation, use of adjuvants-influences the time course and the class of antibody responding t o immunization. it was reported in the earlier literature (e.g., burnet et al., ) that the outcome of a neutralization reaction was not absolute. it may register as a positive or negative result depending on the host cell or animal used as indicator. the importance of this source of variation was reemphasized more recently by the results described by kjellh and schlesinger ( ) , and subsequently by other investigators. this may be an important consideration, since the nature of the neutralization phenomenon may be an alteration in the viral capsid of lesser or greater subtlety. this alteration may, for one cell, represent a qualitatively unrecognizable particle but, for another, an altered particle albeit one that the cell still recognizes and can cope with, perhaps inefficiently. andrewes and elford ( b) , for example, discussed the basis for abnormally small plaques in terms of "incompletely neutralized" virus. it has also been shown that the rate of appearance is delayed when survivors of animal virus neutralization are assayed (mandel, . ; westaway, a; yoshino and taniguchi, b ). an unusual host variation was described by hawkes ( ) . antiserum to some, but not all, togaviruses enhanced infectivity providing ( ) antiserum was obtained from fowl, not rabbits or mice, and ( ) the host was chick cells, not pig kidney cells. enhancement resulted from interaction with low concentrations of serum, but neutralization was observed with higher concentrations. subsequent studies (hawkes and lafferty, ) suggested that enhancement was the result of a modification of the protein capsid that rendered the virus more efficient in its interaction with chick cells. three aspects are of relevance-complexity, stability, and origin of viral antigens. with respect to stability, the genotype of viruses is usually very stable, and one can be assured that a given virus will display the same antigenic characteristics a t all times, irrespective of the host in which i t replicates. for enveloped viruses, the situation is complicated by the fact that the antigens are derived from two sources: ( ) stable virus-coded proteins and ( ) the host-coded lipoproteins that comprise the envelope. it has been thoroughly established that the viral envelope is acquired from the host cell during maturation (e.g., haukenes, ) . although the envelope contains viral proteins, it also retains the antigenic characteristics of the host. consequently, these viruses induce and react with two groups of antibodies with different specificities. nonenveloped viruses may be simple or complex. rna bacteriophages, for example, are constructed of one kind of polypeptide (the one mole-cule of a protein may be irrelevant). although picornaviruses contain four polypeptides, certain members (poliovirus, rhinovirus) induce the synthesis of only one specific antibody, probably because the morphological unit functions as an immunogenic element. with increasing complexity, the number of determinants increases. the interpretation of the effect of antibody on such viruses becomes difficult, since distinctions are required among critical, noncritical, and possibily quasi-critical sites. to illustrate, madeley et al. ( ) showed that the hemagglutinin and neuraminidase antigens of influenza virus induced specific antibodies. the antihemagglutinin antibody neutralized infectivity, while the antineuraminidase antibody had no such effect. they reported that these antibodies did not interfere with each other, suggesting that the antigenic structures were so arranged that there was no steric interference. majer and link ( similarly observed specific antibody responses. in addition, they showed that the nonneutralizing interaction with neuraminidase antibody could lead to neutralization by the addition of antiglobulin serum. a reasonable explanation, in the opinion of these workers, was that steric interference with the hemagglutinin antigen followed the secondary antibody reaction. for other viruses, the complexity is aggravated. adenovirus has three protein antigens, hexon, penton, and fiber, and each elicits a specific antibody. for some strains the hexon antibody has neutralizing consequences. it has, however, been shown that this interaction is rather complicated (kjellbn and pereira, ) . the hexon consists of two antigens, one of which is subsurface. the interaction of antibody with the surface antigen results in a rearrangement of the hexon structure that exposes the second antigen. neutralization is a consequence of the reaction of this antigen with antibody. recently, symington et al. ( ) studied the effect of antisera prepared specifically against the two (el, e ) surface glycoproteins of sindbis virus. each antiserum alone had poor neutralizing activity. antiserum versus the intact virion, however, was more active than the two specific sera, separately or in conjunction. brown and smale ( ) described a complex interaction between fmd virus (a picornavirus) and viral antiserum that suggests three distinct antigenic binding sites. two are associated with the vertex regions, and the third with the planar surfaces. there is some specificity with respect to the interactions of igg and igm with their respective combining sites. since all studies were based on agar precipitin reactions and electron microscopy, the significance of these interactions cannot be assessed with respect to neutralization. subsequent studies (rowlands et al., ) revealed that the antigens located at the vertexes were involved in stimulating the production of neutralizing antibody. recently, cavan-agh et al. ( ) reported that the vp, capsid polypeptide of fmd virus induced neutralizing antibody. studies on another picornavirus, poliovirus, showed that antigenic specificity was related to a structural requirement. subviral s particles had the same specificity as the parent particle, however, the substructure of the s particle was antigenically distinct. proteins with antibody activity encompass a diverse group of molecular species that vary in several parameters: mw, net surface charge, conformation, number of antigen-binding sites (valence), propensity for reacting with antigen (avidity), and force for binding antigen (afhity). the numerically dominant class of antibody, the igg form, is composed of two heavy (h) and two light (l) polypeptide chains bound by interchain disulfide linkages, the two antigen-binding sites are located at the termini of two pairs of l-h chains. the molecule can be dissected enzymically so as to obtain specific fragments. pepsin cleaves the molecule into a fragment which contains both combining sites but lacks a portion of the h chains. the fragment, designated f (ab') , can be dissociated under reducing conditions into two monovalent . s fragments designated fab'. papain cleaves the s molecule into two monovalent fragments designated fab, and one fragment, designated fc, which has no antibody activity. a third method for dissecting the molecule is based on reduction of the interpeptide disulfide bonds to yield free l and h chains. the different classes of antibody vary in valence (bi-, tetra-, decavalent) and, accordingly, in mw froin about , to about soo,ooo daltons. for a comprehensive discussion of this subject, the reader is referred to spiegelberg's review ( ) . the functions of the various segments of the antibody molecule in the neutralization reaction have been studied by isolating the desired fragment and examining its behavior. vogt et al. ( ) demonstrated that the fab fragment of poliovirus antibody retained neutralizing activity but a t reduced efficiency. comparison of the fab fragment with native antibody by kinetic analysis showed a reduced reaction rate for the monovalent fab fragment. vogt et al. ( ) also stated that the virus-fab complex was stable when subjected to dilution. although the kinetic reactions were not observed long enough to be sure, the data indicated that eventually both reactions approached the same final level, although a t different rates. cremer et al. ( ) reported that poliovirus and wee virus were neutralized by the fab fragments of their respective antibodies. these results were obtained with the metabolic inhibition test. when, however, neutralization of wee virus was evaluated by mouse infectivity, there was no evidence of neutralization. these workers considered the possibility that dissociation had occurred or that the fc moiety of the molecule was required. philipson et al. ( ) developecl an interesting system for detecting virus-antibody interaction based on the distribution of virus between two phases of an aqueous polymer system. when poliovirus was neutralized by intact antibody, its distribution pattern was inverted. when, however, virus was neutralized by the fab fragment, its distribution pattern remained characteristic of unneutralized virus (philipson and bennich, ) . it was also reported that virus neutralized by the partially separated but otherwise intact igg molecule (as a result of reduction and alkylation) showed a distribution pattern intermediate between that of virus neutralized by native igg and that of virus neutralized by fab. these results suggest that the distribution pattern of virus-antibody complexes is related to the charge characteristics of both virus and antibody, or antibody fragment, and that both species of the complex may undergo modifications as a consequence of interaction. neutralization reactions of poliovirus by s igg, s f(ab' , and . s fab' were compared (keller, ) . neutralization was similar for all three forms of antibody. the stability of the virus-antibody complexes, however, varied when examined by dilution dissociation and by acid reactivation. shortly after formation, the complex with . s antibody was the least stable, and with antibody the most stable. given sufficient time, all complexes approached a uniform state of stability. inasmuch as and antibodies are both bivalent, keller considered the likelihood that the fc fragment contributed to the stability of the virusantibody complex. in light of this report, a subsequent article by keller ( ) is puzzling. poliovirus was neutralized with s antibody. exposure of the virus-antibody complex to pepsin converted the molecule to the s form without a change in the neutralized status of the virus. further treatment with reducing agent converted the bivalent s molecule to the monovalent . s form with restoration of infectivity. that the antibody fragment was still associated with virus was shown by the sensitivity of the complex to antibody specific for the attached . s fragment. keller had previously ( ) reported that the . s fragment had neutralizing activity. in a study of the effect of neutralizing antibody on poliovirus (mandel, b) it was seen that neutralization was accompanied by a change in the electrophoretic characteristics of the virus. a further analysis of this phenomenon (mandel, ) showed that the fab fragment had the same effects as the native, s antibody, namely, neutralization and electrophoretic modification. the interaction of adenovirus with monovalent antibody fragments was examined by kjellcn ( ) . although the fragments inhibited hemagglutination, there was no reduction in infectivity. antibody fragment was shown to have bound to virus by a blocking experiment. possibly, kjellcn conjectured, the fc portion was essential for neutralization, or the fragment had reacted with a site that was not critical. the latter possibility poses the question, why, then, does it block the action of neutralizing antibody? in studies with influenza virus, lafferty ( b) reported that the fab fragment neutralized infectivity and inhibited hemagglutination. however, in contrast to antibody, the virus-monovalent antibody complex did not acquire stability. lafferty attributed this deficiency in the antibody to its monovalency. neutralization of herpesvirus by fab fragments was reported by ashe et al. ( ) . it was also shown that the fab fragment of anti-antibody neutralized herpesvirus that had been sensitized by viral s or fab antibody. shinkai and yoshino ( ) examined the reactivity of antibody fragments toward herpesvirus. at weeks after initiation of the immune response, f (ab') neutralized poorly, but a t weeks its neutralizing capability equaled that of igg. at weeks the monovalent fab' fragment failed to neutralize, per se, as well as in the presence of anti-antibody. evidence that the fragment had combined with virus was provided by blocking experiments. after weeks into the immune response, fab' displayed a moderate neutralizing capacity which could be enhanced by anti-antibody. several investigations of the interaction of bacterial viruses with antibody fragments have been reported. klinman et al. ( ) used fragments to evaluate the role of antibody valence in the neutralization of an rna bacteriophage, r . a comparison of the neutralization rate constants for bivalent and s molecules with those for monovalent , , and s molecules revealed a -fold higher rate for the bivalent molecules. the and monovalent antibodies were hybrid molecules containing one reactive site. these results stress the contribution of valence while minimizing a role for the fc portion of the antibody molecule. these investigators attributed, speculatively, the lower reaction rate for the monovalent antibodies to a lower binding afkity and a concomitant higher dissociation rate compared with those of bivalent antibody. rowlands ( ) questioned the role of antibody size and its steric effect on neutralization efficiency. neutralization rate constants (molecule-l ml min-l) of , , and . s antibodies were determined using bacteriophage f (closely related to r ). for , , and . molecules the rate constants were . x - , . x -l , and . x lo-", respectively. since there was a considerable disproportionality between rate constant and molecular size, he concluded that size was a relatively minor determinant in efficiency of neutralization. i n studies on the neutralization of two coliphages, t and t , goodman and donch ( ) observed that the efficiency decreased as size and valence were reduced in going from a s to a to a . molecule. i n a later study goodman and donch ( ) analyzed the role of molecular size in neutralization. they examined the neutralizing ability of l and h chains and of the fd fragment (the h-chain portion comprising the antigen-binding region of the antibody molecule). neither l, h, nor fd fragment neutralized bacteriophage t . however, binding to virus occurred, since neutralization resulted when antibody specific for the respective fragments was added to the virus-fragment complex. these workers proposed that lack of neutralizing activity was related to an insufficiently large fragment size. stemke and lennox ( ) compared the neutralization rates of two coliphages, t and t , by bivalent and monovalent antibody. the bivalent s antibody rate was about -fold greater than that of the fsb i fragment, and about -fold greater than that of the fab i fragment. they reported the interesting observation that, whereas the interaction of phage t was single-hit with the antibody, it was multihit (about . average hits) with the fab fragments. however, all reactions with phage t were of the single-hit form. when the reactions were enhanced by the addition of antibody directed against the viral antibody, it was observed that the degree of enhancement was inversely related to the neutralization efficiency. stemke and lennox proposed that several parameters such as size, valence, and binding affinity, separately or in conjunction, contributed to the activity of antibody. they also questioned the significance of the use of anti-antibody. perhaps, they suggested, neutralization that required the secondary effect of anti-antibody was not a bona fide interaction with respect either to the antibody per se or to the viral site to which it binds. stemke ( ) further corroborated the greater importance of valence over size of the antibody molecule. blank et a . ( ) evaluated the relative influence of antibody valence and affinity on the neutralization reaction. based on their studies of the interaction of dnp-t (bacteriophage t coupled with the hapten dnp) with rabbit anti-dnp antibody, they concluded that valence was of greater significance. bivalent antibody was found to be a t least -fold more efficient than monovalent antibody. since the forward reaction was extremely rapid, these investigators considered that the neutralization rate reflected the rate of dissociation of virus-antibody complexes. hornick and karush ( ) studied the neutralization of dnp+x bacteriophage. among other observations, they reported that bivalent antibody was much more efficient (lo*) than monovalent antibody, again stressing the importance of valence. a survey of the literature, or of the reviews of the literature, indicates that several different phenomena are generically grouped as viral neutralization. justification for this is found in the fact that there is a measure of consistency among all insofar as virus, antibody, and host indicator are, in all instances, part of the phenomenon, and that the criterion is, for all, loss of infectivity. a closer examination indicates that the underlying interactions have important differences. that there is an awareness of this consideration is reflected in the use of the plural form when daniels ( ) and burns and allison ( ) refer to "mechanisms" of neutralization in their discussions. it has been shown that neutralization may require the contribution of mediators, e.g., complement, or may depend on secondary phenomena such as aggregate formation. it may therefore be useful for the purpose of analyzing reaction mechanisms to define the various situations. a. primary. at the simplest level, a virion composed of multiple copies of a single antigen is neutralized by one or more molecules of a single molecular species of antibody. this interaction is independent of any "third-party" mediation and of secondary events such as aggregation. the possibility that formation of the virus-antibody complex is the initial stage of sequential rearrangements within the complex is, however, recognized as a segment of a single event, the possibility that the neutralized state is conditional, namely, dependent on the host, is an unavoidable, but possibly explicable and informative, complication. b. mediated. a primary reaction that fails to go to completion may be activated by interaction of the bound antibody with complement, or interaction with an antibody to itself. c. biphasic. two antigen-antibody reactions are involved. the first reaction enables the second reaction to take place, which culminates in neutralization. a. steric hindrance. involvement of a critical antigen does not occur when the virion is neutralized. interaction of noncritical antigens with antibody results in the amassing of antibody that interferes, possibly by preventing adsorption, with viral replication. b. virolysis. interaction of enveloped viruses with envelope-specific antibody (which is also host-specific) in the presence of the complete complement system results in irreversible traumatization of the envelope. since these viruses can also be neutralized by .antibodies that are specific for virus-coded antigens, virolysis represents an alternative neutralization pathway. since viruses are multivalent and antibody bivalent, it is possible to induce aggregate formation by appropriately adjusting the concentrations of the reactants. the interaction of antibody with noncritical sites does not lead to neutralization. however, if the concentrations are equivalent, secondary events will occur, resulting in the formation of a lattice structure. reduction in infectivity may follow simply as a result of the polymerization of several monomeric units into a single infectious unit. one of the conclusions burnet et nl. ( ) arrived a t in their studies of several bacterial and animal viruses was that the two groups differed fundamentally in their interaction with antibody. whereas bacterial viruses formed irreversible complexes with neutralizing antibody, animal viruses (those examined by burnet et al.) formed freely reversible complexes (burnet, ) . the assay inethods employed were at least formally comparable, namely, the plaque method for bacteriophages and the then newly developed pock-counting method utilizing the chorioallantoic membrane of embryonated chick eggs for animal viruses. subsequent studies (dulbecco et az., ) of two animal viruses, poliovirus and wee virus, employing the plaque assay method with animal cell cultures, indicated that the reaction of these viruses with antibody was irreversible. reversibility, in the present context, represents a dynamic equilibrium between reactants and product with the final equilibrium depending upon association and dissociation rates, which in turn are concentration-dependent. the demonstration of reversibility requires that the sole variation in the reaction conditions be a change in the concentration of the reaction components, usually by dilution. such a change should then drive the reaction toward dissociation until reestablishment of the equilibrium characteristic for the particular system. this aspect of viral neutralization has been under examination from the time that neutralization was first subjected to experimental scrutiny. interpretation of early studies indicated that reversibility was characteristic of the neutralization of fowl plague virus (todd, ) , vaccinia virus (andrewes, ; olitsky, \, eee virus (pierce et al., ) , papilloma virus (bryan and beard, )' influenza virus (taylor, ; burnet, burnet, , , and poliovirus (gebhardt and bullock, ) . however, the reaction was considered irreversible for papilloma virus (friedewald and kidd, ) and eee virus (labzoffsky, ) , and partially reversible for influenza virus (taylor, ; burnet, ). it has also been reported that the extent of dilution dissociation decreased with prolonged incubation (andrewes, ; burnet, ) . this aspect of viral neutralization was examined by gard ( ) using a particularly appropriate system, namely, a strain of theiler's virus (fa strain of mouse poliomyelitis) which could be assayed in mice with or without dilution based on the incubation period. he observed that, whereas the undiluted virus-serum mixture indicated neutralization, the diluted mixture did not. however, after hours a t °c or days a t "c, dilution of the reaction mixture showed the expected decrease for a nondissociating complex. gard proposed that the interaction between virus and antibody entailed two stages-a rapid but weakly cohesive union followed by enhancement of the forces binding virus and antibody. it may be relevant that the source of antibody in this study was serum from normal mice. it was shown by olitsky ( ) that normal mice almost universally carry the to strain of theiler's virus. antibody in the sera of these mice is to-specific. the extent of cross-reactivity between the two strains may be a contributing factor to this phenomenon which gard called "immunoinactivation." however, in subsequent studies with human poliovirus and homologous antibody, gard ( ) observed the same immunoinactivation characteristics. using the plaque assay for animal viruses employing animal cell cultures, dulbecco et a . ( ) analyzed the reaction of wee virus and poliovirus with antibody. with respect to dilution dissociation, they reported that the phenomenon did not occur with these viruses. studies of the reversibility of these and other viruses employing cell cultures as the test system indicated no spontaneous dissociation for ndv (rubin, ; rubin and franklin, ; granoff, ) , poliovirus (mandel, (mandel, , ketler et al., ; keller, ; wallis et al., ), adenovirus (kjellh, ), visna virus (thormar, , herpesvirus (yoshino and taniguchi, b ashe and notkins, ) , or vee virus (hahon, ) . the possibility that dissociation occurs but to a minor degree was considered for jev (hashimoto and prince, ; iwasaki and ogura, b) and was proposed for herpesvirus (ide and yoshino, ) and w n virus (westaway, a) . the data from which dulbecco et al. ( ) concluded that the reactions were irreversible were reinterpreted by fazekas de st. groth and reid ( ) to show that the results were consistent with a reaction which is freely reversible and subject to mass-action phenomena. reaffirmation of the view that a freely reversible equilibrium characterizes virus-antibody interaction resulted from studies on neutralization of influenza virus (fazekas de st. groth and webster, ) . the complexity of the neutralization phenomenon has been stressed by burnet ( , p. ) , dulbecco et al. ( , fazekas de st. groth and reid ( ) , and fazekas de st. groth ( ) . it, is clear that neutralization consists of three domains-virus, antibody , and assay h o s t a n d each domain can be divided into subdomains of varying characteristics. to illustrate, svehag ( ) compared the dissociability of poliovirus-rabbit antibody complexes when antibody was either early or late . in neither case was dissociation evident with simple dilution. when dilution was carried out in conjunction with increasing ionic strength, minor but significant dissociation occurred when virus was complexed to s antibody. with antibody no dissociation occurred when salt molarity was increased from . m to . m . the bond strength of the complex is a function of antibody type. the results of svehag ( ) indicate that with late antibody the affinity constant is exceedingly high. in studies with poliovirus (mandel, ) it was shown that, when centrifuged neutralized virus was suspended in antibody-free medium, no evidence for reversibility was obtained over a period of days a t °c. both diluted and undiluted resuspended virus was monitored. neutralized virus was prepared using varying antiserum multiplicities that resulted in surviving fractions as high as %. potentially dissociable virusantibody coinplexes were demonstrated by acid reactivation of infectivity. an analysis of dissociability on a molecular level was described by lafferty ( a,bl . the reaction of influenza virus with antibody was biphasic with respect to time. shortly after initiation of the reaction, the virus-antibody complex was completely reversible by dilution. given sufficient time, however, the complex became irreversible. when monovalent antibody fragments were used, virus-antibody complexes were formed but failed to become irreversible. lafferty ( a,b) proposed that the transition to irreversibility represented the time required for the second combining valence of a bound antibody molecule to bind to an adjacent antigenic site on the same virion. that such monogamous bivalent binding occurred was demonstrated by electron microscopy (lafferty and oertelis, ) . reversibility therefore requires the simultaneous dissociation of both antibody-virus bonds. the probability of such an event is proportional to the square of the probability of the single event. lafferty ( a) also suggested that close scrutiny of the methodologies employed in studies of neutralization could reconcile the two views, i.e., that a t least some features of a freely reversible system are characteristic of virusantibody interaction. the preponderance of recent evidence points to the virus-antibody (late) complex as a firmly bound structure. under certain limited conditions, dissociable complexes can actually be demonstrated. under other conditions dissociation is an artifact inherent in the experimental procedure. the initially formed monovalent complex is readily dissociable. with time, as bivalent binding occurs, the complex becomes stabilized. the degree of stabilization has been estimated to be several orders of magnitude increase in affinity constant. even after stabilization a very small fraction of the neutralized population is readily dissociable by dilution, and the presumption is that virus has reacted with antibody of very low affinity. the rate of neutralization depends on antibody concentration. when testing for dilution dissociation, the procedure is to prepare several dilutions of a virus-serum mixture and assay each for infectivity. if dilutions are made while the reaction is in progress, the rates will diminish in proportion to the dilution. after inoculation of the test host, the reaction continues until vinis has interacted with susceptible cells. moreover, the presence in the host of antiserum in different concentrations tends to bias the results additionally. the net result can be a disproportionality of surviving virus with dilution, which in fact is due t o decreased n e b tralization rates rather than dissociation. one result of the studies of dulbecco et al. ( ) on the neutralization of animal viruses was to focus attention on a phenomenon they called the "persistent fraction." the observation that a fraction of a viral population was refractory to neutralizing antibody had already been described by andrewes and elford ( ) ) for several bacteriophages, and by burnet et al. ( ) for several aniinal viruses. with respect to the phages they had examined, andrewes and elford proposed that failure to be neutralized was not attributable to genotypic resistance, nor to the tendency for virus-antibody complexes to dissociate. a reasonable explanation for this phenomenon, in the view of andrewes and elford, was that combination with antibody had occurred hut without sufficient effect to negate infectivity. this view was supported by observations that ( ) abnormally small plaques were produced, and ( ) infectious particles were nonfilterable in contrast to the control virus. these surviving particles were designated "incompletely neutralized." as andrewes and elford interpreted this phenomenon, a small number of antibody molecules had combined with antigenic determinants of the virus which were distinct from the host combining determinants. the presence of antibody constituted a steric encumbrance, thereby retarding the initiation of replication, hence small plaques were produced. the demonstration of a "persistent fraction" by dulbecco et al. ( ) for animal viruses by means of plaque assay method stimulated renewed interest in this phenomenon. although dulbecco et al. eliminated several explanations, e.g., dissociation, participation of nonantibody serum components, and hereditary heterogeneity, a positive explanation had yet to be found. subsequent studies indicated that this phenomenon was characteristic of a wide variety of viruses (wallis and melnick, ) , e.g., picornavirus (dulbecco et al., ; mandel, ; bradish et al., ; wallis and melnick, ; ozaki, ; fiala, ; lewenton-kriss and mandel, ) , myxovirus (lafferty, a,b) , paramyxovirus (granoff, ), poxvirus (lafferty, a,b ; mcneill, ; majer and link, ) , togavirus (dulbecco et al., ; hashi-mot and prince, ; rawls et ae., ; ozaki and tabeyi, ; westaway, ; hahon, b; symington et al., , herpesvirus (yoshino and taniguchi, b) , equine arteritis virus (hyllseth and petterson, , reovirus (huggett et al., ) , and bacteriophages (hale et al., ; rappaport, ) . kjellh ( ) observed a gradual increase in resistance to neutralization the longer adenovirus reacted with antiserum. he considered this result to be indicative possibly of a requirement for increased antibody multiplicity, but not because of dissociation or refractoriness. the practical significance of this phenomenon was emphasized by several reports showing the presence of infectious virus-antibody complexes in the circulatory system of ldh virus-infected mice (notkins et al., ) , visna virus-infected sheep (gudnad ttir and pblsson, ), (toolan, ) , lcm-infected mice dixon, , ) , mink infected with aleutian disease (porter and larson, ) , and mlv-and msv-infected mice (hirsch et al., ) . the "persistent fraction" has also been referred to as the nonneutralizable fraction. since, as discussed in section iv, this fraction can be neutralized under certain conditions, it seems more appropriate to designate it the nonneutralized fraction. several basically divergent hypotheses have been proposed to explain the presence of a nonneutralized fraction (this designation is used in preference to "persistent fraction"). fazekas de st. groth and reid ( ) ascribed it simply to a reversal of the neutralization reaction, i.e., dissociation. wallis and melnick ( , ) considered that no unusual interaction between virus and antibody occurred. however, the formation of aggregates, either before or during interaction with antibody, resulted in the shielding of some particles from antibody. a somewhat similar view was proposed by rappaport ( ) differing, however, in one respect, namely, that for a single virion, binding of antibodies may occur to most antigenic determinants, leaving others free and inaccessible, hence the particle retains infectivity. bradish et al. ( ) proposed an "amphoteric" state for some virus-antibody complexes such that either viral or antibody function dominated, depending on various contributory factors, e.g., the type of host cell comprising the test system. a similar interpretation was proposed by lafferty ( a) for rabbit poxvirus. heterogeneous antibody populations in a given serum with the capacity to neutralize, or to induce the nonneutralized state, were considered a possibility (lafferty, a,h ; mcneill, ; lewenton-kriss and mandel, ; ozaki et al., ) . all hypotheses, except that of fazekas de st. groth and reid ( ) , envision the nonneutralized fraction as consisting of virus complexed with antibody without absolute loss of infectious capability. recently, ide and yoshino ( ) revised their previous interpretation of the nonneutralized state of herpesvirus and concluded, as did fazekas de st. groth and reid, that a freely reversible equilibrium could account for their findings. wallis and melnick ( ) reported that the previously observed (wallis and melnick, ) resistance of a strain of echovirus to neutralizing antiserum could be overcome by eliminating viral aggregates. in a monodispersed state, virus was readily neutralized. extrapolation of this possibility to representatives of various groups of viruses revealed t,hat in every case filtrates consisting of singly dispersed particles could be completely neutralized, whereas unfiltered virus showed nonneutralized fractions (wallis and melnick, ) . further analysis of this problem disclosed that, when monodispersed herpesvirus was exposed to minimal concentrations of antibody, the resultant virus-antibody aggregates became nonneutralizable. attempts by other investigators to eliminate the nonneutralized fraction by the method of wallis and melnick met with varying results. a reduction, but not complete elimination, was reported for vee virus (hahon, b) and rhinovirus (fiala, ) . in other studies it was reported that filtration had no effect on the nonneutralized level or neutralization characteristics (ashe et al., ; hyllseth and petterson, ; majer and link, ; huggett et al., ; lewenton-kriss and mandel, ) , although elimination of a short lag period (majer and link, ) and a slight increase in neutralization rate (lewenton-kriss and mandel, ) were observed. baughman et al. ( ) described a complex result with respiratory syncytial virus. filtration considerably reduced the nonneutralized fraction when horse antiserum was used. however, when guinea pig serum was used, no difference was seen between filtered and unfiltered virus. several studies focused on the role of the host cell, probably because of the striking illustration by kjell n and schlesinger ( ) that the outcome of virus-antibody interaction was affected by the host cell system serving as the indicator of neutralization. bradish et al. ( ) observed that aliquots of f m d virus-antiserum mixtures assayed in pig kidney cell cultures and in mice showed -fold lower viral survival in the latter host. a similar observation was described by lafferty ( a) with rabbit poxvirus. survival was considerably lower when tested in rabbit skin, compared with mouse brain or chorioallantoic membrane. i n another study on the neutralization of poxvirus (mcneill, ) , the neutralization rate as well as the nonneutralized level varied according to whether assays were done with monkey kidney or hep cells, the latter showing a higher rate and lower final survival level. when the outcome of neutralization of poliovirus was assayed in hela or monkey kidney cell cultures, the results were the same (dulbecco et al., ; lewenton-kriss and mandel, ) . however, the interesting observation was made (lewenton-kriss and mandel, ) that virus propagated in hela cells yielded a considerably higher nonneutralized fraction than virus propagated in monkey kidney cells, both having been assayed in hela cells. it was also shown in the same study that results of mediated neutralization varied with the host cell, i.e., primary neutralization results were the same for hela and monkey kidney cells, but antiglobulin neutralization was seen only with hela cells. hahon ( b) surveyed five different cell lines and found the same level of nonneutraliaed vee virus with each. these studies, as stressed by bradish et al. ( ) , strongly imply that nonneutralizability is a relative condition, a state of limbo, which is dependent on the host cell for the properties that the virus-antibody complex will manifest. considerable interest has centered on the role of antibody in establishing the nonneutraliaed state. evidence was presented for poliovirus (mandel, ) and ldh virus (notkins et al., ) that nonneutralized virus reacted with antibody, since antiserum prepared against the globulin fraction of the viral antiserum neutralized all or part of the nonneutralized fraction. it became of interest to determine ( ) if virus that was not neutralized had reacted with a unique class of antibody, or ( ) if the antibody was conventional but the antigen with which it combined was not essential for infectivity, or ( ) if the characteristics of the interaction per se were unique. recognition of an infectious virus-antibody complex was based on neutralization mediated by antibody specific for the antiviral serum globulin, namely, mediated neutralization. a somewhat puzzling observation was described by capstick et al. ( ) and bradish et al. ( ) . the size of the nonneutralised fraction of fmd virus was inversely related to serum concentration. a recent report (symington et al., ) described in more detail a similar observation, namely, at high-and low-antibody multiplicity the nonneutralized fraction of sindbis virus was small, but at intermediate multiplicities the fraction was large. these workers considered the situation to be complex in that the serum contained antibodies of different avidities in unequal concentrations, and that the different antibodies either neutralized or protected against neutralization. with varying concentrations of antiserum one or the other kind of antibody had a competitive advantage. in studies with poliovirus evidence was presented that implicated antibody heterogeneity. ozaki ( ) , svehag ( ) , and lewenton-kriss and mandel ( ) showed that the level of nonneu-tralized virus was highest with the earliest collected antiserum and decreased the later this antiserum was collected during the immune response. mcneill ( ) suggested the possibility of a kind of antibody that interfered with neutralizing antibody but did not elaborate on the nature of this putative antibody. in studies on rabbit poxvirus, lafferty ( a) concluded that the nonneutralized state was attributable to the interaction of virus with either of two kinds of protective antibody. in one case virus reacts with nonavid antibody. such a complex is readily reversed by washing or by dilution. i n the second case, virus binds firmly and retains its protected status, in each case, the complex is conditionally infectious, depending on the host system. ozaki ( ), svehag ( ) , and lewenton-kriss and mandel ( ) showed that the level of the nonneutralized fraction of poliovirus decreased as antisera were collected a t later times during the immune response. westaway ( ) and ozaki et az. ( ) reported similar observations for several togaviruses. it has been shown (svehag, ) that the chronology of the immune response can be characterized by successive changes in the class of antibody produced, as well as qualitative changes within a class. antibodies produced early are predominantly of the macroglobulin ( s) type with low binding affinity. these are succeeded by the type which tends, with time, to increase in binding affinity. a similar relationship was shown to exist in the response of individuals actively infected with poliovirus (brunner and ward, ; ogra et al., ) , arbovirus (bellanti et al., ) , mumps virus daugharty et al., ) , coxsackievirus (schmidt et al., ) , or influenza virus (brown and o'leary, . these observations tend to incriminate a specific type of antibody as the cause of the nonneutralized state. hahon ( b) prepared nonneutralized vee virus with human antiviral antiserum. for mediatcd neutralization he prepared anti-human igg, iga, and igm in goats. the anti-igg and anti-iga sera were effective, the latter somcwhat less. the anti-igm had no secondary neutralizing activity. it was also shown that goat anti-human fab and anti-fc sera had secondary neutralizing activity, the latter quite weak. these results implicated igg and iga as nonneutralizing antibodies. the absence of anti-igm activity indicated cithcr that this class of antibody was not involved, or that it was absent in the virus-specific antiserum. a similar result was described by notkins et az. ( ) in their studies on ldh. virus was sensitized with mouse immune serum. goat anti-mouse globulin neutralized the sensitized virus. rab-bit anti-mouse igg, iga, and igf neutralized sensitized virus, but rabbit anti-mouse igm was ineffective. in another aspect of these studies, it was shown that the monovalent fab fragment of goat anti-mouse globulin partially neutralized and blocked the remainder against neutralization by the undigested anti-mouse globulin. ozaki et al. ( ) obtained antisera to jev early ( days) and late ( days). neutralization showed nonneutralized fractions in each case, the level being higher with the early serum. neutralizing antibody in the early and late sera were igm and igg, respectively. goat anti-rabbit igm serum neutralized % of the nonneutralized fraction when early serum was used, whereas anti-igg was without effect. when virus was neutralized with late serum, anti-igg reduced the nonneutralized fraction slightly, but the anti-igm not a t all, the same specificity was seen when direct neutralization was carried out by fractionated antiviral igm and igg then challenged by the fractionated anti-antibodies. the possible occurrence of a unique class of antibody that can specifically induce the nonneutralized state was investigated by attempting its isolation (lewenton-kriss and .mandell ). an early ( -day) rabbit poliovirus antiserum was fractionated by three unrelated procedures: ( ) gradient centrifugation, ( ) chromatographic separation using sephadex for molecular sieving, and ( isoelectric focusing electrophoresis. i n all instances in which antibody activity was detected, neutralization was accompanied by a nonneutralized fraction. the igg and igm fractions of a horse anti-rabbit -globulin each had secondary neutralizing activity, and the igm fraction was the more active on a molar basis. a further implication of the role of antibody in nonneutralizability was described by hashimoto and prince ( ) . the resulting nonneutralized fraction when bev reacted with rabbit antiserum in excess could be neutralized in part by guinea pig antivirus antiserum. however, the resulting nonneutralized fraction when guinea pig antiserum in excess was used was not affected by the rabbit antiserum. perhaps the guinea pig antiserum contained a low level of functional complement. it has also been shown for poliovirus (svehag, ; eewenton-kriss and mandel, ) and for jev (ozaki st al., ) that the nonneutralized fraction induced by an early rabbit immune serum cannot be neutralized by a late immune serum from the same rabbit. i n all instances direct use of the late serum reduced infectivity to a considerably lower level than the level of the nonneutralized fraction resulting from the use of early serum. this observation is not consistent with a dissociation hypothesis, since the later appearing antibody with its greater binding affinity should be able to replace the weaker early antibody. rappaport ( ) proposed that the nonneutralized condition was the result of incomplete binding of antibody to all antigenic determinants. he showed that the nonneutralized fraction of ms (an icosahedral rna bacteriophage) represented particles which were about half-saturated with antibody. hence these particles were protected against additional binding of antibody, yet they retained infectious capability. i n their studies with herpesvirus, yoshino and morishima ( ) arrived a t a similar interpretation. fazekas de st. groth and reid ( ) proposed that the data of dulbecco et nl. ( supported the dissociation hypothesis for the nonneutralized fraction. ide and yoshino ( ) reevaluated their interpretation of the nonneutralized state for herpesvirus in favor of the dissociation hypothesis. the experimental basis was the observation that the level of surviving virus diminished when the filtrate of a virus-serum mixture was incubated. these workers considered this evidence for reequilibration, since virus-antibody complexes, but not antibody or free virus, were retained on the filter. failure to alter the nonneutralized fraction by altering the concentration of antibody was explained on the basis that equilibrium was established between antibody and the total number of critical reactive sites rather than the number of viral particles. hence, for example, considerable dilution and possibly considerable time may be required for detectable signs of dissociation (i.e., increase in infectivity). the correlation between a high nonneutralized fraction and early serum, together with the known low affinity of early antibody, either igm or igg, suggested the possibility that the nonneutralized fraction of poliovirus was the result of a defective reaction (lewenton-kriss and mandel, ) . there is evidence that neutralization is the culmination of sequential events beginning with the union of virus and antibody. it has therefore been proposed (lewenton-kriss and mandel, ) that, either because of a defect in antibody or because of improper binding of antibody to virus, the reaction does not go to completion. addition of antibody specific for the virus-associated antibody activates the aborted reaction. a similar interpretation has been applied to mediated neutralization of the nonneutralized fraction of herpesvirus (yoshino and isono, ) . studies by stemke and lennox ( on the neutralization of bacteriophage t disclosed that the fab fragments derived from hyperimmune rabbit antiseruni had neutralizing activity. although the fab i fragment had a higher neutralizing capacity than the fab i fragment, the addition of antiglobulin (goat anti-rabbit globulin) increased neutralization of both to the same final level. it was therefore concluded that both fragments bound equally well quantitatively. however, the fab i fragment had a lower neutralization potential. the possibility was considered that distinctive binding sites existed for the two fragments. antiglobulin was used by goodman and donch ( ) to demonstrate that l chains derived from rabbit anti-tl serum reacted with t bacteriophage. per se, i, chains had questionable neutralizing activity, but activity was enhanced to an appreciable level by goat anti-rabbit globulin. a kinetic examination of the effect of antiglobulin on bacteriophage neutralization (dudley et al., ) showed that the rate of neutralization of bacteriophage f (an icosahedral rna virus by s antibody, pepsin-derived fragment, or . fragment derived from the fragment was enhanced about three-fold by the addition of antiserum against the respective globulin fractions. evidence has been presented in support of the various hypotheses, although interpretations and attempted confirmations have not in some instances led to the same conclusions. i n a broad sense, the hypotheses reduce to two: ( ) the nonneutralized fraction is a result of the dissociation of virus-antibody complexes; and ( ) virus has reacted with antibody (a without losing infectivity and (b) acquiring a protected status. in support of the latter, evidence. has been presented that clumping may be responsible. evidence has also been presented that single particles are involved, since monovalent fragments of viral antibody as well as monovalent fragments of the anti-antibody participate in this phenomenon. although the cluinping hypothesis provides a possible explanatipn for the protected status, no explanation is evident when monodispersed virus is in the nonneutralized state. possibly, on the abortive reaction hypothesis, a stage in the reaction sequence has been reached which is short of ncutralization but renders the viral capsid nonantigenic. studies on bacterial viruses have shown that a nonneutralized fraction occurs. from the early studies of andrewes and elford ( s) to recent studies (e.g. , hale et al., ) the presumption has been that the non-neutralized fraction is the result of an unusual virusantibody reaction. as with animal viruses, the nonneutralized fraction is subject to neutralization by anti-antibody. also, as with animal viruses, some reports indicate that igm antibody is involved, whereas other reports state that igg antibody is involved. however, there is unanimity in the observation that antibody synthesized early, rather than late, in the immune response is more responsible for the nonneutralized state. since it has also been a common observation that early antibodies have a relatively weak binding capability, it is this characteristic that is most likely directly concerned with nonneutralization. studies with antibody fragments (stemke and lennox, ) have shown that, although fab i has a higher neutralizing capability than fab i, both interact with virus to the same degree as shown by mediation with anti-antibody. the interaction of fab i with antigen may be less likely to result in neutralization than the fab i interaction. it is also worthy of note that aggregation cannot be the basis of this interaction, since these reactions involve monovalent fragments. at the present time there are no experimental data that can account for the refractory state of the nonneutralized fraction. possibly the infectious virus-antibody complex is sterically hindered, or the antigenicity of the capsid has been modified. that virus could react with antibody without loss of infectious capability was suspected by andrewes and elford ( a) . evidence in support of this presumption was reported for poliovirus (mandel, and ldh virus (notkins et al., ) . poliovirus that survived exposure to rabbit antiserum, and ldh virus that survived exposure to mouse antiserum, were neutralized by anti-rabbit globulin and anti-mouse globulin sera, respectively. notkins et al. ( ) referred to infectious virus-antibody complexes as "sensitized virus." the appropriateness of this designation is indicated by the observations that such complexes are sensitive to several other factors, sensitivity being manifested by loss of infectivity. the involvement of the complement system in viral neutralization was suspected as early as . gordon ( ) reported the loss of neutralizing activity when sera from rabbits immunized with vaccinia virus ~ were heated. however, the loss could be restored by the addition of fresh normal guinea pig serum. similar observations were reported by douglas and smith ( ), tanaka ( ) , and imagawa ( , implicating the presence of a thermolabile accessory substance in sera derived from animals immunized with vaccinia virus. a more detailed investigation of this phenomenon was described by mueller ( ) . the presence of a thermolabile substance that participated in the neutralization of rous sarcoma virus was shown to be present in the sera of geese, ducks, chickens, and rabbits that had been immunized with rous virus. neutralizing activity was lost as a consequence of heating ( °c for minutes) but could be restored by the addition of guinea pig serum if fresh, but not if heated. these results prompted mueller to suggest that the thermolabile substance was alexin (i.e., complement). aware of a probable role for complement in the neutralization of mumps virus by human sera, leymaster and ward ( ) stressed the need to preserve such activity by appropriate handling procedures. of interest was their observation that sera from individuals as long as years after inuinps infection still showed a dependence on a thermolabile serum component. a similar caution was expressed by pollikoff and sigel ( ) with respect to sera with neutralizing activity for lcm virus (an arenavirus). another instance of dependence of antiserum on a thermolabile serum factor was reported by adams and imagawa ( ) involving sera from ferrets immunized with a mouse-adapted strain of distemper virus. howitt ( ), however, was unable to detect a neutralizing accessory substance in sera of horses that had been immunized with equine encephalomyelitis virus. neutralizing activity was affected neither by heat ( °c for minutes) nor by addition of complement. these results do not necessarily challenge the credibility of previous observations since, in the light of more recent knowledge, the stage in the antibody response that is complement-dependent probably had been passed since, as howitt stated, the animals had been hyperimmunized. the independence of neutralizing activity was also reported (strong, ) for human sera capable of neutralizing a presumable herpes-like virus, virus w. neither heat nor known complement-deactivating procedures had any effect on neutralization. another study that questioned the role of complement, but not the presence of an accessory substance, was reported by ginsberg and horsfall ( ) . sera from normal (i.e., not knowingly exposed or immunized to the viruses under study) humans, rabbits, guinea pigs, and mice with neutralizing activity for influensa virus, mumps virus, and ndv lost activity upon heating ( °c for minutes), storage ( °c for weeks), depletion of ca +, or decomplementation. sera devoid of activity as a result of heat or storage were reactivated by the addition of fresh serum from a child with no prior history of mumps or influenza infection. although these investigators were cognizant of the similarities in characteristics of complement and the accessory substance, they considered the latter to be distinct from complement because of certain quantitative discrepancies. whitman ( ) suspected the presence of a neutralizing accessory substance in serum from an individual convalescing from infection with wee virus when neutralizing activity decreased disproportionately with serum dilution. suspecting the presence of an accessory factor, he confirmed his suspicions by demonstrating its thermolabile character and the augmentation of neutralizing activity by the addition of guinea pig serum to heated ( °c for minutes) immune serum. in spite of the similarities between complement and the accessory substance, whitman conservatively eschewed a specific label for the active material. somewhat earlier, morgan ( ' had reported that discrepancies in the quantitative aspects of the neutralization of wee virus by immune rabbit sera were related to the use of fresh or heated serum, and that the addition of complement to the latter tended to eliminate the discrepancies. another togavirus (dengue) was reported (sabin, ) to be neutralized by complement-dependent antibody present in the serum of a convalescent monkey. noting that previous studies had led to discrepant conclusions about the presence and nature of substances in immune sera that acted in conjunction with specific antibody, leymaster and ward ( ) attempted to refine the experimental system by using a virus, mumps, capable of being assayed in a host devoid of complement, i.e., an embryonated chicken egg. sera from immunized monkeys, or from human cases of mumps infection either in the acute or convalescent stage, contained neutralizing activity that was nullified by heating or by decomplementation. the addition, in either case, of fresh sera obtained from children less than years of age restored neutralizing activity. these workers attributed the restoration to complement. i n the studies cited thus far, the viruses under investigation were myxoviruses, togaviruses, or poxviruses, all of which are virions containing lipid-rich envelopes. that the presence of an envelope is not required for complement-dependent neutralization was shown by several studies involving envelope-free bacterial viruses. i n , hershey and bronfenbrenner described the participation of complement in the neutralization of two coliphages. an enigmatic aspect of this brief report indicated that in some instances a virus-serum mixture that had reacted to completion showed partial reactivation upon subsequent addition of complement. participation of complement in the neutralization of bacteriophages has also been reported by barlow et a . ( , cowan ( ) , toussaint and muschel ( ) , pernis et al. ( ) , and adler et al. ( ) . harris et al. ( ) reported the induction in humans and mice of complementdependent antibody following immunization with bacillus megatherium bacteriophage. of interest was their observation that the addition of normal, fresh mouse serum in moderate concentration to heat-inactivated mouse antiserum restored activity. however, a t a high concentration no reactivation ensued. in order to establish more rigorously that the identity of the thermolabile accessory factor was complement, dozois et al. ( ) fractionated a rabbit serum containing antibody to wee virus as well as accessory factor. removal of the complement system depleted the serum of neutralizing activity. by rcconstituting the serum the neutralizing activity was restored, and the details of the reconstitution procedure indicated that components , , and of the complement system were essential, whereas component was either not required, or required in trace concentrations. studies on two other togaviruscs, murray valley encephalitis and wn encephalitis, were described by westaway ( a). unlike wee, murray valley encephalitis virus was neutralized only slightly more efficiently with added complement, whereas wn virus neutralization was perhaps slightly inhibited by the addition of guinea pig serum. another instance of complement-independent neutralization of a togavirus, jev, was reported by hashimoto and prince ( ). the addition of fresh guinea pig serum to either early ( -day) or late ( -day) immune rabbit sera affected neither the rate nor the degree of neutralization. an unusual observation in these studies was the spontaneous partial reactivation of neutralized virus relatively late in the kinetic analysis of the reaction. ozaki and tabeyi ( ) observed marked enhancement of neutralizing actvity of jev by fresh guinea pig serum. the rate and extent of neutralization were both increased. this result is in contrast t o the results reported by hashimoto and prince ( ). of possible relevance to the different results are the observations of iwasaki and ogurs ( s) that jev grown in porcine cells induced complement-dependent antibodies specific for viral antigens as well as antibodies specific for host lipoproteins comprising the viral envelope. however, virus grown in mouse brain was insensitive to complement-dependent antibody. whereas ozaki and tabeyi ( ) used virus cultivated in porcine cells, hashimoto and prince ( ) used virus derived from infected suckling mouse brain. baughman et al. ( ) reported that antibodies of guinea pigs and ferrets immunized with respiratory syncytial virus were markedly complement-dependent. although antibody from horses appeared to be inde-pendent by end point analysis, a complement effect was observed as an increase in the neutralization reaction rate. it has been a common observation that sera obtained from normal individuals contain neutralizing substances, probably immature antibody, against an array of viruses, a rule of thumb to distinguish normal antibody from other inhibitory substances is the greater thermolability of the latter and the inability to replace the latter with complement. mccarthy and genner ( ) examined normal sera from guinea pigs, rabbits, and humans for neutralizing activity against variola virus, herpesvirus, and cowpox virus. positive sera lost some, but not all, activity after being heated a t "c, and activity was regained upon the addition of fresh serum. these investigators proposed that the positive sera contained two separate neutralizing factors acting independently. perhaps they envisioned two classes of antibody, one requiring complement and the other independent of complement, howitt ( ) surveyed the sera from various animal species for the presence of normal neutralizing activity against ndv. the species examined included some known to have high levels of complement, e.g., human, monkey, rabbit, and guinea pig, and species with little or no complement, e.g., ferret, hamster, and chicken. sera from human, monkey, rabbit, and guinea pig neutralized ndv when tested fresh, but lacked activity after heating ( °c for minutes). the absence of neutralhing activity in fresh sera from ferret, hamster, and chick, and the ability to negate activity by heating the sera from complement-rich species, suggested a role for complement in the neutralizing activity of normal sera. studies on the nature of the bacteriophage-neutralizing substance in normal human sera led muschel and toussaint ( i to conclude that t,he substance was antibody which was qualitatively indistinguishable from antibody appearing early during the immune response. in both instances, neutralizing antibody was complement-dependent. more recently, bendinelli et al. ( ) reported on the distribution of normal antibody among various species of animal. i n the presence of complement (endogenous or added) the sera from various species, but not from mouse, rat, and sheep, neutralized friend leukemia virus. bccause of sensitivity to heat ( oc for minutes), a requirement for divalent cation, and restoration of deactivated sera by the addition of complement, these workers considered thc neutralizing activity the result of complement-dependent antibody. several studies on rubella and equine artcritis virus (togaviruses of the alphavirus subgroup) have shown that antibodies to these viruses may be complement-dependent (neva and weller, ; parkman et al., ; leerhoy, ) . rawls et al. ( ) examined sera from an infant with rubella infection, from the mother of the infant year after the infection, and from an adult more than years after infection. i n each instance the addition of complement to the heated sera increased the degree of neutralizing activity. decomplementation resulted in reduced activity. it was of interest to these investigators that, unlike herpesvirus, rubella virus induced complement-dependent antibody long after infection. leerhoy ( ) observed that, whereas guinea pig and horse sera effectively enhanced the neutralizing activity of human serum for rubella virus, rabbit and calf sera were relatively poor. since horse sera lack c (leerhoy, ) , this component of complement does not participate in this system. however, maess ( considered c the limiting factor in the complement-dependent neutralization of equine arteritis virus. this conclusion was based on the distribution of activity following dialysis of a guinea pig serum with complement-effected activity. hyllseth and petterson ( ) also reported complement-dependent neutralizing antibody for equine arteritis virus in the serum of immunized rabbits as well as in the postinfection serum of horses. kinetic studies, using horse serum as the source of antibody, showed a very slow rate of neutralization. the addition of complement (i.e., fresh guinea pig serum) , . , or hours after the start of the homologous reaction resulted, in each instance, in rapid extensive neutralization. all reactions, homologous as well as complement-effected, were kinetically single-hit. radwan and burger ( a,b) demonstrated that antibody from horses, guinea pigs, rabbits, hamsters, and mice immunized with equine arteritis virus was complement-dependent. such dependent antibody was found to be late-appearing igg. early igg and igm were nonneutralizing, either per se or with complement. further studies (radwan et al., ) showed that the addition of complement to virus complexed with dependent antibody eventually resulted in lysis of the viral membrane. however, through the use of trypsin it was shown that neutralization preceded viral lysis. in previous studies on the neutrahation of a coronavirus, avian infectious bronchitis (berry and almeida, ), it was shown that complement-dependent neutralization occurred in the absence of viral lysis if the antiserum was devoid of antibody to the lipoprotein envelope. in the presence of such antibody, lysis was observed by electron microscopy. recent studies on complement-effected neutralization have focused on the role of the various components of the complement system. it is perhaps appropriate a t this point to outline the salient features of complement. for a very comprehensive recent exposition of this subject, the reader is referred to osler's review ( ) or, for a briefer discussion more oriented to the subject at hand, to . two reaction pathways have been defined-classic and alternative. the classic system consists of glycoproteins, of which are functional only when aggregated, for which caz+ is required. i n the presence of an antigen-antibody complex the first component, c , (a conglomerate of clq, clr, and cls), binds to the antibody of the complex. thereupon, components c , c , c , c , c , c , c , and c , in that order, bind successively to the forerunner complex. some of the components exist in a precursor state and are activated as a result of binding (e.g., c acquires esterase activity) or by an activated component (e.g., c undergoes partial cleavage by the activated c esterase) . for coniplement-induced cell lysis, the entire sequence of reactions is required after the initial reaction between antibody and a cellular surface antigen. as shown below, the entire complement system may not be required for complement-eff ected neutralization. the alternative pathway involves activation of properdin (a basic glycoprotein) and several additional cofactors present in serum. the final product of the alternative system is c and therefore is functional in the absence of c , c , and c . biochemical studies have elucidated the characteristics and isolation methods of the individual components, thus allowing an analysis of their individual roles. the discovery of specific inhibitors has added to the investigative versatility of these systems. for example, zymosan or cobra venom can destroy c , thus the classic pathway is nonfunctional. differential thermolability allows for selective inactivation of the alternative pathway ( °c for minutes) and the classic pathway ( °c for minutes). finally, sera can be obtained from animals with genetic defects, so that a specific component is absent. the complexity of virus-antibody-complement interaction is further compounded by the molecular heterogeneity of the different classes of antibody, their diverse reactions with viral antigens, and their differential capacity to activate the complement system. the first stage in complement activation is the binding of the c l q component to the fc hinge region of the antibody molecule. igm, being a pentamer of igg, has a far greater potential for activating complement. variation in capacity to activate complement exists among the subclasses of igg, e.g., igg , but not igg , from the same rat is capable. a study of human myeloma proteins (spiegelberg, ) revealed that iggl and igg are more active than igg , whereas igg is incapable of activating complement. the complexity of this system is further ramified by the variable sensitivity of a given complement system to antibodies of different origins. for example, guinea pig complement reacts maximally with homologous antibody, half as well with rabbit antibody, feebly with dog antibody, and not at all with chicken antibody. furthermore, the homologous reaction is not necessarily optimal, as shown by the threefold greater reactivity of rabbit complement with guinea pig antibody than with rabbit antibody (gigli and austen, ) . i n view of the complex nature of complement activation, failure to effect viral neutralization by complement may be due as much to an inappropriate system as to real insensitivity. in several of the previously cited studies, it was shown that the ability to effect viral neutralization with complement was related to the antibody developmental stage in the immune response. antibody that appeared early reacted with virus either without neutralizing or neutralizing poorly, but in either case the addition of complement enhanced the degree of neutralization. in contrast, late antibody was independent of complement. studies on the chronology of appearance of the different classes of antibody (uhr et nl., ; uhr, ; uhr and finkelstein, ; svehag and mandel, , a,b; graves et al., ; brown et al., ; mckercher and giordano, ; ogra et al., ) have indicated that igm ( s) antibody precedes igg ( ). however, osler ( ) indicated that, by the use of appropriate methods, the response to a nonviral iinmunogen (human serum albumin) in rabbits and guinea pigs within the first week was predominantly igg antibody. although the igg antibody exceeded by -fold the amount of igm (on a molar basis), the igg antibody was not detectable by the more conventional serological rcactions. it is likely therefore that the affinity of the very early igg antibody is of a very low order of magnitude. cowan ( ) has also qucstioned the interpretation of such data. i n some studies, detection of the early igm was based on its sensitivity to -mercaptoethanol. it has, however, been recognized recently that early igg has a similar sensitivity. in other studies recognition of the type of antibody was based on separation by density gradient centrifugation or by molecular sieving. yoshino and taniguchi ( ) reported that early antibody in rabbits elicited by immunization, or after corneal infection, with herpesvirus could neutralize only when complement was present. antibody appearing later was complement-independent. further studies (yoshino and taniguchi, al showed that dependent and independent antibodies were of the igg class, that both resisted thermal inactivation a t "c, or ooc for minutes, and resisted inactivation by reduction ( -mercapto-ethanol). it was also shown (yoshino and taniguchi, ) that antibodies induced in guinea pigs by immunization, and in humans following herpes infection, were initially dependent and later independent of complement for neutralizing activity. the additional observation was made that herpes antibody in normal individuals (i,e., not currently, or recently infected) is complement-independent. hence the presence of complementdependent neutralizing antibody is of diagnostic significance. a more detailed characterization of the antibody response was described by shinkai and yoshino ( a) . early sera from herpes-immunized rabbits and guinea pigs were fractionated. rabbit sera contained complement-dependent antibody of the igm, fast igg, and slow igg classes. the dependent antibody in guinea pig serum was principally slow jgg (i.e., igg ). late rabbit sera contained very little igm, and the igg antibody was no longer complement-dependent, unlike the guinea pig serum which continued to synthesize dependent igg. reimmunization of rabbits elicited a brief response of dependent igm and a burst of independent igg. following primary herpes infection in humans, three categories of antibody were described (heineman, )-totally complement-dependent, potentiated, and independent. the data do not permit an interpretation of "potentiated," but conceivably it refers to the presence of antibodies of both other categories. basically similar results have been described for bacterial viruses. hbjek ( , ) immunized newborn rabbits with bacteriophage t . sera collected , , and days later contained complement-dependent, thennolabile s antibody on day , complement-dependent antibody on day , and partially dependent antibody on day . this investigator suggested that the transition indicated the gradual enhancement of binding strength of antibody with time after immunization. in related studies hhjek ( ) and hltjek and mandel ( ) showed that, whereas normal or early immune antibody t o t was complement-dependent, normal or early immune antibody to bacteriophage x was independent. as a possible explanation for the difference in behavior of the two viruses, it was proposed that binding affinity of antibody to antigen was the determinant characteristic, and that affinity for the relatively simple, small, icosahedral x bacteriophage was sufficiently high not to require augmentation. these investigators stated that, when the source of normal serum was newborn pigs that had not been allowed to receive colostrum, the sera contained no neutralizing activity against either t or x . pernis et al. ( ) compared antibody to bacteriophage t obtained from normal newborn, normal adult, and immunized adult rabbits. neutralization with newborn serum required complement, unlike both adult sera. irreversible (by addition of fresh serum) loss of activity by heating ( °c for minutes) occurred with newborn and adult normal sera. treatment with edta or zymosan eliminated activity from both normal sera. these workers attributed complement-dependent neutralization to thermolabile s antibody. the results of treatment implicate the early components (cl, c , and c ) of the classic pathway in complement-effected neutralization. a recent study by schrader and muschel ( ) on phage tzrabbit antibody interaction indicated that antibody (collected over a period of - days after a single immunization) was dependent on the c component only. interestingly, they observed that the addition of both c and c was less effective than the addition of c alone. neutralization by s antibody was not activated by c but only by the complete complement system (i.e., whole guinea pig serum). studies on the complement dependence of herpes antibody induced in rabbits revealed the interesting observation that antibody obtained after hyperimmunization was independent of complement when tested for end point titer. however, the same antibody when assayed by kinetic analysis showed an increase in reaction rate constant in the presence of complement. in their studies on the serological relationship between human and simian herpesviruses, stevens et al. ( ) observed, as others have, the dependence on complement of early and s antibodies. late antibody of the type "matured" to independence, unlike late antibody. previously, westaway ( b) had examind the serological relationships among group-b arboviruses (flaviviruses) , inclusion of fresh normal rabbit or guinea pig sera in the neutralization reactions enhanced the quantitative results. however, westaway questioned the desirability of this procedure, in this case, inasmuch as the effect of enhancement on the specificity of the reaction was not known. several studies on neutralization of cmv (currently classified as herpesvirus) have yielded divergent results. in rabbits immunized with a strain specific for humans, andersen ( ) observed the early appearance of complement-fixing antibody which was incapable of neutralizing even in the presence of complement. although late sera contained neutralizing antibody, complement was required as late as weeks after hyperimmunization. pursuing these studies, andersen ( ) described the failure to detect neutralizing antibody, either complement-dependent or independent, in early sera of infected human subjects, although complement-fixing antibodies were present. antisera in rabbits were prepared with two human strains (ad and c ) and two simian strains (davis and t ). with the human strains, late sera contained complement-independent antibody, but with the simian strains antibody was dependent on complement for neutralization. in another study graham et d . ( ) prepared antisera in primates (baboons and monkeys) to two human strains (c and ad ) and two monkey strains (gr and gr ). the hyperiminune sera to the human viruses contained complement-dependent antibody (of and classes), whereas antibody to the monkey strains was independentthe reverse of the results obtained by andersen ( ) . graham et al. ( ) have also indicated that antibody from goats hyperimmunized with the human strains was complement-dependent, as with the primate antisera. these workers have stressed the need for inclusion of freund's adjuvant to obtain reasonably high levels of circulating antibody. whether the use of adjuvant had any effect on the quality or class of antibody was not discussed. minamishima et al. ( ) characterized cmv antibodies obtained from subhuman primates in the wild. complement-independent antibody was detected against a simian strain of cmv. when the sera were tested for neutralization of human cmv strains, one strain (c ) was neutralized only when complement was present, while a second strain (ad ) was not neutralized at all. finally, ablashi et al. ( ) described the successful immunization of rabbits with a simian strain (sa ). success, in this case, was indicative of the production of and s antibodies, both of which were complement-independent. with the elucidation of the nature and mode of action of complement, attempts were undertaken to determine the basis for complement participation in neutralization. shortly after the discovery in of properdin, but before its role in the alternative complement pathway was understood, van vunakis et al. ( ) attempted to determine its function in the neutralization of phage t r+ by normal human sera. heating ( °c for minutes), the addition of edta, or the addition of aymosan nullified neutralizing activity. zymosan-treated sera, however, retained hemolytic activity. in the light of more recent knowledge, these data implicate one or more of the early-acting components (cl, c , or c ) in neutralization, inasmuch as the alternative pathway was functional. the early suspected implication of the role of properdin in viral neutralization was discussed by ginsberg and wedgwood ( ) . taniguchi and yoshino ( ) examined the role of complement in the neutralization of herpesvirus by early rabbit antibody. the immune serum was deprived of neutralizing activity by heating ( oc for minutes) , zymosan, and ammonium hydroxide treatments. individual components of complement (i.e., c , c , c , and c ), obtained by fractionation of guinea pig sera, were added singly and in combination to the deactivated rabbit serum. only when all four components, in optimal concentrations, were added did the rabbit serum reacquire neutralizing activity. an earlier study of this phenomenon (dozois et al., ) , in which rabbit antibody to wee virus was examined, resulted in a somewhat different conclusion; whereas components c , c , and c were required, component c was either dispensable or required in trace amounts. a t least one difference in the methodologies employed in the two studies involved the source of the complement components, namely, guinea pig sera and rabbit sera, respectively. yet another interpretation of this phenomenon was proposed by daniels et al. ( , ) . virus was allowed to react with isolated igm antibody from early rabbit antiserum. virus-antibody complexes were isolated by centrifugation and shown to be infectious and neutralizable by complement. the addition of either activated c or c was without effect, but the addition of both resulted in neutralization. the c and c components manifested activity when c was present a t an optimal concentration and c a t a suboptimal concentration. a dependence on the first four complement components for neutralization of ndv by early rabbit igm antibody was described by linscott and levinson ( ) . neutralization enhancement by fresh guinea pig serum was negated by such treatments as heat, divalent cation depletion, and cobra venom. activity was restored when all of the first four components were added to the treated serum. a similar conclusion with an interesting ramification was reported by oldstone et al. ( , ) . antibody to polyoma, a nonenveloped virus, was induced in rabbits with the aid of freund's incomplete adjuvant. antibody of the igg class was isolated from heated antisera. dependence on complement was a function of the multiplicity of bound antibody: below to molecules per virion required complement, above did not. at a multiplicity of , virus was sensitized and therefore neutralizable by fresh serum. neutralization was also effected by a c -deficient serum but not by a ccdeficient serum. neutralization was effected by c l q alone, or by c , c , c , and c in combination, but not by combinations of c and c , or c , c , and c . the positive result with c l q is attributed to its ability to aggregate the virus-antibody complexes. as these investigators indicate, this effect is of academic interest only, since c l q is not normally found in the free state in serum but rather as the conglomerate clq-clr-cls, i.e., c . in the various studies on complement-effected neutralization, several mechanisms to account for this phenomenon have been proposed. ( ) the accumulation of complement on a virion-antibody complex produces a shield that, for steric reasons, prevents viral function. ( ) complement components cross-link juxtaposed virion-antibody complexes, resulting in the formation of aggregates. ( ) for enveloped viruses, interaction of a virion-antibody complex with the complete complenient system results in lytic lesions in the membrane. ( ) based on the hypothesis that neutralization is the terminal stage of a multistage phenomenon, complement supplements a reaction which in its absence fails to go to completion. ( ) complement stabilizes a virus-antibody complex which otherwise is readily dissociated by dilution. daniels et al. ( daniels et al. ( , observed that complement component c failed to effect neutralization of herpesvirus that had combined with dependent antibody. however, the subsequent addifion of c resulted in neutralization. these workers proposed therefore that thc additive accumulation of both components was sufficient to interfere sterically with viral infectivity. a similar interpretation was proposed by linscott and levinson ( ) for complement-effected neutralization of ndv. the latter workers stressed the exponential manner in which the successive components of complement accumulate, e.g., one molecule of bound c may initiate the binding of several thousand molecules of c . direct support for this proposal of the amassing of complement on virion-antibody complexes was provided by electron microscopy. berry and almeida ( ) evidence to substantiate the aggregation hypothesis was based on filtration and sedimentation analyses. in studies on herpesvirus (wallis and melnick, ; wallis, ) it was shown that filterable complexes of virus and dependent antibody became nonfilterable after reacting with complement. this was attributcd to the formation of relatively large aggregates. if, however, the virion-antibody complexcs were highly diluted a t the time of addition of complement, neutralization without loss of filterability resulted. hence, under the latter conditions, complement exerted its effect through stcric hindrance. in speculating on the nature of complement-dependent antibody, wallis and melnick suggested it was, or functioned as, a monovalent molccule. oldstone et al. ( oldstone et al. ( , demonstrated the aggregation of polyoma virus-antibody complexes by the increase in sedimentation ratc. the addition of an average of two molecules of antibody per virion increased its sedimentation ratc from s to s. when the first four components of complement were added, the sedimentation rate increased to , indicating aggregation through the cross-linking action of complement. in these studies virus was doubly labeled radioisotopically, thercby allowing individual tracking of capsid and genome. although the sedimentation rate was increased and the infectivity was neutralized, the virion remained intact based on the cosedimentation of the capsid and genome radioactivity markers. it should be noted that polyoma is a simple nonenveloped virus. notkins et al. ( ) examined the effect of complement on herpesvirus complexed with igm complement-dependent antibody. although the addition of complement (pooled guinea pig sera) reduced infectivity by % , these investigators observed "little or no effect" on the sedimentation rate. the reasons for the different results for presumably similar systems (i.e., herpes-antibody-complement) are not discernible. yoshino and taniguchi ( b) demonstrated the stability of complexes of herpesvirus and complement-dependent antibody. neither extensive dilution nor sonication promoted dissociation. it was also shown that complexes that had adsorbed to host cells continued to show sensitivity to complcment, hence cellular induced dissociation did not occur. yoshino and colleagues reject steric hindrance as the basis for complement-effected neutralization based on filtration studies (yoshino and kishie, ) and on kinetic analyses in which single-hit kinetics were observed for both sensitization and complement-effected neutralization (yoshino et al., ) . it has been shown for avian infectious bronchitis virus (berry and almeida, ) and for equine arteritis virus (radwan et al., ) that under appropriate conditions irreversible damage to the viral envelope accompanies neutralization in the presence of complement. however, it has also been shown that neutralization can occur in the absence of envelope damage. as the culmination of extensive studies of the complement-effected neutralization of herpesvirus by yoshino and colleagues, the following hypothesis .has been proposed (yoshino and isono, ) . antibodies are distinguishable functionally, if not physicochemically, by their neutralizing or sensitizing effect. neutralizing antibody per se is capable of initiating the sequential reactions that terminate in the neutralization of infectivity. in contrast, the reactions initiated by dependent antibody fail to go to completion. they proposed that, whereas the earliest stages of the neutralization pathway cannot be influenced by complement, the later stages require progressively decreasing amounts of complement. in their studies, yoshino and isono ( ) discerned differences in antibodies with respect to how far the antibody could drive the reaction; complement-independent antibody (e.g., late igg) drove the reaction to completion, and the decreasing order of capability was: dependent late and early igg, late igm, and early igm. the above proposal was based on a concept (mandel, ) of neutralization in which one molecule of antibody initiates con formational transitions between neighboring capsid subunits. the net result is an overall capsid alteration. evidence underlying this concept was obtained with a picornavirus. yoshino and isono ( ) recognized the risk in extending this concept to an enveloped virus such as herpesvirus. however, they considered the possibility that the reactive surface antigens were protrusions of an underlying protein continuum. the role of complement in the in vivo interaction of herpesvirus and antibody was recently examined by strunk et al. ( ) . guinea pigs genetically deficient in c synthesis were compared with genetically competent guinea pigs. following infection with herpesvirus, both groups showed the same c and c through c levels before and after infection. the normal animals showed no utilization of c during infection. these investigators suggested therefore that the classic pathway was not involved in viral clearance, and possibly the alternative pathway was activated. in styk et al. reported the presence in normal sera of a substance that acted in conjunction with antibody to inhibit influenza virus. subsequent investigations by styk and colleagues ( styk and colleagues ( , styk and colleagues ( , styk, styk, , styk, , hana et al., ; kociskova, et al., ; styk and hana, a were carried out to characterize the substance which they called cofactor. an a strain of influenza virus was used, and antibody activity was based primarily on inhibition of hemagglutination, and occasionally on neutralization. as to the nature of cofactor, it was found to be distinct froin any of the complement components, although the distinction between it and c was in some instances equivocal. electrophoretically, it migrated as a @-globulin. based on exclusion chromatographic analysis it was a macroglobulin. it was found to be thermolabile and resistant to trypsin but not to periodate, and was considered a lipoprotein. norinal sera of humans, mice, horses, pigs, and cattle contain cofactor. unlike cofactor from these sera, cofactor from guinea pigs requires divalent cation. the proposed mode of action resembles that of complement and antiglobulin, namely, it combines with antibody that has reacted with antigen. i n doing so, cofactor reduces the probability of dissociation of the virus-antibody complex. additionally, its presence increases the degree of steric hindrance to normal viral function. there was no evidence that cofactor reacts directly with virus. the relationship of co-factor to antibody class was studied. antibody appearing early in the immune response was more sensitive to cofactor than late antibody. characterization of antibody class showed that antibody, but not late s antibody, was sensitive. these investigators considered the interesting possibility that, the early immune response may consist of a antibody that is cofactor-dependent. however, the procedures usually employed for separation of antibody types also segregate cofactor with the s antibody. hence, although s antibody may be present, the demonstration of its presence requires cofactor from which it has been separated. based on this conjecture, these investigators found that in some instances a very early response consisted of and s antibodies, both requiring cofactor. because of this dependence, these antibodies were considered "imperfect" in contrast to "classic" antibodies. polyak et al. ( ) confirmed the conclusion of styk and his colleagues that cofactor and complement were different entities. smorodintsev and yabrov ( extended the observation to other strains of influenza virus, as well as to sendai virus. i n their interpretation, a nonspecific thermolabile stimulator of antibody functions by stabilizing complexes of virus and nonavid antibody. the involvement of rheumatoid factor in mediated neutralization has been described recently for herpesvirus (ashe et al., , vaccinia virus (gipson et al., ) , and hepatitis virus (markenson et al., ) . rheumatoid factor is a circulatory igm antibody that appears in response to r variety of pathological conditions. it is considered specific for the host's own igg which may have undergone alteration as a result of binding to a foreign antigen. it was shown by ashe et az. ( ) that, when herpesvirus was sensitized by igg antibody, the complex bound rheumatoid factor. however, the tripartite complex was still infectious but had acquired sensitivity to complement or to anti-human igm with loss of infectivity. it was also shown through the use of fab fragments of viral antibody that> the fc portion of the antibody molecule was the binding region for rheumatoid factor. it has also been reported (austin and ) that staphylococcal protein a can effect secondary neutralization or mediate secondary neutralization of herpesvirus and vaccinia virus. like rheumatoid factor, protein a hinds to the fc region of igg that is complexed to virus. i n some instances neutralization is effected, or in other instances requires the addition of anti-protein-a antibody. failure of rheumatoid factor and protein a to effect neutralization has been attributed (austin and to their mws, about , and about , , respectively. a lower-mw preparation of protein a, i.e., about , daltons is effective. it was therefore suggested that too few molecules of the higher-mw substances bind to the infectious virus-antibody complex. a survey of other physicocheinical parameters indicated to austin and daniels ( that net charge and shape were irrelevant. this interpretation is somewhat puzzling, since these workers consider sterie hindrance to be the basis for neutralization. it seems that, if fewer molecules of the higher-mw substance bind to the virus-antibody eoinplex. it is because the complex has been saturated and therefore is as completely sterically hindered as by the lower-mw compounds. way and garwes ( ) hsve reported that neutralization of semliki forest virus by guinea pig antiserum can be enhanced by the addition of serum from various animal species. the nature of the enhancing factor and its mode of action were not characterized. in studies on the neutralization of poliovirus by rabbit antiserum (mandel, ) it was observed that the degree of neutralization was enhanced by the subsequent addition of an antiserum against rabbit -globulin. antiserum induced in one species of animal (e.g., goat) to whole globulin, or a specific fraction thereof, of another animal (e.g., rabbit) is designated goat anti-rabbit globulin, or goat anti-rabbit fab, and so on. in their studies on mice chronically infected with ldh virus, notkins et az. ( ) suspected the presence of infectious virus-antibody complexes in the sera of these mice. neutralization by goat anti-mouse globulin confirmed this suspicion. the generality of this phenomenon was extended when it was seen that herpesvirus could be neutralized by an antiglobulin serum (ashe and notkins, ) . after exposure of herpesvirus to a hyperimmune rabbit antiserum, a considerable proportion of virus survived. goat anti-rabbit globulin neutralized more than . %. the same gcneral phenomenon has been described for adenovirus (kjellh and pereira, ), poliovirus in human stool specimens (keller and dwyer, ) , vaccinia virus (majer and link, ) , influenza virus (majer and link, ) , vee virus (hahon, a) , equine arteritis virus (radwan and burger, a) , ndv (b. mandel, unpublished observation), and bacteriophages t and t (goodman and donch, ) , f (dudley e t al., ) , and t (adler et al., ) . following the suggestion of notkins et al. ( ) , the term "sensitization" indicates infectious virus-antibody complexes. the neutralization of sensitized virus by anti-antibody is designated "mediated neutralization." notkins et az. ( ) compared the effectiveness of antisera against unfractionated as well as fractionated globulin for mediated neutralization. viral antiserum was obtained from mice chronically infected with ldh virus .and was used for sensitization. rabbit anti-mouse globulin and anti-mouse igg were capable of inducing neutralization, unlike anti-igm which was inactive. i t was also shown that the fab fragment derived from a goat anti-mouse globulin serum had secondary neutralizing activity, albeit weak, and also had blocking activity against intact globulin. the fc fragment, however, had no activity. these studies suggest that igm either did not participate in sensitization, or could sensitize but was not accessible for mediated neutralization. there is the third possibility that the antiviral serum had little or no igm. inasmuch as monovalent fab was able to mediate neutralization, its mode of action could not have been due to the aggregation of sensitized virus. poliovirus obtained from human stool specimens was neutralized by anti-human iga, but not by anti-human igm, as reported by keller and dwyer ( ) . these workers suggested that virus was complexed with the l-chain moiety of antibody. i n another report, keller ( ) demonstrated sensitization of poliovirus "in reverse." virus that had been neutralized by rabbit antiserum was reactivated by peptic digestion and sulfhydryl reduction. the presence of fragmented antibody (fab) complexed to virus was ascertained by mediated neutralization with goat anti-rabbit globulin. keller ( ) proposed that neutralization was a consequence of an altered structural configuration, rather than aggregation. hampar et al. ( ) examined the sensitizing potentialities of the different classes of antibody appearing early or late in rabbits immunized with herpesvirus. early and s antibodies lacked appreciable neutralizing activity. although complement neutralized or s-complexed virus, goat anti-rabbit globulin neutralized only s-bound virus. late s antibody had neutralizing activity which was enhanced by antiantibody. neutralization of late s-bound virus was enhanced by antiantibody but not by complement. ashe et al. ( ) investigated the possibilities for sensitization and mediated neutralization using various combinations of antibody frsgments. herpesvirus-specific fragments were derived from hyperimmu-nized rabbits and anti-antibody fragments from sheep anti-rabbit globulin serum. virus-specific fab i and fab i fragments neutralized, and survivors where shown to have been sensitized. the fc fragment did neither. virus, sensitized by intact antibody (class not indicated) was neutralized by sheep anti-rabbit globulin as well as by the fab i and fab i fragments therefrom, virus, sensitized by either fab i or , was effectively neutralized by antiglobulin. when virus was sensitized by fab i, anti-fab i was moderately effective but anti-fab i was not. when sensitized by fab , neither anti-fab i nor anti-fab i was effective. hence virus that had been sensitized by univalent antibody fragments could be neutralized by univalent anti-antibody fragments. basically similar findings were reported for ldh virus . in the same study (ashe et al., ) , it was shown that, when virus was sensitized with antibody of a specific allotype, it could be neutralized only by an antiserum directed against the same allotype. a virus-specific and antiglobulin sera were induced in rabbits of the appropriate allotypes. mediated neutralization required that the anti-antiserum be induced in a rabbit that lacked one allotype determinant present in the virus-specific serum. for example, antibody from rabbit of allotype , / , was sensitive to mediated neutralization by antiserum from rabbit of allotype , / , that had been immunized with the , / , globulin. these workers attribute particular significance to this result because these allotypic determinants specify the characteristics of the variable regions (i.e., the combining site) of the antibody molecule. shinkai and yoshino ( b) recently examined the sensitizing capabilities of antibodies produced a t various stages in the immune response, and the fragments derived from these antibodies. antibody to herpesvirus was elicited in rabbits and collected weeks (early) and > weeks (late) after immunization. antiglobulin was prepared with chromatographically isolated normal rabbit globulin. immune -globulin was purified and fractionated by appropriate procedures to yield f (ab') , fab' fab i, and fab i fragments. tests to determine sensitization and neutralization showed the following results. early igg, igm, and late igm had little or no neutralizing activity, unlike late i&. of these, only early igg had sensitizing activity. all fractions [i.e., f(ab'),, fab, fab i, and fab ii] of early igg neither neutralized nor sensitized. of the late igg fractions, f(ab'), neutralized almost as well as igg itself, fab' and fab i neutralized nioderately but could be enhanced t o near full activity by anti-antibody. fab i neutralized weakly and sensitized moderately. although fab' derived from early igg neither sensitized nor neutralized, it was shown to have reacted with virus in blocking experiments. vee virus showed a strong susceptibility for sensitization by human antiserum (hahon, b) . goat anti-human globulin, specific for igg or iga, neutralized sensitized virus, while igm was ineffective. it cannot be decided if the ineffectiveness was real or if it was due to the absence of igm antihody in association with virus. further studies (hahon, b) revealed that the fab fragment of the antiglobulin had mediated neutralizing activity but was less effective than the intact igg. hahon ( a) expressed the opinion that assaying for neutralization by the use of specific serum and antiglobuh when carried out by the quenching of fluorescence of infected cells was more sensitive than by the quenching of infectivity in mice. majer and link ( ) studied the kinetics of neutralization of vaccinia virus. neutralization by human antiviral igg showed a slight lag, followed by an exponential decline and leveling off. when sheep antihuman globulin was included in the reaction, the lag and leveling off were eliminated. filtration of virus to exclude aggregates eliminated the lag but not the tendency for the reaction to level off. radwan and burger ( b) presented data indicating that the fab region of the bound antivirus antibody is the target site for mediated neutralization. they sensitized equine arteritis virus and then cleaved the bound antibody with trypsin. virus complexed to the fragmented antibody was insensitive to neutralization by complement but retained its sensitivity to antiglobulin, hence this evidence indicated absence of the fc region. notkins et al. ( ) attributed neutralization of sensitized virus to aggregation of the complexes by antiglobulin. herpesvirus complexed with hyperimmune antibody was isolated by gradient centrifugation and then treated with antiglobulin. compared with untreated complexes, treatment caused an increase in sedimentation rate. influenza virus has two discrete surface antigens, hemagglutinin and neuraminidase. the interaction of hemagglutinin with antibody results in neutralization, unlike the reaction of neuraminidase with antibody. a study of each antigen-antibody reaction (majer and link, ) showed that the hemagglutinin reaction was insensitive to antiglobulin. however, addition of antiglobulin to the neuraminidase reaction resulted in neutralization. it was theorized that the sites were, respectively, critical and noncritical for infectivity. neutralization via the noncritical site mediated by antiglobulin could be explained on the basis of steric interference with the adjacent hemagglutinin. or, less likely in the opinion of majer and link, the antiglobulin stabilized the neuraminidaseantibody reaction. presumably, these workers considered that stabilization could have an effect on the neighboring hemagglutinin structures; the term "stabilization" was not defined. sensitization of adenovirus and neutralization by anti-antibody was i described by kjellh and pereira ( ) in studies to determine which of the viral antigens induced neutralizing antibody. guinea pig antibody to the hexon antigen of type- adenovirus had neutralizing activity. antibody specific for other adenovirus types, e.g., and , reacted with type but without neutralizing. addition of goat anti-guinea pig globulin resulted in neutralization. there is no evidence that antibody can influence the viral replicative process after viral uncoating has occurred. hence the focus of interest on how antibody functions is restricted either to the direct effect of antibody on the integrity of the virion, or to the subversion of one of the early stages in the replication cycle. as indicated previously (section i ,e) , neutralization can be the final manifestation of several phenomena that are fundamentally different. at the most elementary level, one can envision a morphologically simple virion (with several copies of a single antigen uniformly distributed throughout the capsid) at a very low concentration interacting with one or several molecules of a homogeneous antibody population, and as a result losing its infectious capability. at the other extreme, one can envision a morphologically complex virus, composed of several diff went antigens in a topographically complex distribution, some antigens being virus-specific and others host-specific, some reacting with one class of antibody and others with a different class (e.g., herpesvirus; see miyamoto et al., ) . in addition, if the relative concentrations of virus and antibody are adjusted appropriately, secondary interactions leading to the formation of large aggregates may occur. which of the concurrent reactions represents the critical neutralizing event cannot readily be isolated from those that may also lead to neutralization but for superficial reasons. a systematic analysis of the intrinsic neutralization mechanism should entail an examination of the direct effect of antibody on the virion, and an examination of the distinct early stages of the viral growth cycle. it has been shown that irreversible damage may ensue when certain viruses are exposed to antisera in the presence of complement (almeida and waterson, ) . in all instances, including more rccent studies (oroszlan and gilden, ; radwan et al., , the viruses were enveloped. analysis of the interacting antigen-antibody systems revealed that the host-specific envelope antigens, reacting with antibody in the presence of the complete complement system, resulted in the formation of lesions in the envelope (berry and almeida, ). similar results were described by radwan et az. ( ) . visible holes and leakage of internal genomic material clearly represent irreparable damage. however, the interaction of such viruses with antibody to nonenvelope antigens resulted in neutralization that could be reversed (radwan et al., ) . it has been shown that exposure of neutralized poliovirus to acidic conditions restored infectivity (mandel, (mandel, , . similarly, acid treatment of neutralized tobacco mosaic virus resulted in reactivation (rappaport, ) . subsequent studies with many viruses, both simple and complex, have shown that reversal of neutralization can be achieved by a variety of procedures (for a review of this topic, see mandel, a) . these results indicate that any alteration in the capsid induced by antibody binding to it is fully reversible. it has been reported that reactivation by acid did not necessarily entail dissociation of the virus-antibody complex when adenovirus was neutralized by rabbit antibody (kjellh, a,b) . although it has been reported that under certain conditions neutralized virus is incapable of adsorbing to host cells, this is not the basis for neutralization in every case. adsorption of neutralized virus has been described for several bacterial and animal viruses (hultin and mckee, ; nagano and oda, ; tolmach, ; rubin and franklin, ; mandel, ; dales and kajioka, ; joklik, ; silverstein and marcus, ) . moreover, it has been reported that virus is still susceptible to neutralization after adsorption, providing penetration has not yet occurred (nagano and mutai, ; ishida and' ackermann, ; mandel, mandel, , yoshino and taniguchi, b; stinski and cunningham, ) . the above observations, particularly the latter, implicate a stage in the replication cycle subsequent to adsorption as the antibody-sensitive stage. the capability of neutralized poliovirus to adsorb to hela cells was found to vary according to several conditions (mandel, s) . at low multiplicities of antibody, and soon after virus-antibody interaction, the virus-antibody complexes adsorbed as well as unneutralized virus. at high multiplicities, adsorption was impaired but not totally negated. when neutralized virus was held at "c, the capability to adsorb improved until it exceeded that of the control about three-fold. the timedependent improvement was seen with but not with s antibody. it was first shown (joklik and darnell, ) that poliovirus particles, adsorbed to hela cells at low temperature, have about an equal chance either to penetrate or to elute at a higher temperature. the possibility that anybody might bias these alternatives in favor of elution was examined (mandel, b) . it was found that neutralized virus had a decreased probability for elution-about % compared with % for unneutralized virus. this observation, together with the enhanced adsorptive capacity, implies that when virus has reacted with antibody the virion has acquired a greater binding affinity for the receptor. with neutralized avian infectious bronchitis virus, there was no effect on elution (stinski and cunningham, ) , but with ndv (silverstein and marcus, ) and rabbit poxvirus (joklik, ) elution was greater for neutralized virus. several studies have bcen concerned with microscopic observation of the fate of adsorbed neutralized virus (dales and kajioka, ; joklik, ; silverstein and marcus, ) . where penetration had occurred, the uncoating process appeared to be unusual, leaving the impression that the intracellular particles were being randomly degraded or incompletely uncoated. in biochemical studies of poliovirus (mandel, b) , it was observed that the fraction of cell-associated virus that was uncoated was smaller for neutralized than for unneutralized virus-about % compared with about %. in the uncoated unneutralized virus, the viral rna was distributed between intact rna and degraded (i.e., acidsoluble) rna. in the uncoated neutralized virus about half as much degraded rna was seen, but no intact rna was detected. basically similar results were described by stinski and cunningham ( ) for a quite different virus-avian infectious bronchitis, an enveloped virus. several studies have indicated that the presence of antibody as a ligand to the antigenic binding site causes an alteration in the biochemical characteristics of the capsid. a dna coliphage, x , is incapable of infecting bacterial protoplasts. bowman and patnode ( ) reported that, when virus was treated with highly diluted antiserum, infectivity was not demonstrable with intact bacteria but was with bacterial protoplasts. since dnase did not affect the phage, infectivity could not be attributed to released dna. however, an anti-dna serum (lupus erythematosus) neutralized infectivity for protoplasts. these workers suggested that the capsid had undergone a rearrangement rendering the dna accessible to interaction with the external environment. kjellkn and pereira ( ) have proposed that the hexon structure of adenovirus is composed of two antigens, one of which is internal. interaction of the external antigen with antibody causes a rearrangement that exposes the internal antigen, thus enabling it to react with neutralizing antibody. as a possible interpretation of the enhanced infectivity of arboviruses by antibody, hawkes and lafferty ( ) suggested that the effect may be attributed to a "particle that has, in effect, a new protein coat" as a result of binding antibody. a study of the electrophoretic characteristics of poliovirus (mandel, b) showed that the capsid structure was in a metastable conformation, oscillating between two distinguishable isoelectric states. when neutralized, irrespective of antibody multiplicity, the capsid was stabilized in one of the conformational states. studies on the topographic arrangement of the structural elements of picornavirus capsids have been carried out by labeling procedures. it has' been shown (e.g., carthew, ; beneke et al., ) that the vp polypeptide is most highly labeled, and vp and vp are relatively inaccessible. vp appears to be entirely shielded, although in the free state it can bind the labeling compound. carthew ( ) showed that, after reacting with antibody, the vp polypeptide became accessible, indicating that a rearrangement in the spatial organization of the capsid had occurred. in light of this observation, it is of interest to consider the results described by breindl ( ) . after neutralisation of poliovirus, the virus-antibody complex was fractionated, yielding a fragment consisting of the atibody bound to the vp polypeptide, this result, together with the labeling rcsults, suggest the interesting possibility that antibody initially combines with the reactive site which may not include ' the vp polypeptide. conformational rearrangements may ensue that allow the antibody to transfer its allegiance to a more compatible receptor, the vp polypeptide. a schematic proposal of how such a rearrangement might occur has been suggested by lonberg-holm and yin ( ) . an in vitro system for uncoating poliovirus has been developed (de mandel, , ) . when neutralized poliovirus was examined in this system, it was seen to be resistant to uncoating. i n the previous studies on the intracellular fate of neutralized poliovirus (mandel, b), evidence for a low level of uncoating was seen in the form of degraded viral rna. however, a similar result was seen with native virus but, unlike neutralized virus, undegraded rna was also seen. the degraded rna possibly reflects an aberrant, abortive uncoating process to which neutralized virus as well as native virus is susceptible. perhaps the most basic problem of the neutralization phenomenon concerns the mechanism whereby antibody deprives a virion of its infectious capability, the second most basic problem is perhaps the quantitative requirement for antibody to exert its effect. i n all likelihood, the solution of either problem will point to the solution of the other. the early studies of andrewes and elford ( a) and burnet et al. ( ) began to suggest some ways of thinking about the latter problem. in their studies of the kinetic characteristics of thc neutralization reaction, andrewes and elford ( a) summarized their findings as the "percentage law." i n essence they observed that a given concentration of antiserum neutralized the same fraction of virus per unit time, irrespective of the concentration of the initial viral input. the law, however, had to be qualified: the concentration of antibody had to be in considerable excess over the concentration of virus. the law has been found to apply to virus-antibody interactions in general. the basis for this relationship can be understood in terms of collision frequency and relative concentrations of the reactants. the number of successful collisions is a function of the concentrations of virus and antibody. if the concentration of antibody is in great excess, the number of collisions per unit time will not decrease as a result of removal of antibody by binding to virus. since the collisions are indiscriminate, the number involving unneutralized particles will diminish by the same proportion for each unit of time. the result is a reaction of the first order. if virus concentration is increased, the number of collisions will increase accordingly, but the fraction of virus particles undergoing neutralization will be the same as for the lower concentration. from these observations it follows that the rate of neutralization is a function of t,he concentration of free antibody, and the rate will be constant, providing the decrease in free antibody concentration during the course of the reaction is extremely small relative to the total concentration. when, under these conditions, the reaction is plotted on a semilogarithmic plot, a linear relationship is seen of the kind that can be described by the expression for a first-order reaction. it is, however, clear that the reaction in actuality is second-order, involving two reacting molecules. as indicated in section ii,e it has been proposed that neutralization is the result of sequential bimolecular interactions culminating when a critical number of antibody molecules are associated with the virion. in most instances a plot of the logarithm of the surviving fraction of virus as a function of time is linear for the early phase of the reaction. in their analyses of the neutralization reaction, dulbecco et al. ( ) interpreted this observation as evidence for a single-hit phenomenon. there has been cri,ticism of this interpretation on purely speculative grounds, and occasionally a reaction has been recorded that shows evidence, based on a time-dependent delay of neutralization, for a multihit reaction (e.g., lafferty, a; philipson, ) . in accordance with the target theory of inactivation, the proportion of surviving virus can be expressed by the equation where v t and vo are the concentrations of virus at times t and zero, k is a constant, and n is the number of targets per virion that must be hit in order for the virion to be inactivated. in the case in which n = the equation reduces to a semilogarithmic plot of a reaction that fits eq. ( ) shows a linear course from time zero, indicating that one successful encounter inactivates that particle. where more than one successful encounter is required, a period of time has to elapse to allow for the total necessary encounters before the first particle will be inactivated. the period of time is indicative of the number of hits required, which is the value of n in eq. ( ) . when portrayed graphically, this period is seen as a lag or shoulder on the curve preceding the linear course of inactivation. a single-hit mechanism does not rule out the possibility that more than one molecule combines with a particle prior to inactivation. it does rule out the participation of these molecules in the inactivating event. otherwise their participation would be manifested by a lag in the time course of inactivation. the data of rappaport ( ) , which showed four or five molecules of antibody per inactivated virion, indicated that all but one molecule were not involved in causing neutralization. other studies (yoshino and taniguchi, b; ashe and notkins, ; krummel and uhr, ; dudley et al., ; hahon, b; lewenton-kriss and mandel, ) showed that several molecules of antibody reacted with one virion (animal or bacterial) prior to inactivation. their presence was detected by the use of antiglobulin to mediate neutralization. i n several of these studies the mediated inactivation showed a more rapid rate but, nonetheless, also characteristically a single-hit mechanism. for example, in the studies of dudley et al. ( ) and krummel and uhr ( ) , the reaction rate of the mediated reaction was three-to fourfold more rapid than the direct reaction, suggesting that the earliest binding molecules were noneffective but could be made effective by the antiglobulin. in instances in which multihit kinetics were observed (e.g., lafferty, a.b; philipson, ) either low temperature or antibody of the igm class, or sera a t very low dilutions, were involved. conceivably, an effective interaction under these conditions might require the cooperation of several molecules of antibody binding to neighboring antigens. a further possibility for a multihit phenomenon with single-hit kinetics would require that the necessary hits all occur simultaneously, not sequentially. recently, della-porta and westaway ( ) reviewed the question of the minimum number of molecules of antibody required for neutralization. their view, as previously proposed by westaway ( b) , is that neutralization results from a multihit reaction. the conclusion is based on several phenomena: ( several examples of multihit inactivation have been reported in which a lag is seen in the kinetic analysis. ( ) the existence of infectious virus-antibody complexes that can be neutralized by complement or antiglobulin has been reported. ( ) the outcome of virus-antibody interaction is variable, depending on the host or cell indicator. ( ) neutralization of infectious virus-antibody complexes may ensue after prolonged incubation. ( ) synergistic effects, either within a native virion composed of several distinct antigens, or particles with hybrid antigenicities resulting from mixed infection that produces phenotypically mixed particles (i.e., capsids with specificities of both parents) have been observed. until more definitive knowledge about neutralization is available, some of the above illustrations can as well be used to fortify the single-hit hypothesis. at the outset, if these investigators consider the few instances in which a shoulder was evident (in kinetic analyses) as support for a multihit mechanism, the same logic can support the single-hit mechanism where no shoulder was evident. there can be no argument that on the average several molecules of antibody may combine with a virion when it is neutralized. whether all, or only one, have a function in neutralization is at present undetermined. possibly all but one may be functionally deficient, or they may have reacted with sites that do not respond appropriately. a relevant observation has been made in several studies; neutralization of infectious virus-antibody complexes by antiglobulin also shows a single-hit mechanism, and at a rate that exceeds the rate of the homotypic reaction. if a virus requires n hits to be neutralized but has received nz hits, it should not be neutralizable by antiglobulin. it is also pertinent that monovalent fragments of the antiglobulin can mediate neutralization. these observations can also be explained on a single-hit basis, if it is assumed that. a virus-antibody interaction may be ineffective but subject to activation. the view that neutralization is dependent on a multihit reaction was also expressed by daniels ( ) . he indicated that neutralization was the outcome of sequential reactions involving initially free virus and then infectious virus-antibody complexes. not until the final required molecule reacted did the virion lose its infectivity. however, short of the full requirement, anti-antibody could achieve the final effect. it is difficult to envision an explantory mechanism, since the anti-antibody does not bind to the virion but only to the antiviral antibody. in essence, the number of virus-bound antibodies has not been increased by the antiglobulin reaction. a single-hit mechanism implies either that there is only one critical site for neutralization, or that one molecule of antibody can cause neutralization a t any of several sites. an infectious phenotypically mixed particle is in a sense two particles, since the capsid is composed of the antigens of both progenitors. it is not unexpected that the reaction shows the need for more than one molecule. it is relevant that an excessive amount of antiserum to either parent has little neutralizing effect, whereas a mixture of antisera to both parents neutralizes effectively. as a result of extensive studies of the serological reactions of togaviruses, della-porta and westaway ( ) have proposed a multihit model to account for neutralization. as previously reported (westaway, b) , neutralization requires that several antigen-antibody complexes be strategically disposed over the viral surface. since these viruses also hemagglutinate, and since neutralization is not absolute but is host-dependent, the topology of the bound antibodies determines for which hosts the complex is neutral or infectious, and whether the virion retains its ability to hemagglutinate. further, these viruses contain two antigens of type and group specificity. the extent t o which cross-reactions occur, based on neutralization or hemagglutination-inhibition, depends on how the cross-reacting antibodies are disposed on the viral surface. recently, a hypothesis was proposed to account for the neutralization of poliovirus and to suggest a mechanism whereby one molecule of antibody achieved this end (mandel, ) . in this hypothesis the assumption is made that neutralization is the result of one effective collision. justification for this assumption is based on the interpretation of kinetic data. an effective collision is dependent either on reaction with a critical site, or on reaction with any antigenic site but in a critical manner. previous studies (mandel, ) ) showed that the poliovirus capsid undergoes spontaneous oscillations between two conformational states characterized by distinct isoelectric points. inactivation by antibody stabilized the capsid in one of the conformational states. subsequent studies (mandel, ) with highly diluted antiserum showed the redistribution of the viral population from the normal to the neutralized conformational state by an all-or-none mechanism. the two states differed by more than ph units. under no conditions were particles of intermediate isoelectric point seen. moreover, the proportion of virus neutralized and the proportion of virus conformationally stabilized were equal. it was also shown that, in an in vitro system capable of carrying out the early stage of viral uncoating, neutralized virus was refractory. the above observations have been integrated into the following hypothesis. the metastable nature of the viral capsid suggests intersuhunit cooperative transitions. effective attachment of one molecule of antibody to an antigenic subunit stabilizes that subunit in a specific nonfunctional conformation. through cooperative transitions all neighboring units are similarly stabilized until the entire capsid becomes conformationally homogeneous. these expectations are based on the similarities of the structure of the viral capsid and the structure of proteins subject to allosteric transitions (see monod et al., , for a discussion of allosterism) . particles in this state are incapable of undergoing normal interactions with plasma membrane of susceptible cells. it was pointed out earlier that neutralized virus not only retained its capability to adsorb, but did so with increased affinity, probably because of the more highly charged nature of the abnormal conformation. after extensive studies of the neutralization of herpesvirus, with particular emphasis on sensitization and mediation by complement, yoshino and his colleagues have proposed a hypothesis to account for neutralization and sensitization (yoshino and isono, ) . crucial to this hypothesis is an appreciation of the variability inherent in antibodies with respect to their class and to their binding affinity. according to their hypothesis the most efficient antibody is capable of initiating a series of physicochemical changes in a virion that culminates in neutralization. less efficient antibody can induce some, but not all, of the changes. i n this case, complement (and presumably antiglobulin) can mediate the reaction. very inefficient antibody-"slow-reacting" in their terminology -c a n bind to virus but cannot drive t,he reaction to the stage where complement can complete the reaction. in terms of how far the various antibodies can alter the virion toward the neutralized state, these workers classify the antibodies as slow-reacting, early igm, late igm, early and intermediate igg (all incapable per se of neutralizing, and, finally, late igg antibody that is independent of complement. they have also subdivided the essential changes in the virion leading to neutralization with respect to ( ) dependence on time (the early stages) and ( ) dependence on temperature (late stages). they propose that one molecule of antibody is sufficient to initiate all reactions whether they fail or succeed in neutralizing. the satisfying feature of this hypothesis is that it allows an interpretation of several distinct phenomena that characterize neutralization: ( ) single-hit inactivation, ( ) role of antibody diversity, ( ) mediated neutralization and, possibly, ( ) the nonneutralized state. in order to understand the neutralization phenomenon, trautman has stressed the necessity for ascertaining two parameters: the minimum number of critical sites required for infectivity, and the various small virus-antibody complexes that are infectious. to derive this information, trautman ( ) has developed a computer assisted program. based on data for fmd virus, trautman and harris ( ) have determined that a t least two or three critical sites (the number varies according to the host assay system) that react with igg are required, whereas only one that reacts with igm is required for infectivity. the total number of either type of critical site is as yet not known. a reasonable summation of available data have led trautman and harris ( ) to propose that fmd virus contains one unique vertex involved in infectivity. this structure consists of one igm, and five to ten igg reactive sites. the authors have indicated that "complications" may arise that would require investigation in order to derive valid interpretations. they have discussed six possible complications. in , smith reported data that clearly showed the in vitro interaction of viruses (vaccinia and herpes) with homologous antibody. somewhat later, sabin ( ) was unable to demonstrate such in vitro interaction and questioned the basic nature of antibody action. however, subsequent studies established beyond reasonable doubt that virus and homologous antibody do combine. the only other aspect of viral neutralization about which there is a similar degree of unanimity is that the mechanism is not yet understood. this state of uncertainty has recently been deplored: ", . . remains undefined" (della-porta and westaway, ), ". . . remains unknown" (yoshino and isono, ) , and ''. . . questions remain to be clarified" (daniels, ) . when consideration is given to the known variables of this system (i.e., when each component is recognized as a population within which there is broad variation) and to the realization that neutralization encompasses several distinct phenoma, it is not unexpected that the fundamental nature of the neutralization reaction has not yet been elucidated. burnet's eloquent assessment of this situation ( , p. ) is as true today as then. however, the ability to cope with this difficulty has improved, at least technologically. daniels ( ) has summarized the neutraliztion phenomenon as representing three pathwaye: ( ) saturation of viral antigens, ( aggregation by cross-linking of multivalent virions by bivalent antibodies, and ( ) virolysis. although the net result in each case is loss of or reduction in infectivity, the underlying mechanisms may be basically different. virolysis, for example, is the result of an enveloped virus reacting with antibodies specific for the envelope proteins and depends on participation of the complete complement system. aggregation may reduce the number of infectious units without literally neutralizing the individual virions. saturation may or may not involve intrinsic neutralization. in order to understand the fundamental mechanism, it seems preferable to select as a paradigm the simplest available system-a simple virus (e.g., one composed of several copies of one antigen directly accessible to antibody) reacting with a homogeneous population of high-affinity antibody (e.g., late igg) capable of neutralizing without the aid of accessory substances. interaction of virus at a very low concentration with the minimum multiplicity of antibody is most likely to reveal why virus has been neutralized. since under some circumstances neutralized virus can still adsorb to cells, or conversely, since adsorbed virus can still be neutralized, the implication is that the viral capsid has undergone an alteration that subverts subsequent interaction with cells. the nature of the alteration is most likely to be understood on the basis of protein-protein interactions. some clues have already been described indicating that each reactant has undergone some change from its native state. antibodies can interact with complement, or bind to cells, only after they have reacted with antigen. feinstein and rowe ( ) have proposed that antibody undergoes a conformational rearrangement upon binding t o antigen (for an extensive discussion of this proposal see feinstein et al., ) . several examples have been cited that indicate conformational or degradative changes in virion capsids following interaction with antibody. it is of interest that, whereas early antibody reacts with virus but requires mediation, late antibody can neutralize independently. it has also been shown that one difference between early and late antibody is the greater binding affinity of late antibody. the greater the binding affinity, the greater the stress, involvement, and disorganization in the capsid. such disorganization may be reversible (e.g., by causing dissociqtion) but in such a state the capsid may not be acceptable t o a cell. an attempt has been made in this article to delineate some of the problems: ( ) the mechanism of intrinsic neutralization, ( ) the singlehit versus multihit requirement for neutralization, ( ) the nature of the nonneutralized or sensitized state, ( ) the nature of mediated neutralization by complement or ant.iglobulin, ( ) the differences in the characteristics of virus-antibody complexes according to antibody type and affinity. it hardly needs to be stated that solution of these problems will represent the groundwork for considerations of problems at the next level-in vivo interactions. proc. soc arch. gesamte virusjorseh arch. gesamte virvsforsch proc. natl. acad. sn sn'ence j . i m m u o l . , principles of animal virology the antigens virology , proc. natl. acad virology , folia mkrobiol. (prague) , . hijek folia microbwl. (prague) nature (london) , . hornick virology , arch. gesamte virusforsch. , . kjellkn, i,. ( ~) virologv , . . . fed a d a proc. natl. acad. sci. usa. , . lonberg-holm arch. grsamte virusforsch cold spring harbor symp methods virol. me) complement-mechanisms and function arch. gesamte virus-parkman nature (london) , virozogu , virology , proc. soc acta virol. (engl inject. zmmun. , . englewood cliffs. new jersey. jorsch. , . immunol. acfa virol. (engl i n "antibodies to biologically active molecules tokyo iji shimhi proc. soc r o c . natl. acad. sci viruses affecting man and animals" (m. sanders and m intert rology , . way j p n key: cord- -z b mb authors: diamant, eran; torgeman, amram; ozeri, eyal; zichel, ran title: monoclonal antibody combinations that present synergistic neutralizing activity: a platform for next-generation anti-toxin drugs date: - - journal: toxins (basel) doi: . /toxins sha: doc_id: cord_uid: z b mb monoclonal antibodies (mabs) are among the fastest-growing therapeutics and are being developed for a broad range of indications, including the neutralization of toxins, bacteria and viruses. nevertheless, mabs potency is still relatively low when compared to conventional polyclonal ab preparations. moreover, the efficacy of an individual neutralizing mab may significantly be hampered by the potential absence or modification of its target epitope in a mutant or subtype of the infectious agent. these limitations of individual neutralizing mabs can be overcome by using oligoclonal combinations of several mabs with different specificities to the target antigen. studies conducted in our lab and by others show that such combined mab preparation may present substantial synergy in its potency over the calculated additive potency of its individual mab components. moreover, oligoclonal preparation is expected to be better suited to compensating for reduced efficacy due to epitope variation. in this review, the synergistic neutralization properties of combined oligoclonal ab preparations are described. the effect of ab affinity, autologous fc fraction, and targeting a critical number of epitopes, as well as the unexpected contribution of non-neutralizing clones to the synergistic neutralizing effect are presented and discussed. since the emergence of hybridoma technology [ ] , the research and the development of monoclonal antibodies (mabs) has rapidly progressed. mabs, well-characterized individual full or partial immunoglobulin molecules, are currently being developed for a broad range of indications, from diagnostics and imaging to the treatment of medical conditions such as cancer and infectious diseases. in recent years, mabs and related products have been the fastest growing class of therapeutic agents [ ] . the use of antibodies as therapeutics was conceived long before the development of mab technology; passive immunity, i.e., the transfer of antibodies from the serum of an immunized person or animal to a non-immunized one, was first demonstrated more than years ago in a pioneering experiment by behring and kitasato in which serum therapy protected against diphtheria [ ] . serum therapy was commonly used to treat infectious diseases until the discovery of chemotherapy in the form of sulfonamide in the s [ ] . antibiotics were less expensive and difficult to use, had significantly fewer adverse effects, and were not pathogen-specific, rendering the diagnosis of the microbial infection unnecessary before treatment; thus, serum therapy was pushed aside. however, the emergence of multi-drug-resistant forms of new and old pathogens and the recent explosion of the immunocompromised population worldwide have led to a necessary resurgence in the development of antibody-based therapeutics to treat and prevent this new wave of drug-resistant infectious diseases [ ] . furthermore, whereas antimicrobial drugs can kill the microbes but cannot eradicate pre-formed toxins, a specific antibody is the only compound that can neutralize a given toxin [ ] . therefore, antibodies remain attractive for therapeutic purposes. however, modern polyclonal antibody (pab)-based therapeutics, although improved compared with past serum therapies, continue to suffer from major limitations that might be elegantly overcome by mab-based preparations. first, treatments with hyperimmune equine-or other animal-derived antisera are associated with substantial side effects, including hypersensitivity-related reactions such as serum sickness and anaphylaxis [ , ] ; in addition, the use of human antisera involves the risk of blood-borne disease [ ] . these safety limitations can be addressed by using human-derived or humanized mab-based preparations that display a substantially decreased incidence of side effects and viral contamination [ , ] . humanization, the replacement of mouse constant regions and variable framework with human sequences, results in a product displaying significantly reduced immunogenicity and improved in vivo tolerability [ ] . human or humanized mabs exhibiting enhanced pharmacokinetics enable the administration of a lower protein load and a reduced frequency of administration during the course of treatment. second, pab-based preparations exhibit significant batch-to-batch variability, and their supply is limited. in contrast, mabs can be produced in vitro, thereby ensuring an unlimited supply of highly purified, well-characterized products that are devoid of contaminating proteins [ ] . despite the potential of mabs as powerful tools in the fields of infectious diseases and toxins, during the past years, following the hybridoma revolution, out of the more than immunoglobulins (iggs) and their derivatives that have been approved for use for various indications [ ] , only one mab has been approved for the prevention of a viral infection (rsv) [ ] , and one mab has been approved in the u.s. for the treatment of bacterial toxin (anthrax) [ ] . all others were clinically designed for the treatment of cancer and autoimmune or allergic conditions [ ] . although many mabs have been purified and characterized for their protective efficacy against different toxins [ ] , some of which are under investigation in clinical trials [ , ] , pabs remain almost the only available preparations for this indication. mab-based therapy has some limitations. first, mabs exhibit unprecedented specificity to their antigenic target, but this extreme specificity may hamper the efficacy of any individual mab in the case of the absence or modification of its target epitope in a mutant or subtype of the infectious agent. second, the potency of mabs, especially those that play a role in the neutralization of pathogens and toxins, remains relatively low compared with pab-based preparations. this inferior neutralizing potency of individual mabs may be attributed to the differences in the functional impact of specific antigenic epitopes, to their low affinity to the target epitope, to the biochemical stability of each mab molecule, or reduced clearance of the mab-antigen immune-complexes (ics). these two major limitations of individual mab-based preparations, their low neutralizing potency and potential failure to treat mutants or subtypes, can be overcome by combining several mabs to form a mab cocktail. moreover, as will be further discussed in details, recent studies show that such mab cocktails exhibit synergistic neutralization; i.e., mab cocktails display substantially elevated neutralizing activity that exceeds the level of improvement expected by the contribution of each mab in the mixture. in fact, the neutralizing activity of mab cocktails can be similar to the potency of pab-based preparations. this phenomenon was demonstrated in our lab as well. we generated mabs against botulinum neurotoxins (bonts). we not only simultaneously obtained neutralizing mabs against different bonts in a single automated process but also successfully demonstrated significant synergistic neutralization by the combined anti-bont mabs [ ] . bonts, which serve as an excellent model for complex antigens against which mabs are required, are produced by clostridium botulinum strains and are considered the most lethal toxins identified, with an estimated human median lethal dose (hld ) of ng/kg body weight [ , ] . bonts are the only toxins classified by the cdc as category a agents [ ] . however, the standard treatment for botulism relies on equine or limited amounts of human pab-based antitoxin therapy, together with supportive care [ ] . thus, there is a need for safe and reliable anti-bont mab-based pharmaceutical drugs. in this review, we describe the mechanisms underlying the neutralizing synergy of oligoclonal-based preparations, with an emphasis on biological toxins. the importance of ab affinity, the molecular structure of iggs involved in mab-antigen ics clearance, and the unexpected contribution of individual non-neutralizing mabs to the synergistic neutralizing effect are discussed. natural human polyclonal responses are elicited by the concerted action of antibodies that display multiple specificities and bind to several epitopes. using carefully assembled cocktails consisting of recombinant human mabs, it might be expected that these mechanisms would be more efficiently recruited than using single mabs [ ] . the concept of combining several mabs to increase their therapeutic efficacy and to overcome the limitations of pabs appears to be very logical. indeed, in the last years, many studies have evaluated two or more mabs in various combinations to improve the efficacy of these potential therapeutics for infectious disease, primarily viruses and toxins (see table for a list of oligoclonal antibody-based preparations against various toxins). most of these mab combinations exerted additive or synergistic neutralizing effects, demonstrating their potential value as future therapies. when combining two or more drugs, such as anti-toxin mabs, four outcomes can be expected: an additive effect, i.e., the summation of all individual neutralizing activities; antagonism, defined as a reduction in efficacy; indifference, i.e., one drug does not enhance or reduce the efficacy of the second drug; and synergism, in which the protective properties of the combined mab preparation significantly exceed the additive effect of the components in the mixture. attempts to estimate the combined effect of multiple drugs have been documented for more than a century [ ] . chou and talalay proposed a general diagnostic function to determine the applicability of experimental data and to distinguish between mutually exclusive and nonexclusive drugs and provided equations for analyzing and quantifying the synergism, summation and antagonism of multiple drugs [ ] . the authors designated a "combination index" (ci), in which a value of < , = , or > indicates synergism, an additive effect (summation) or antagonism, respectively, for each drug combination. similarly, pohl et al. applied the fractional inhibitory concentration (fic) indices approach for the evaluation of drug combinations to characterize the interactions among multiple mabs; similar to the ci, the interaction between two mabs is considered to be synergistic if the fic index is < . , additive if the fic index is equal to , indifferent if the fic index is between and , and antagonistic if the fic index is > [ ] . a synergistic drug combination should facilitate the reduction of drug doses while maintaining efficacy. the fold-reduction in the dose of each drug included in a synergistic combination at a given effect level, compared with the dose of each drug alone, can be calculated as the dose reduction index (dri) [ ] . the ci and the dri also enable a useful comparison of the extent of efficacy enhancement between neutralizing mab combinations in different studies. although many of the studies involving mab cocktails do not provide direct data regarding these values, in most of these studies (table ) , an enhancement in the neutralization of mab combinations was detected compared with that of the individual mab components. this enhancement is often referred to as 'synergism' by authors; however, although enhanced neutralizing effects are reported, it is difficult to compare the extent of this synergy among different studies due to various factors, including toxin biology and the nature of the neutralization assay, which can be conducted either in vivo (for instance, a mouse protection assay) or in vitro (cell toxicity assays, using different cell types). thus, the information in the table was estimated after careful analysis in light of this 'fold-enhancement' (the terminology that we selected to describe the superiority of a mab combination compared with its components). in some cases, inferring the difference in neutralization was not applicable. in a previous study conducted by our lab, specific monoclonal antibodies against the three botulinum serotypes a, b and e were generated by immunizing mice with a trivalent mixture of the recombinant c-terminal half of the heavy chain (hc) of botulinum neurotoxins a, b, and e [ , ] . following immunization, we conducted parallel differential robotic hybridoma screening to identify specific mabs against each of the three toxins. the integration of automated robotic liquid-handling systems can significantly improve the overall screening capacity of hybridoma [ ] . within each serotype-specific group, we produced neutralizing mabs, the majority of which were protective against a toxin dose of mouse lethal dose % (msld ). encouraged by these results, we sought to determine whether the mab combinations in each serotype-specific group exhibited increased neutralizing activity. the best combination for anti-serotype e mabs displayed a very high synergistic neutralizing activity of up to -fold over that of its components (figure ). the cocktail of anti-serotype a mabs, which protected against , msld of toxin a, had a -fold improvement over the calculated additive effect of the mixture's components. strikingly, the removal of three non-neutralizing mabs from the -clonal anti-bont/a cocktail dramatically reduced the neutralizing activity. the importance of non-neutralizing individual mabs was also demonstrated for anti-b mabs, confirming that addition of a single non-neutralizing mab to the neutralizing antibodies may induce synergism. others also found that a combination of seven apparently non-neutralizing mabs with one neutralizing mab directed to the heavy chain of the tetanus neurotoxin (tent) induced synergistic neutralization [ ] . the induction of synergistic neutralizing effects by a combination of non-neutralizing mabs is critical to our comprehension of the mechanisms underlying synergism and may have important consequences for the future screening and selection of potent neutralizing mab cocktails. what are the mechanisms underlying this antibody-mediated synergy? toxin neutralization depends, at least in part, on functional "neutralizing" epitopes recognized by antibodies that interfere with the interaction of the toxin with the target cell, on the binding affinities of the antibodies, and on the quality of antibody-toxin complex clearance from the bloodstream. accordingly, the induction of neutralizing synergism might be explained by at least three putative mechanisms: first, the simultaneous binding of interfering mabs to multiple functional sites on the toxin; second, an increase in the affinity of the mixture of mabs compared with each mab component; and third, enhanced fc-mediated clearance of antibody-toxin complexes from the serum due to multimeric antibody decoration of the toxin. each of these three mechanistic explanations for this synergy may exert a partial influence in different mab combinations and may be considered a different aspect of a tri-partite phenomenon; together, these three mechanisms may induce synergistic neutralization. the humoral response to evading pathogens involves a series of effector mechanisms of antibodies that include direct binding to specific epitopes and indirect antibody-mediated events primarily consisting of antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cell phagocytosis (adcp), and complement-dependent cytotoxicity (cdc) [ ] . for more than a century, toxins have been believed to be neutralized by antibodies not by indirect mediated measures but rather by a direct "interfering" antibody-dependent mechanism in which antibodies bind to toxins and interfere with their interactions with host cells, thus preventing the attachment of the toxin to the target cell or its enzymatic activity that might otherwise damage the target cell. theoretically, for an antibody to interfere with a toxin, only the variable region of the antibody is required because it is the moiety that specifically recognizes and binds to its epitope on the target toxin. thus, this binding alone is generally considered as "neutralization". the constant region (fc) of an antibody is known to exert effector functions (adcc, adcp, cdc) and is thought to become activated by bacteria and tumor cells [ , ] , but the involvement of these mechanisms in humoral anti-toxin responses is unclear. alternative roles of fc in the clearance of ics and in antibody pharmacokinetics are discussed in section . . toxin neutralization has been considered to be primarily due to the binding of the ab to the toxin, blocking its activity [ ] . to design a combination of mabs that mimic a polyclonal response, the principal rationale would be to select mabs that bind to distinct or non-overlapping epitopes. such a combination may facilitate the binding of multiple igg molecules to the target toxin, thus potentially blocking various essential functional domains. this simultaneous blockade increases the probability that these mabs will interfere with interactions between the toxin and its target cell, ultimately resulting in either additive or synergistic neutralization. it has been demonstrated that particular domains on different toxins are associated with enhanced neutralization, depending on their structure and mechanism of action. for instance, hc ( kda) is considered to be the receptor binding domain of the bont to the target neuron, whereas the n-terminal region (hn) serves as the translocation domain; the catalytic domain is a zinc endopeptidase that is localized to the light chain (l). the surface residues of hc vary dramatically among different bont serotypes [ ] . the hc fragment itself is non-toxic, but most neutralizing epitopes have been mapped to this domain [ ] , and the 'neutralizing' epitope-rich hc has been used as a subunit vaccine to produce neutralizing antibodies against bont [ , ] . bacillus anthracis, the causative agent of anthrax, exerts its toxicity via the dissemination of a tripartite exotoxin composed of protective antigen (pa), lethal factor (lf) and edema factor (ef). pa plays a critical role in anthrax pathogenesis, as it associates either with lf to form the lethal toxin (letx) or with ef to form the edema toxin (edtx) [ ] . vaccines composed of pa showed that pa-binding antibodies effectively limit b. anthracis pathogenicity [ ] . moreover, it was demonstrated both for pa and for lf that binding of specific protective epitopes is required for neutralization [ , [ ] [ ] [ ] . pertussis is primarily a toxin-mediated disease caused by bordetella pertussis. pertussis toxin (ptx) is a major virulence factor, and chemically or genetically detoxified ptx is a major component of all acellular vaccine formulations in combination with up to four additional virulence factors. it has been shown that ptx-neutralizing abs bind to its s and s / subunits, which are necessary for its translocation to the cytoplasm and endocytosis, respectively [ ] . ricin (molecular weight, , ) is a relatively simple toxin consisting of an enzymatic a subunit (rta) and a binding b subunit (rtb) joined by a disulfide bond [ ] . o'hara et al. found that epitope specificity is the primary determinant of the ability of an antibody to neutralize ricin. these specific epitopes interfere with the enzymatic activity of rta. moreover, neutralizing mabs were primarily directed against α-helices situated within rta folding domains and , whereas non-neutralizing mabs targeted random loops and coils largely localized to domain [ ] . in rare cases, a single mab can be as protective as a pab-based preparation [ , ] via the blockade of a pivotal epitope that results in complete neutralization. this evidence for the existence of 'hot spot' epitopes that display a unique functional impact suggests that interfering with more than one such epitope might be highly beneficial for toxin neutralization. for most of the reported neutralizing mabs, the potency of an individual mab is substantially lower than that of a pab. various studies have reported that the potency of protective mabs can be augmented additively or synergistically via the addition of other protective mabs and that the combination of protective mabs targeting different epitopes on a toxin molecule can synergize protective efficacy [ ] . however, the exact mechanism underlying synergistic rather than merely additive neutralization following the blockade of multiple significant epitopes is not fully understood. such synergy may stem from interference with two or more epitopes that, although not essential individually, are vital in combination. the concurrent blockade of these types of epitopes might synergistically "shut down" essential functional pathways of the toxin, such as binding to its receptor target. this phenomenon is expected to be more prominent when important neutralizing epitopes perform redundant functions, such as binding to cellular receptors, cleaving a target substrate, etc. (figure ). the following findings support this possibility. schematic illustration of the simultaneous interference with multiple functional epitopes. synergistic neutralizing effect may be the result of antibody interference with two or more distinct epitopes that, although not essential individually (a), are vital in combination (b). important functional pathways of the toxin, such as binding to its receptor target, might be eliminated by this concurrent blockade. abbreviations used: e-epitope, r-receptor on a target cell. for bonts, two classes of binding sites exist, proteins and ganglioside receptors, both of which reside in the hc domain. thus, a double-receptor concept is prevalent [ ] . in a study in , chen et al. predicted that two mabs, which recognized the protein and the ganglioside receptor binding domains, respectively, synergistically neutralized bonts. in this study, the investigators selected three neutralizing mabs that recognized different non-overlapping epitopes of bont/b and compared the neutralizing effects of various antibody combinations. they found that a mab that responded to the ganglioside receptor binding site synergized with the other two mabs that recognized non-overlapping epitopes in syt ii (the protein receptor). however, the combination of the latter two mabs did not exert a synergistic effect [ ] . this example demonstrates that the combination of two mabs that recognize distinct functional domains on bonts may represent the general principle for a potent synergistic effect. interestingly, chen et al. further analyzed the results generated by the lab of james marks [ ] , which developed mabs for bont/a. they found that the neutralizing potency of one pair of mabs (c + d ), which bind to the translocation domain and the sialoganglioside binding domain, respectively, was times higher than that of another pair (s + d ), whose epitopes are located at the same functional domain. this finding provides additional evidence that a combination of mabs that recognize different functional domains is more likely to synergize than a combination of mabs recognizing the same functional domain [ ] . the rtb subunit of ricin contains two galactose binding sites that function independently and are distant from one another [ , ] . hu et al. generated four mabs directed against the rtb subunit and found that all of them bound to conformational epitopes with a high affinity. when testing pairs of mabs, they demonstrated a synergistic neutralization of ricin in vitro and in vivo. hu et al. hypothesized that because the two rtb domains are homologous and are structurally similar, a single rtb-specific mab may block both galactose binding pockets by binding to similar conformational epitopes sharing both domains and that the simultaneous binding of two mabs likely synergistically interferes with ricin attachment to cells. in addition, the authors speculated that mabs might bind to domains that interrupt the transport of rta from the er to the cytosol. they also determined that the synergistic effect of the mab pairs depended on different ricin neutralization activities that were more related with their epitope specificities than their ricin-binding affinities and that were not related to their antibody isotypes [ ] . in another ricin study, mcguiness et al. identified and characterized a murine igg anti-rtb mab ( b ). using a vero cell cytotoxicity assay, they compared b with a known anti-rta mab (r ) and found that b was approximately two-fold more effective at neutralizing ricin. the investigators studied the mechanism of action of these two mabs and found that both b and r blocked ricin attachment to galactoside receptors, although r was directed against rta [ ] . active tent, a close relative of bont, is a di-chain toxin, in which the light chain (lc) and heavy chain (hc) are linked by a single disulfide bond. the kda lc acts as a zinc-dependent endopeptidase, and the kda hc is composed of two distinct functional domains: a translocation domain (n-terminal half) and neuronal ganglioside binding domain (c-terminal half) [ ] . similar to bont, it is accepted that tent acts via a dual receptor model in which gangliosides and glycosylated proteins, such as the synaptic vesicle proteins sv a and sv b, are involved [ ] . in one pioneering study of mab combinations against toxins, volk et al. evaluated the effect of mabs on different tent subunits and found that all neutralizing mabs bound to epitopes on the hc of tent (approximately half of them to the c-terminal region and half to the n-terminal region). when combining these mabs, it was found that different cocktails of - mabs exerted synergistic neutralization in vivo, resulting in up to -fold greater protection than the individual neutralizing mabs. the authors hypothesized that the large number of epitopes found to be involved in neutralization suggested that different antibodies block distinct steps in toxin function and that the simultaneous binding of multiple epitopes is required for such neutralization [ ] . synergistic oligoclonal antibody interference may not necessarily be associated with known neutralizing epitopes but may rather be based on either adherence to epitopes that individually play no apparent role but together perform a significant function or the blockade of functional epitopes due to steric hindrance caused by the valence of a nearby bound antibody. for instance, cheng et al. demonstrated that a combination of two mabs, one directed against the lc (catalytic domain) of bont/a, and the other directed against the translocation domain, did not inhibit endopeptidase activity but, instead, blocked toxin entry into primary and cultured neuronal cells in a synergistic manner. the authors suggested that, via steric hindrance, mabs may have interfered with bont/a receptor binding, subsequent neuron internalization, or interaction with intracellular snap [ ] . similarly, synergistic effects of the combination of protective and non-protective mabs were demonstrated for a pair of a protective mab and a disease-enhancing mab against anthrax by the lab of arturo casadevall. chow et al. investigated whether the combination of a protective and a disease-enhancing mabs act in concert to abrogate pa oligomerization, thus preventing the translocation of lf and ef into the cell to cause subsequent toxicity. individually, the protective mab inhibited furin cleavage and subsequent oligomer formation, whereas the enhancing mab did the opposite. however, the igg mab combination resulted in mab-pa complex formation that completely abolished pa oligomerization via a furin cleavage-independent mechanism [ ] . evidence supporting the concept that synergistic activity is partially derived from the combination of mabs that simultaneously block different functional epitopes was provided by the neutralization of staphylococcal enterotoxin b (seb), one of several potent exotoxins secreted by staphylococcus aureus that cause toxic shock syndrome (tss) [ ] . similar to other superantigens, seb simultaneously binds to major histocompatibility complex class ii (mhc-ii) molecules on antigen-presenting cells (apcs) and to t-cell receptors (tcrs) that incorporate v chains [ ] . blocking antibodies may prevent seb from facilitating the formation of the mhc-ii/seb/tcr complex, thereby inhibiting this toxin. according to the crystal structures of complexes consisting of seb and mhc-ii or tcr, the two binding sites are spatially distinct from the contact areas [ ] . varshney et al. generated mabs against seb and evaluated different pairs of mabs in which only one or none of the mabs individually protected against seb toxicity. the authors reported that protection was achieved when these mab combinations were administered simultaneously. in fact, this was the first report in which enhanced protection against seb-induced lethal shock (sebils) was demonstrated in mice by the combination of two individually non-neutralizing mabs. these mabs recognized non-continuous residues that likely contribute to conformational epitopes [ ] . the phenomenon of synergistic neutralization due to blockade of functional non-overlapping epitopes is not restricted to toxins, and has also been demonstrated in the study of viruses and bacteria. li et al. evaluated different combinations of human mabs directed against various epitopes on human immunodeficiency virus type (hiv- ) envelope glycoproteins for their ability to neutralize a chimeric simian-human immunodeficiency virus (shiv-vpu ). in accordance with the putative synergistic mechanism, the most potent neutralization of triple combinations evaluated was attained using three mabs directed against three different domains, one each on v , gp , and gp . the authors further evaluated a combination of four mabs (the previous three mabs and one that recognized the cd binding site) and found higher neutralization potency and a higher degree of synergy than any of the triple combinations [ ] . similarly, meulen et al. presented experimental animal data showing that protection against severe acute respiratory syndrome coronavirus (sars-cov) infection was feasible using a mixture of two human mabs (cr and cr ). this mab combination neutralized sars-cov in a synergistic manner by recognizing different epitopes on the receptor-binding domain. [ ] . faleri et al. recently reported the functional characterization of two mabs that recognize factor h binding protein (fhbp) on neisseria meningitidis serogroup b (menb) surface [ ] . fhbp has proved to be a virulence factor for n. meningitidis and a target for functional bactericidal antibodies. fhbp binds to human factor h (hfh), that serves to downregulate the host alternative complement pathway, and helps the organism evade host innate immunity [ ] . although the mabs recognized two overlapping epitopes that were part of the hfh binding site on the n-terminus of the hfbp, they did not elicit complement mediated bactericidal activity against menb, when tested either individually or in combination. interestingly, when tested in combination with another mab, that recognized a conformational epitope located on the c-terminus-opposite to the hfh binding site, the two mab combinations exerted different levels of synergistic activity, depending on the orientation of the mabs on fhbp. the authors suspected that while the concomitant binding of one combination may result in a reciprocal orientation suitable for correct engagement of c q, the relative position of the other combination might not be optimal to promote the same effect [ ] . in summary, combining mabs that bind to functionally distinct epitopes of a toxin may result in synergistic neutralization. moreover, even the combination of mabs that individually do not display neutralizing activity may exert a synergistic neutralizing effect due to either the steric hindrance of adjacent neutralizing epitopes or partial or full epitope redundancy that is overcome by simultaneous binding. another mechanism that may underlie the synergistic neutralizing activity of a mab combination is the increased affinity of the cocktail compared with each mab component. various findings indicate that neutralization potency is associated with the affinity of the antibody to the toxin. as demonstrated in the following section, additional findings have indicated that the simultaneous binding of multiple mabs to a multisite antigen may result in a substantial increase in overall affinity. taken together, these findings suggest that certain combinations of mabs bind to a toxin much more strongly than each mab alone, thus inducing synergistic neutralization. the correlation between antibody binding affinity and toxin neutralization is somewhat intuitive, as toxin blocking and the subsequent clearance of the formed ics depend on the binding of antibodies to the toxin. thus, one can easily conceive that adherence to the toxin would be more efficacious if the binding of the antibody to its epitope were stronger, thus prolonging the duration that this epitope is 'occupied', leading to more potent inhibition and clearance from the bloodstream. there are several indications that the neutralizing activity of antibodies is associated with their affinity to the target epitope. indeed, in vivo assays conducted in our lab revealed that the neutralizing potency of mabs against the three different bonts was associated with an increased affinity to the toxin, as all neutralizing mabs exhibited the highest anti-toxin elisa titer [ ] . pless et al. also reported that a common characteristic of all anti-bont/a-hc neutralizing mabs examined was a very high affinity [ ] . in another anti-bont study, amersdorfer et al. showed that the most potent anti-hc scfv displayed the highest affinity for its epitope [ ] . in accordance with this concept, maynard et al. showed that the protection against anthrax toxin by recombinant antibody fragments was correlated with antigen affinity [ ] . likewise, scott characterized a panel of anti-tetanus toxin scfvs and found that / clones neutralized toxin activity based on a ganglioside binding assay and that this effect was strongly related to the affinity of these clones [ ] . nevertheless, we found that some mabs exhibited no neutralizing activity despite displaying a high elisa titer. these results suggest that high affinity may be a necessary but not sufficient parameter of neutralizing potency [ ] . the findings described in the previous section demonstrate that the affinity of an antibody to its epitope correlates to its neutralizing activity. thus, if the simultaneous binding of multiple mabs increases the overall affinity of the mabs to the antigen, the neutralizing activity of the combined mabs may be enhanced. what is 'overall affinity', and how can it be increased? affinity, a measure of the strength of the noncovalent interaction between an antibody and its antigen [ ] , is mathematically represented by the equilibrium constant (k) that characterizes the reversible binding of an antibody to an epitope on an antigen [ ] . affinity can be divided into 'intrinsic' and 'functional'. the term 'intrinsic affinity' characterizes systems involving monovalent interactions between one paratope and one epitope, while the term 'functional affinity', also known as 'avidity', refers to higher order complexes between bivalent or multivalent abs and a number of binding sites per antigen [ ] , and reflects the intrinsic affinity, the valence and the topological relationship between the antigenic determinants and the antibody combining sites [ ] . the strength of multivalent interactions (functional affinity or avidity), usually exceeds, sometimes greatly, the corresponding intrinsic affinity [ , ] . multivalent linkages within ics can be formed between either the same epitope expressed on separate antigens and ab molecules of the same specificity (mab), or between diverse epitopes and abs with two or more specificities (such as in the case of natural immune reactions), which may have cooperative interactions between them, eventually leading to increase in the functional affinity. two basic mechanisms may underlie such cooperative interactions between mabs that bind to diverse epitopes. first, the binding of one bivalent mab to two epitopes on two soluble antigens, thereby facilitating their cross-linking, increases the probability that a second mab, specific for an independent epitope, could bind to two sites instead of to only one site [ ] . this phenomenon was demonstrated years ago by moyle et al. for antibodies against human chorionic gonadotropin (hcg). the authors found that some mab pairs but not others displayed a higher affinity to the hormone than either mab alone and that this increased affinity was associated with the formation of a stable circular complex consisting of two antibody molecules (each one of the two mabs) and two hcg molecules [ ] . second, the increase in functional affinity observed for multiple mabs may be due to a conformational change in the antigen that occurs upon the binding of the first mab, resulting in a higher affinity of the second and third mabs [ , ] (illustrated in figure ) . thus, the binding of the first antibody might induce a more stable conformation of the antigen that thermodynamically or entropically favors the binding of a subsequent antibody [ , ] . in evidence supporting the influence of affinity on the synergistic neutralization of toxins was recently reported in a tetanus study. scott et al. characterized scfvs to tent and found that certain scfv combinations exhibited synergistic binding based on competitive binding studies. these findings agreed with their results that some scfvs reduced toxin activity without recognizing the hc domain, which is known to attach to gangliosides. thus, the authors suggested that the synergy in binding stems from conformational changes in the toxin upon binding of one scfv that favors binding of the next and that the targeted epitope may be less important when identifying toxin-neutralizing mabs [ ] . in accordance with these findings, yousefi et al. detected synergistic neutralizing effects of one pair of mabs, but not other pairs, that recognized two distinct but very close or overlapping epitopes within the gt b-binding fragment of tent. this pair of mabs displayed an in vivo potency several times higher than the commercial tig antibody preparation [ ] . the authors speculated that the observed cooperative binding effects were caused by conformational changes in the tent protein in which binding of the first antibody thermodynamically facilitated the binding of the second antibody. ngundi et al. investigated the mechanism involved in the synergistic neutralizing effects of combined anti-pa mabs against anthrax. the authors found that one mab promoted the bivalent binding of another mab to pa, presumably by bridging two pa monomers, and proposed that bridging via one mab promotes bridging via the other. thus, any transient dissociation of one antibody arm from the toxin would result in rapid rebinding, as the other antibody bridge would prevent the antigen from diffusing away. this phenomenon would substantially increase antibody avidity, i.e., the combined strength of multiple bonding interactions, resulting in synergistic neutralization [ ] . all of the evidence described above suggests that the increased affinity of mab cocktails might result in a synergistic enhancement of toxin neutralization. low-affinity fcrs do not bind to monomeric immunoglobulins at a detectable affinity; nevertheless, they bind to aggregated antibodies complexed to multivalent antigens with a high avidity. thus, a multivalent fc-decorated antigen, efficiently recognized by fcγr, is the basis for the effective clearance of ics rather than unbound antibody or monomeric antibody-antigen ics [ ] . low affinity fcrs on resident macrophages may be more relevant than are high affinity fcrs with respect to the clearance of ics [ ] . in human liver, low affinity fcγrii and fcγriii were detected on both kupffer cells and liver sinusoidal endothelial cells, whereas high affinity fcγri is not expressed in liver macrophages [ , ] . additionally, it was demonstrated that the rate of removal of complexes to the liver is proportional to the size of the complex [ ] . consequently, the adherence of individual mabs to an antigen (such as a toxin) would be expected to form relatively small ics because one antigen molecule can bind to no more than one mab (except for rare instances involving repetitive epitopes). because larger igg-toxin ics are much more efficiently cleared from the bloodstream [ , ] , it is anticipated that by combining two or more mabs that bind to distinct toxin epitopes, thus mimicking a polyclonal reaction, larger ics are formed and the clearance of combined neutralizing mabs is substantially enhanced. thus, toxin clearance is considered to be much more effective for a mab combination than for a single mab treatment. ics are formed when soluble iggs bind to a target antigen. ics, when directly administered to non-immunized animals or formed following the administration of low doses of antigens to immunized animals, are removed rapidly in part by the reticuloendothelial system (fixed mononuclear phagocyte system) [ ] and by certain circulating leukocytes that express receptors for the ics [ ] . generally, the clearance of ics following a polyclonal response occurs via two pathways, both of which involve antibody fc. one clearing mechanism is mediated by fcγ-receptor (fcγr) binding. the second mechanism is mediated by the complement system, which plays a crucial role in ic handling [ ] . these two mechanisms can act independently [ ] . thus, antibody fc-mediated clearance is thought to play an important role in toxin neutralization, as well. accordingly, it has been demonstrated that the association between pabs and bont leads to enhanced clearance from the circulation of rats, particularly to the liver and spleen [ ] . notably, other roles of the igg fc might obscure its involvement in toxin-igg ic clearance. for instance, fc substantially contributes to igg stabilization in the bloodstream primarily due to protection by the neonatal fcr (fcrn). fcrn plays a critical role in regulating the levels of circulating iggs in rodents and higher species via strict ph-dependent igg/fcrn binding [ ] . the fcrn-bound antibody is thought to be protected from degradation, thereby increasing the half-life of igg in serum. therefore, the use of antitoxin agents that do not bear fc (such as fab, scfv, etc.) and thus display a shorter half-life than intact igg would be expected to exhibit reduced efficacy due to a rapid decrease in the concentration of the antitoxin agent in the bloodstream, as demonstrated by bont toxicity studies [ , ] . nevertheless, in addition to the inferior potency of the f(ab') antitoxin, mazuet et al. showed that in spite of its shorter half-life ( -fold), one f(ab') retained its neutralization efficacy compared with intact igg. interestingly, the mab that was not affected by fc deletion displayed a much higher affinity than the other mab [ ] . the elimination of foreign fc from equine antitoxin preparations serves to lower immunogenicity; nevertheless, polyclonal f(ab') -based preparations protect against toxin challenge without activating fc-mediated effector functions. this protection suggests that a high dose of f(ab') may compensate for the lack of fc-mediated mechanisms and may induce neutralization solely via toxin blocking. furthermore, it is plausible that the addition of human fc fragment to the equine f(ab') preparation would improve antitoxin efficacy and reduce the total amount of foreign protein administered to a patient. however, such manipulation is not applicable for polyclonal preparations and may be difficult to evaluate in animal models due to the variance in fcγrs and complement receptors (crs) among species. manipulation of fc fragments is being performed on mabs, enabling the examination of the role of fc in neutralization in vivo. recently, the generation and characterization of an fcγr-humanized mouse that lacks all murine fcγrs was described; this model may enable the accurate prediction of the consequences of linking human fcγrs to iggs for a particular biological response [ ] . recapitulation of the human-specific fcγr expression pattern in this mouse model was used recently to assess the activity of anti-anthrax toxin mabs containing specific fc variants that selectively enhanced their affinity for particular human fcγrs [ ] . accumulating evidence from recent years supports the involvement of igg fc (for mabs and pabs) in toxin neutralization. vitale et al. showed that f(ab') fragments of an anti-pa mab were -to -fold less potent than the complete igg in neutralizing anthrax toxin in vitro, even though they retained equivalent affinity for pa. the addition of fcr-blocking antibodies greatly reduced the activity of this mab, and the neutralizing activity of mouse, rabbit, and human antisera elicited by pa vaccines was effectively abrogated by blocking fcrs. however, in vivo, significantly higher concentrations of f(ab') did protect against anthrax toxin cytotoxicity; thus, in vivo protection cannot be unambiguously attributed to the fc-enhanced neutralization activity [ ] . it is unclear whether this potency reduction in vivo is also attributed to the shorter half-life of the f(ab') fragments. this neutralizing activity was abrogated by fcr blockers for pab and mab treatment, implying that their potency is indeed fcr-dependent. experiments conducted by arturo casadevall and colleagues have broadened our understanding of the function of fc in anti-pa antibodies [ ] . abboud et al. generated identical variable regions and specific igg a and igg b variants of an igg anti-pa mab and found that the efficacy of antibody-mediated neutralization was dependent on the isotype and that this neutralization activity required a competent fcγr. furthermore, they showed that the igg a mab prevented lethal toxin cytotoxicity more efficiently than the igg mab and that passive immunization with igg and igg a mab protected wild-type, but not fcγr-deficient, mice against b. anthracis infection [ ] . nevertheless, the precise toxin neutralization mechanism after fcγr engagement was unclear. the authors hypothesized that mab pa-fcγrs ics are endocytosed and sorted to lysosomes for degradation but suggested that the differences in efficacy between the isotypes implied a difference in fc-mediated effector functions rather than defective endocytosis in mice deficient in fcγrs. the function of fc was also evaluated using other anti-toxin systems. akiyoshi et al. evaluated mabs directed against the a and b subunits of shiga toxin (stx ), which is the primary virulence factor for hemolytic uremic syndrome (hus). the investigators generated and evaluated (in vitro and in vivo) isotype variants (igg , igg , igg , and igg ) and fab and f(ab') fragments of a mab specific for the a subunit of stx . these isotype variants exhibited protection in vitro, and the igg and igg variants exhibited the highest protection in vivo. although the fab and f(ab') fragments exhibited protection in vitro, they did not exhibit protection in vivo. the authors obtained similar results for a mab directed against the b subunit of stx [ ] . pincus et al. examined the role of the fc region in mediating protection from ricin toxicity and found comparable results to those for stx . they compared the in vitro and in vivo effects of intact ig and of fab fragments derived from pab and anti-a chain mab preparations. the results revealed little difference between ig and fab in terms of antigen binding and in vitro neutralization in hela cells but found relatively large differences in their protection of animals. in addition, in vivo experiments demonstrated that the murine mab protected more strongly than its chimeric mouse/human variant, indicating that mouse fc regions perform better than human fc in mice. moreover, the investigators explored the role of fcγr in the protection of cells from ricin toxicity using a panel of hela-derived cells carrying no fcr or human fcγri, fcγriia, fcγriib, or fcγriiia and found that the presence or absence of fcr did not influence mab-mediated protection. these findings indicated that neither the fc region nor the presence of an fcr on the target cell influence the ab-mediated protection of individual cells. however, in vivo, the presence of fc enhanced their protective efficacy [ ] . varshney et al. compared the neutralizing and protective activity of anti-seb mabs in vitro and in vivo. the authors demonstrated that changing the isotype of already protective mabs, without affecting their antigen specificity or sensitivity, enhanced their protective ability, suggesting a therapeutic role for fc [ ] . to directly determine the role of fc, we recently measured the protective properties of igg mab and its f(ab') form in mice exposed to bont/a. equal amounts of igg mab and its f(ab') fragment ( pmol/mouse) were incubated with increasing amounts of bont/a ranging from to msld . then, these mixtures were injected into mice, and their survival was monitored. the intact igg mab protected % of the mice from a challenge of up to msld bont/a, whereas its f(ab') fragment protected against only msld of the toxin. thus, the protective efficacy of f(ab') in mice was -fold lower than that of its igg form, demonstrating the important role of fc in protection against bont toxicity (paper in preparation). recently, several studies conducted by the lab of charles shoemaker directly investigated the phenomenon of synergistic neutralization mediated by increased fc valence (illustrated in figure ). sepuldeva et al. proposed a novel concept in which bont neutralization is exerted by combinations of high-affinity epitope-tagged scfvs against bont/a, serving as the 'blocking' arm, and anti-tag mabs containing fc, serving as the 'clearing fc' arm. in this manner, the investigators elegantly illuminated the critical contribution of fc-mediated clearance of bonts to synergistic neutralization and dissected the function of fc from other putative mechanisms. the authors showed that for individual non-neutralizing scfvs, the addition of additional clearing fcs synergistically increased the level of protection against bont/a. thus, when an scfv contained two copies of the tag (i.e., two 'clearing fcs' for each 'blocking arm') instead of one, protection against the toxin was approximately -fold enhanced, approximately the same fold-change as the addition of a different scfv. the improvement achieved by the inclusion of an additional tag likely resulted from further fc decoration of the toxin. following that study, the definitive role of fc-mediated clearance in synergistic activity was demonstrated by pharmacokinetic studies showing that bont/a was rapidly cleared from the sera of mice administered a pool of anti-bont/a scfvs and a clearing fc but not from the sera of mice administered either the scfvs or the clearing fc alone. moreover, the efficacy and clearance profile of this mixture of a neutralizing dose of scfvs combined with an anti-tag clearing fc was comparable to that of a polyclonal antitoxin serum [ ] . . schematic illustration of the fc-mediated clearance of antibody-toxin complexes via multimeric antibody decoration of the toxin. the clearance of ics occurs via fcr and cr pathways, both involve antibody fc. low-affinity fcrs on resident macrophages bind to antibody-antigen complexes with a high avidity but do not bind to monomeric antibody-antigen ics at a detectable affinity. in addition, larger igg-toxin ics are much more efficiently cleared from the bloodstream. thus, a mab cocktail might mimic a polyclonal reaction by forming large ics with multiple fc toxin decoration, resulting in a substantial enhanced antibody-toxin complexes clearance. the figure illustrates binding of multiple fcs with high avidity to fcrs (b), as opposed to low affinity binding of a single mab fc (a). additional experiments were performed by the same lab to further investigate the role of toxin blocking and clearance in determining antitoxin efficacy. the authors generated small ( kda) and stable camelid heavy-chain-only ab vh (vhh) domains against bont/a. these vhhs were used as antitoxins via the co-administration of recombinant high-affinity anti-bont tagged vhh and an anti-tag clearing fc. the authors compared neutralizing or non-neutralizing binding vhhs administered with or without clearing fc to evaluate the relative contributions of toxin blocking and clearance to antitoxin activity. a cocktail of up to three vhhs, each containing one tag, administered with an anti-tag clearing fc was insufficient to induce efficient clearance. however, a heterodimer consisting of two non-neutralizing monomeric vhh units, each containing two tags instead of one, enabled the simultaneous binding of four fc domains to the toxin. the resulting toxin clearance dramatically enhanced the protection of mice from virtually no protection in the absence of the clearing fc to complete protection against msld of bont/a. furthermore, a comparison of the antitoxin efficacy between neutralizing and non-neutralizing vhh agents in the presence or absence of the anti-tag clearing fc revealed that the neutralizing vhhs were highly effective in the absence of clearance (and were improved in the presence of the clearing fc), whereas non-neutralizing vhhs depended on the clearing fc for efficient neutralization [ ] . a similar technique utilizing a vhh-based neutralizing agent (vna) and a clearing fc agent was demonstrated to be effective for stx. tremblay et al. showed that co-administration of a vna and an anti-tag fc substantially increased the prevention of death or kidney damage in mice following challenge with stx or stx [ ] . however, in a recently conducted anti-ricin study, contradicting results were obtained. vance et al. showed that on one hand, the addition of a clearing fc agent to double-tagged vhh heterodimers (enabling up to four fc molecules to decorate each toxin molecule) significantly increased the protection of mice from ricin toxicity, likely via the promotion of toxin clearance [ ] . on the other hand, the presence or absence of an anti-tag clearing fc linked to a high affinity heterodimer consisting of two non-neutralizing vhhs afforded the mice no protection against ricin challenge. because ricin is toxic to all cell types and preferentially targets macrophages, including kuppfer cells in the liver [ ] , the authors postulated that the accelerated fcγr-mediated clearance of ricin in the absence of the inhibition of functional ricin domains may not improve the clinical results and might even enhance its toxicity [ ] . the significance of fc valence for toxin clearance from the circulation was recently shown using red blood cells (rbcs) mediated toxin sequestration [ ] . opsonization of particulate pathogens by antibodies and complement can lead to their binding to the complement receptor (cr ), specific for c b, on primate erythrocytes. this immune adherence may prevent pathogens from leaving the bloodstream and facilitate their destruction by liver macrophages [ ] . ronald taylor and colleagues used mabs specific for cr cross-linked with pathogen specific mabs to generate heteropolymers (hps) which can bind a wide range of substrates to primate erythrocytes, and imitate the natural clearance mechanism of c b-opsonized substrates bound to erythrocyte cr . in this way, fc receptors on the phagocytic cell engage the erythrocyte-bound complex, cr is removed by proteolysis, and the entire immune complex and cr are internalized while sparing the erythrocyte [ ] . the hp methodology was recently applied by sharma et al. that used bont as a model system in an attempt to enhance toxin rather than particulate pathogen neutralization. they converted a pair of bont specific mabs into hps and tested them in transgenic mice expressing human cr on rbc membranes. the authors reported that two hps given in combination had -fold greater potency than un-modified mabs, capable of neutralizing msld bont/a. the ics formed with an hp and an un-modified mab were less potent than those formed with two hps. in addition, peritoneal macrophages internalized bont better when it was bound to two hps rather than to an hp + mab or mab + mab combination, independent of whether the hp pair contained a cr -binding or nonbinding control mab. this may indicate that enhanced bont clearance from the blood circulation by fixed tissue macrophages is attributed to the opsonization of multiple fc domains in the hp complexes [ ] . currently, modern biotechnological advancements have enabled the manufacture of an unlimited supply of human or humanized mabs, as well as other mono-specific biopharmaceuticals, that display exquisite specificity and unprecedented affinity for life-threatening toxins and that exhibit a low risk of immunogenicity, low batch-to-batch variability, and a long serum half-life of up to month, reducing the frequency of drug administration [ , , ] . these characteristics are highly valuable and advantageous compared with pab-based preparations. however, the antigenic variability of toxins (and pathogens in general) and the relatively low efficacy of individual antitoxin mabs limit their utility as therapeutics. one rational solution to overcome these limitations would be to combine mabs into therapeutic cocktails to mimic safe polyclonal responses. until recently, this option was difficult to implement due to regulatory and cost concerns [ ] . nevertheless, efforts in recent years to produce effective mab cocktails for post-exposure rabies and botulinum prophylaxis [ , ] have established an important precedent by increasing the relevance of the use of mab combinations against toxins and infectious diseases. many studies have shown ( table ) that different combinations of mabs against various toxins and other infectious agents indeed exhibit enhanced efficacy and that in many cases, this enhancement is synergistic, occasionally exceeding the efficacy of the pab-based preparations that are generally used. in this review, we described a body of evidence supporting three potential mechanisms underlying synergistic neutralization by mab combinations: ( ) the simultaneous binding of interfering mabs to multiple functional sites of the toxin, facilitating the significant blockade of two or more essential epitopes that react with the target cell, thus dramatically reducing the potency of the toxin; ( ) a dramatic increase in the affinity of the combination of mabs compared with each mab component, correlating with neutralization; and ( ) fc-mediated clearance of antibody-toxin ics from the serum, which is enhanced by the multimeric antibody fc decoration of the toxin. it is plausible that these three mechanisms complement each other and that the combination of these mechanisms mediates the enhanced cooperative activity of mab cocktails. these mechanisms may also clarify the contribution of individual non-neutralizing mabs to the synergistic neutralization of a mab cocktail, a phenomenon that was observed in previous studies by our lab and by others [ , , , ] . this synergism can be facilitated by increasing the valence of fc molecules decorating the toxin, thereby enhancing toxin clearance, by targeting a critical number of epitopes, by increasing the avidity of the mab cocktail, or via the interaction of two or more mechanisms. one conclusion may be that screening for toxin-neutralizing mabs should involve high-affinity mab combinations to avoid overlooking mabs that individually display low or no neutralizing activity. mab cocktail-based drugs that display enhanced neutralization efficacy can significantly lower the amount of drug administered in comparison with the efficacy of a single monoclonal antibody, which will likely reduce cost and side effects. the synergistic activity of mab combinations may depend on the presence of fc and on the fc isotype [ , , ] . thus, an in vitro assessment of the neutralizing capacity of a mab combination might not fully reflect its in vivo potency. moreover, the evaluation of a mab from a certain animal origin might not exert its full potency due to the inter-species variance of fcrs and crs. therefore, special care should be taken when selecting the animal model for evaluating the synergistic effect of a mab combination. understanding the mechanisms underlying synergistic neutralization is crucial for combining mabs to form potent antibody cocktails. future experiments should focus on developing analytical tools that will enable further investigation of these mechanisms. it is tempting to speculate that control of the enhancement of neutralization might become feasible via the rational selection and combination of mabs. eran diamant, amram torgeman, eyal ozeri and ran zichel conceived the review and wrote the manuscript. continuous cultures of fused cells 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broadly protect against meningococcal disease. microbiol high-affinity, protective antibodies to the binding domain of botulinum neurotoxin type a genetic and immunological comparison of anti-botulinum type a antibodies from immune and non-immune human phage libraries protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity characterisation of a panel of anti-tetanus toxin single-chain fvs reveals cooperative binding affinity, complementarity, cooperativity, and specificity in antibody recognition affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory b-cell responses the affinity of antibody: range, variability, and the role of multivalence the influence of polyvalency on the binding properties of antibodies a circular antibody-antigen complex is responsible for increased affinity shown by mixtures of monoclonal antibodies to human chorionic gonadotropin fc receptor biology blockade of clearance of immune complexes by an anti-fc gamma receptor monoclonal antibody receptor-mediated endocytosis of immune complexes in rat liver sinusoidal endothelial cells is mediated by fcgammariib immunohistochemical phenotyping of liver macrophages in normal and diseased human liver studies of reticuloendothelial function in the mouse with model immune complexes. i. serum clearance and tissue uptake in normal c h mice studies on antigen-antibody complexes. i. elimination of soluble complexes from rabbit circulation liver cell uptake and degradation of soluble immunoglobulin g immune complexes in vivo and in vitro in rats divergent roles for fc receptors and complement in vivo an initial assessment of the systemic pharmacokinetics of botulinum toxin monoclonal antibody clearance. impact of modulating the interaction of igg with the neonatal fc receptor mouse model recapitulating human fcgamma receptor structural and functional diversity human igg fc domain engineering enhances antitoxin neutralizing antibody activity prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity a requirement for fcgammar in antibody-mediated bacterial toxin neutralization evaluation of fab and f(ab') fragments and isotype variants of a recombinant human monoclonal antibody against shiga toxin a single vhh-based toxin-neutralizing agent and an effector antibody protect mice against challenge with shiga toxins and stepwise engineering of heterodimeric single domain camelid vhh antibodies that passively protect mice from ricin toxin heteropolymer-mediated clearance of immune complexes via erythrocyte cr : mechanisms and applications development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans humanized staphylococcal enterotoxin b (seb)-specific monoclonal antibodies protect from seb intoxication and staphylococcus aureus infections alone or as adjunctive therapy with vancomycin the authors declare no conflict of interest. key: cord- - h authors: späth, peter j.; granata, guido; la marra, fabiola; kuijpers, taco w.; quinti, isabella title: on the dark side of therapies with immunoglobulin concentrates: the adverse events date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h therapy by human immunoglobulin g (igg) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. the success of igg concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of frontiers in immunology. a part of the success is the adverse event (ae) profile of igg concentrates which is, even at life-long need for therapy, excellent. transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. the cornerstone of the regulatory network is current good manufacturing practice. non-infectious aes occur rarely and mainly are mild to moderate. however, in recent times, the increase in frequency of hemolytic and thrombotic aes raised worrying questions on the possible background for these aes. below, we review elements of non-infectious aes, and particularly focus on hemolysis and thrombosis. we discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence ae profiles and efficacy of igg concentrates. i.e., cold-ethanol or ion-exchange chromatography, contaminants, route of application, i.e., intra muscular (imig), intravenous (ivig), or subcutaneous (scig), the rate of increase of the exogenous igg in the circulation of the recipient over time and, last but not least an eventually existing risk factor from patients' side ( figure ) as well as incorrect handling of the concentrate are factors having a role in inducing non-infectious aes related to administration of igg concentrates ( table ) . igg concentrates represent a defined part of the adaptive immune system, are isolated from pooled human plasma of at least donors, which contribute to the repertoire diversity in the final product. therapies with igg concentrates manufactured according to regulators requirements are acknowledged to be safe in general. this does not exclude the occurrence of aes which in their majority are rare and clinically mild to moderate. below, we like to give a few insights into various aspects and possible mechanisms of aes. table . contemplating on the active ingredient of the concentrates, the administration of the igg molecules inevitably results in the interactions of the exogenous igg with the various parts of the immune system of the recipient and vice versa. this interaction in its principle generates an inflammatory condition. a key parameter deciding on the intensity of such a condition is the rate of increase over time of exogenous igg in the circulation of the recipient. insert to figure (shadowed): ∆igg in the circulation over ∆time is dependent on the combination of infusion rate (ir) and the strength of the solution applied. main figure: the mode of application, i.e., intravenous or subcutaneous, are additional factors being decisive for kinetics and the area under the curve of generation of pro-inflammatory mediators and for passing the individual threshold (short-dashed line) for a clinically noticeable ae. the threshold in turn depends on eventually exiting risk factors form the patient side. although the area under the curve might be the same for low (long-dashed line) and high (solid line) irs, at high ir, the system might not be able to cope with the extent of reactions. at low ir (long-dashed line), all the events might remain below the threshold of clinically observable ae. subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent than with ivig. in contrast, local mild to moderate aes are more frequent with scig. virus inactivation and virus elimination methods by validating an already performed step of the fractionation process or by introduction of dedicated steps (figure ) . a hallmark of virus elimination introduced in the late s in berne by the team of christoph kempf is the large-scale virus filtration technique (formerly also termed "nanofiltration") ( ) . meanwhile, virus filtration became a versatile tool to eliminate a variety of pathogens, the suspected agent of variant creutzfeldt-jakob disease included. thanks to the tightly implemented regulatory framework, pathogen safety of plasma products is at a level never reached before. this is well supported by the fact of reports missing in the last decade of transmission by igg concentrates of emerging viruses (sars coronavirus, west nile virus, mers coronavirus, and others), zoonotic pathogens, or the agent of variant creutzfeldt-jakob disease (vcjd). furthermore, the development of specific mass screening techniques might help to eradicate in any blood product the transmission of vcjd in the future ( ) . the human immune system is in charge of controlling invading organisms and mediates homeostasis. the immunoglobulin pools in mammals (igm, igg, and iga) to its smaller part provide defense and to the larger part homeostasis. human igg has a role in both. efficient host defense is supported by "immune antibodies." these have undergone somatic hypermutations and have in their vast majority narrow specificities and high affinities. antibodies, generated in absence of external stimuli are termed "natural antibodies" (nabs). they occasionally recognize self structures. in general, the v(d)j genes of nabs are in germ-line configuration or have undergone a few somatic hypermutations only. furthermore, they have broad specificity, are of low affinity (with exceptions) and high avidity ( ) . these nabs can participate in primary host defense, i.e., at a time point when an immunologic reaction has not provided the specific antibodies, react, e.g., with repetitive structures which can be found on bacteria or viruses ( ) . the self-reactive nabs, which we like to term physiologic autoantibodies, comprises various populations of antibodies such as (i) those able to interact through their complementary variable regions (v-regions) with the v-regions of circulating and membrane-bound (bcr) immunoglobulins and the t cell receptor (tcr) β-chain variable region, providing a peripheral immune network (v-connected network) ( ); (ii) the nabs reacting in a non-idiotypic manner with the hinge region of immunoglobulins ( , ) ; (iii) populations of nabs showing a wide variety of specificities toward growth factors, cytokines, or anaphylatoxin ( ); (iii) and the population reacting with the soluble or membrane-bound forms of cell surface molecules having immunological importance, the last described being the fc receptors cd and cd ( ) . antibodies reacting with docking structures for viruses or bacteria can have additional first-line defense potential ( ) . these populations of nabs were described having a peripheral immune network homeostatic and anti-inflammatory function ( , ) . although the primordial humoral proteins comprising the complement and lectin-like proteins in the plasma play a definite role, another population of self-reactive nabs reacting with, e.g., epitopes conserved over the evolution apparently has tissue homeostatic function and might support the efficient removal of roughly altered/senescent cells of the body per day (for references see below). the signal for research on nabs in ivig was the description of igg autoantibody-mediated immune thrombocytopenia (itp) being corrected by infusion of a polyclonal, polyspecific igg concentrate ( , ) . this research has expanded ever since. the populations of immune antibodies and nabs in igg concentrates upon infusion/injection inevitably react with occasional pathogens, toxins, or superantigens and concomitantly infusion/injection also results in recognition of a wide array of tissue antigens and v-regions of the recipient's immune system. reactions with tissue antigens and v-regions are conveyed by the self-reactive antibodies of the many donors in the igg concentrate. vice versa, the recipient's immune system reacts with the infused igg. a bewildering wide range of possible reactions can occur which primarily are dependent on the immune status of the recipient at the time of therapy and to a smaller part on the igg concentrate(s). the therapeutic effect achieved depends on the disease treated, and can depend on the concentration reached locally, i.e., can have agonistic or antagonistic effects ( , ( ) ( ) ( ) . in summary, it is our opinion that igg concentrates always provide more or less the same "bouquet" of igg specificities (similarity); however, it is the recipient's actual immune condition which decides from which igg specificities the patient's derailed immune system is profiting (diversity). parameters of igg-mediated aes are: (i) the content in the product of biologically highly active likely beneficial ingredients that have to be kept under control (e.g., content of "dimers" devoid of remarkable complement activation in vivo; see below), and the content in unwanted active ingredients that have to be discarded during manufacturing (alloantibodies); (ii) impurities such as iga (anaphylactoid reaction); (iii) activated coagulation and contact activation factors (thromboembolic events) and; (iv) excipients such as sucrose (osmotic nephrosis). below, we like to add and contemplate on how fully native igg molecules not harmed by the manufacturing process might add to aes. the above mentioned inevitable interaction of the exogenous igg with the immune system of the recipient and vice versa in its principle might evoke an inflammatory condition. the sum of the potentially beneficial reactions might overshoot and lead to aes (figure ) . the principle of induction of mild inflammatory www.frontiersin.org figure | staying within a regulatory network is mandatory for remaining at the sunny side of the moon. handling of blood/plasma products to obtain therapeutic goods has to be performed within a regulatory framework. manufacturing of stable blood product has to adhere strictly to current good manufacturing practice (cgmp). application of cgmp starts from the moment of collecting whole blood and isolation of plasma thereof (r = recovered plasma) or the machine-supported collection of plasma (s = source plasma, apheresis plasma). application of cgmp ends at delivery of blood/plasma products to health care professionals. not following cgmp can lead to withdrawal of a plasma product from the market from one to the other day. conditions upon each infusion of a well-tolerated ivig was confirmed when several dozen normogammaglobulinemic volunteers in all cases except one, showed a more or less moderate inflammatory reaction as indicated by the increase of tumor necrosis factor alpha (tnfα) at . h post initiation of infusion. the only person in the cohort not showing a measurable tnfα increase was a woman caring at home for her brother with full blown aids ( ) . subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent compared to ivig but they are not absent ( - ) (a case of unintentional i.v. application of scig is not considered). in contrast, local mild to moderate aes are more frequent with scig ( ) . in summary, the intensity of the resulting aes is depending on the immune status of the recipient, the infusion rate (ir), e.g., how rapidly the active ingredients (the various igg specificities), the impurities, and the excipients reach the circulation of the recipient. thus, the i.v. application has the highest chance for the occurrence of aes. in the early days of ivig therapy, complement-mediated "anaphylactoid" (i.e., immediate) and "phlogistic" (i.e., inflammatory) aes were distinguished ( , ) . the complement-mediated aes were considered to be caused by aggregates in the product ("spontaneous complement activation" or anti-complementary activity or aca) or by in vivo formation of immune complexes (ics, patient's condition related; e.g., subclinical infections or the unnoticed presence of anti-iga antibodies) and therefore only igg concentrates with low or absent aca is accepted by authorities for human use. below, we present one instructive case of each type of reaction. the first reports of rapid onset aes concerned either the application of complement-activating fractions in an igg concentrate or the in vivo formation of complement-activating ics ( ) ( ) ( ) . a very rare but potentially fatal condition is the formation of iga/anti-iga complexes in patients being initiated on replacement therapy and having serum igg antibodies against infused iga not recognized before the start of the ivig infusion ( ) . prerequisite for the presence of anti-iga antibodies is the most common primary immunoglobulin defect, i.e., selective iga deficiency (sigad) or igad associated with diminution of other immunoglobulin classes. igad is defined by serum levels of < . or < . g/l (depending on laboratories). a marked diminution of serum iga consistent with igad in various ethnic groups is estimated being : to : , ( ) . the mean frequency in caucasians is approximately : ( ) . up to % of patients with igad have been reported having anti-iga antibodies in the serum with titers ranging between : and : , . in approximately % of patients with common variable immunodeficiency (cvid), and occasionally in patients with other primary immunodeficiency diseases, measurable anti-iga can be detected ( , ). these antibodies are predominantly of the igg class, but anti-iga antibodies of other immunoglobulin classes have been described as well ( , ) . the reason for their emergence remains unknown. taken the above numbers, the infusion of human-derived products containing iga resulting in severe anaphylactoid type aes should be considerable. this is not the case ( ) . questions about the clinical relevance of above numbers emerge as soon as blood banks (i) estimate the theoretical risk of iga anaphylactic reactions ( ); (ii) assess the relation of severe igad with the presence or absence of anti-iga antibodies ( ) ; (iii) screen donors for very low iga levels in order to become able to provide blood and plasma-derived products free of iga and find a considerably lower frequency than expected ( ) . alternatively, the test systems may not reliably detect anti-iga antibodies being as yet insensitive and inaccurate or -at least -do not correspond to the clinically relevant fraction of antibodies. this comes to mind when a more close look to "anti-iga" gives "unexpected" results, including "anti-iga" in blood donors with normal serum iga level or "anti-iga" that cannot be neutralized with purified iga ( ); or when blood products containing proven anti-iga do not elicit severe aes ( ) . among patients on replacement therapy, those with cvid may rarely develop severe immediate aes ( ) . the discrepancy between anti-iga positive patients and frequency of aes raises the question about the nature of the many reported anti-iga antibodies and also raises the question about the immunologic condition which allows the formation of anaphylactoid anti-iga antibodies. there might be some logic in supposing that anaphylactoid anti-iga cannot evolve at iga levels otherwise fulfilling the definition of igad. such a condition would constantly generate ics which in turn could activate complement, react with immune cells, and be deposited in lung and kidney. indeed, horn et al. found anti-iga frontiers in immunology | primary immunodeficiencies antibodies in cvid patients missing iga + b cells and presenting with iga levels < . g/l, a level which is more than -to fold lower than the threshold for igad ( ) . however, a possibility for an iga-mediated anaphylactoid reaction at measurable iga serum levels might exist. serum iga contains approximately % subclass of iga (iga ) and only % subclass of iga (iga ). selective deficiency of iga and -although evidence is lackingthe presence of a highly specific anti-iga antibody theoretically could elicit a severe ae. the kinetics of anti-iga after infusion of blood products have been studied in a few cases. in these patients, a fall in anti-iga titers has been noticed followed by an increase during subsequent weeks or months. this suggests that at appropriate proportions, iga of the infused material and anti-iga present in the patients' serum combine with each other to form ics. in turn, ics activate complement that are bound and eliminated by macrophages most likely leading to cytokine release. the increase in anti-iga titers over time indicates that the infused iga-containing product has a booster effect ( , , ) . such boosting effect together with the presence of anti-iga before the application of an igg concentrate can be taken as the ultimate confirmation of a supposed iga/anti-iga reaction. figure depicts a well-documented case of iga/anti-iga reaction in a patient who progressed from sigad to cvid. the events during the first h at occasion of the first infusion of ivig were as follows (shadowed area in figure ): min after the start of the infusion, having received eight drops of an igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped. after approximately h, the reaction has weaned, and h later the patient felt well again, and the infusion of total g igg could be continued without further complications. although the patient fairly assured having never received any blood or plasma product in the past, the follow-up of her anti-iga titers from before infusion to year later confirmed a true anaphylactoid reaction mediated by anti-iga, as the anti-iga became undetectable immediately after the infusion and showed a boosting phenomenon during the following months. true anaphylactoid reaction was further confirmed by follow-up of total complement hemolytic activity (ch ) on the day of infusion. interestingly enough, the ch value reached its nadir at the end of the infusion when the patient had no complains. although a single case only, the events during the first infusion call for the following remarks: (i) severe aes most likely occur at concomitant complement and cell activation with cytokine release; (ii) infusion of minute to low amounts of ivig hours before the main infusion can "anergize" cells and stop release of pro-inflammatory www.frontiersin.org figure | an individual's slithering into the dark. a female patient has been suffering from recurrent airway infections since adolescence, occasionally complicated by pneumonia. at age , selective iga deficiency was diagnosed. ten years later, she was hospitalized with pneumonia. within these years, her serum igg had dropped from to . g/l (long-dashed line) and iga was undetectable. the diagnosis was corrected into cvid, and ivig replacement therapy was initiated (shadowed area). two minutes after the start of the infusion of a % igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped and later continued without further complications (see text). the confirmation of a true anaphylactoid reaction due to anti-iga in the serum of the patient was achieved by follow-up of anti-iga (solid line) and ch (short-dashed line). cytokines; (iii) "anergized" cells loose reactivity toward ongoing formation of ics and complement activation products. a non-complement-mediated anaphylactoid reaction was ascribed to the unforeseen release of elastase and other proinflammatory substances from neutrophils activated by the formation of in vivo iga/anti-iga complexes. complement activation or mast cell-dependent release of vasoactive substances was excluded as pathogenic mechanisms. although the iga/anti-iga complexes usually do not cause clinically relevant neutrophil degranulation within the circulation, the presence of a rare genotype encoding a novel gain-of-function igg receptor on neutrophils may provoke premature degranulation by these complexes. this phenomenon was only relevant in hypogammaglobulinemic patients in the presence of in vivo iga/anti-iga complexes ( ). the low prevalence of this genotype combined with an igad or cvid may add how to explain the rarity of serious anaphylactoid reactions in newly ivig-treated patients. authors share the opinion of janne björkander who at occasion of a discussion panel "dilemmas in diagnosis and management of antibody deficiencies: ask the experts" held at occasion of the th annual meeting of the american academy of allergy, asthma & immunology (aaaai), new york city, march - , came to the following conclusion: a clinician has to be aware of the risk, particularly at occasion of first infusions, but otherwise iga is not a major concern (from tape record). in the early days of ig-therapy, the nature of the "phlogistic" aes was obscure. however, it was already known that an ae can be prevented or its evolution halted when the patient receives a low dose of ivig first or the infusion is stopped early and is continued several hours later. hours later the infusion can be (re)started at high rates without further problems ( figure ) . one of the authors had a particular opportunity to get an insight into what a "phlogistic reaction" might be. at the occasion of a voluntary infusion of an investigational liquid ivig, he encountered a severe flu-like ae of more than h duration. before injection, the investigational liquid preparation had passed all release criteria for human use, including spontaneous complement activation assessed by aca and was free of prekallikrein activator (pka). in those days, assays for cytokines in biological samples just began to become available and were included into the parameters assessed in the study. infusion was stopped after h because of a drop of pulse rate and heavy discomfort provoking the laconic comment by the proband's technician who was taking samples:"you look green." the infusion was continued after another min when the heart rate had almost normalized. the infusion could be completed within an additional . h (a total of . g/kg b.w.) without further aggravation of malaise. the leukocyte count transiently had dropped to a nadir of % at h followed by a leukocytosis peak at h. complement activation, as assessed by generation of c a/c a[desarg] and the formation of the terminal complement complex c b- , apparently did not occur: the c a/c a[desarg] value reached a maximum of ng/ml (norm: < ng/ml) at h while the c b- value never moved outside the normal range. instead, a sequence of rapid transient massive increases of pro-inflammatory cytokines was observed: (i) tnfα started to increase min post initiation of infusion from a value of pg/ml to a peak value at h which was above the calibration range of the test kit of pg/ml; (ii) interleukin (il- ) increase started after h from pg/ml and peaked at . h with pg/ml post initiation of infusion; (iii) interleukin (il- ) secretion started after h with an undetectable level and peaked at h with pg/ml. all pro-inflammatory cytokines fell sharply while the second part of the infusion was still ongoing. the day after infusion, the pro-inflammatory cytokine profiles were back to normal and the flu-like syndrome was gone. in contrast, interleukin receptor antagonist (il- ra) values started increasing at . h ( pg/ml), peaked at h (> , pg/ml), and decreased slowly to reach a value of , pg/ml h after initiation of the infusion. soluble tnf receptor p level started at . ng/ml, reached a peak with . ng/ml at the same time as il- ra, and h after initiation of infusion was still at . ng/ml. thus, in this normogammaglobulinemic subject similar cytokine profiles and leukocyte number changes were observed as reported for hypogammagobulinemia under replacement therapy ( , ) . a series of further experiments with investigational and marketed ivigs was performed. all igg concentrates were analyzed for their molecular weight (mw) distribution. the most remarkable differences emerged in the mw range of dimers while the presence of minute amounts of higher oligomers could not be excluded with certainty. below, we will use the term "dimers" for that fraction of igg with higher mw. subsequent findings indicated that levels of "dimers" > % were responsible for complement-independent cell activation and cytokine release. the tnfα peaks assessed at . h post initiation of infusions correlated with "dimer" content of the ivigs and mirrored a clinical score of aes ( ) ( ) ( ) . frontiers in immunology | primary immunodeficiencies a few years before a complement-independent induction of a hypotensive factor by igg di-and oligomers was reported in animal experiments ( ) . a key role for macrophages in the generation of the hypotensive lipid factor was identified as plateletactivating factor, being induced by dimers and polymers ( , ) . several years later, the dimer-mediated aes in animal experiments were confirmed ( , ) . yet, at the same time, the dimer content of ivig apparently correlated with the clinical efficacy in a murine itp model ( , ) . variables such as "ir," "genetic background," "endogenous immunoglobulin levels," or "proportional fraction of polymers versus dimers" may impact on the balance between the phlogiston (being cytokines, active lipid substances, or a combination of factors) and the therapeutic efficacy (blocking igg receptors on liver/spleen macrophages to prevent clearance of "opsonized" material such as platelets in itp). as of today, reports on release of cytokines in humans in association with aes or tolerability toward dimers remain scarce and to the best of our knowledge studies in humans of causative factors/fraction in an igg concentrate has not been adequately addressed ( , , ( ) ( ) ( ) ( ) . in igg preparations, various forms of dimers might be present: formed through covalent binding ( ) by denaturation, hydrophobic interactions of the fc-parts, and by idiotype/anti-idiotype interactions, as part of the v-connected network of peripheral immune homeostasis ( ) . for a commercially viable fractionation process, pooling of donated plasma is mandatory in order to obtain a volume of starting material large enough to cover ever increasing costs for documentation, in-process, and batchrelease testing as it is required by cgmp. pooling also intends smoothing the batch-to-batch differences in antibody titers, a goal apparently difficult to achieve to levels as theory might imply ( ) . consequences of pooling are on the one hand the enrichment of public/common immune antibodies while diluting out individual specificities; on the other hand, the antibodies of the immune network of an individual donor are exposed to those of many other donors. the more individuals contribute to the pool, the more complex the possible "immune-network" interactions among iga, igg, and igm will become. the subsequent fractionation process has far-reaching effects on immunoglobulins from a given pool: only trace amounts of iga and igm are retained in the final product, i.e., igg is deprived of its counterparts of the v-connected immune network. the igg molecules of the homeostatic network "naked" at their v-regions can interact with each other at random combining site-interactions of single donor-derived monomeric igg ( ), otherwise not existing in vivo. this interaction is largely reversible. with increasing numbers of donors included into the pool, the immune network recognition among the "naked" igg molecules of the v-connected network becomes more and more complex, and the dimer and lower oligomer content in the resulting igg concentrate increases ( ) ( ) ( ) . in lyophilized igg concentrates, the dimer formation is "frozen" at a low level while in liquid preparations an equilibrium between monomers and dimers is achieved over time reaching a dimer content of % or more if not hampered by stabilizers. specificities, as far as they have been addressed, in the dimer fraction considerably differ from the monomeric fraction ( ) ( ) ( ) ( ) ( ) . in conclusion, the immunomodulatory efficacy of igg concentrates in part depends on the capacity and extent to form "dimer" fractions devoid of remarkable complement activation in vivo. the "art" of manufacturing a liquid igg concentrate is not to eliminate the monomeric igg having potential for "dimer" formation but to inhibit extensive"dimerization."in summary,aes might be associated with the induction of pro-inflammatory cytokines in absence of measurable complement activation in vivo where all regulatory mechanisms and removal processes of a body are at disposition. at reasonable irs in the open system of the human body, clinically relevant systemic complement activation apparently needs oligomers formed of three or more igg molecules. there are multiple reports of ig-induced hemolytic anemia (ha) in patients receiving high doses of ivig ( , - ) (table ; figure ; www.adrreports.eu). by spontaneous reporting, risk factors recognized for ig-induced hemolysis include beside high doses (more than g ivig over - days), female gender and histo-blood group type a, b, or ab of recipients. www.frontiersin.org a significant proportion of patients receiving ivig develop a positive direct antiglobulin test (dat) detectable after h for up to days after the ivig infusion ( , ) . however, it should be underlined that the dat positivity due to the factors mentioned above ( , ) is not sufficient per se to diagnose hemolysis and dat positivity does not necessarily imply the presence of active hemolysis. dat-positive mild hemolytic reactions can be easily missed and the true incidence of such reactions is difficult to document without careful clinical and laboratory follow-up. in the majority of reports on ha, intravascular red blood cell (rbc) destruction via complement activation or extravascular rbc sequestration and removal by the reticulo-endothelial system was proposed to result from igg alloantibodies with specificity for rbc antigens a, b, d, or c. hemolytic anemia induced by high-dose ivig has an average incidence of . % ( ) . low-dose igg replacement therapy is considered universally as safe, and only few cases of hemolysis following low-dose ivig or scig administration have been described ( , , ) . a baseline wbc and rbc count prior to ivig initiation and a close clinical and laboratory follow-up was suggested as a useful tool for early diagnosis and treatment. a possible work up might be to check hemoglobin (hb) level prior and - h after ig infusion. in case of a drop of hb, the presence of dat, an increase in unconjugated bilirubin, lactate dehydrogenase (ldh), and reduced haptoglobin level, followed by a rise in reticulocyte count should be assessed (figure ) . we systematically reviewed case reports related to ivig-induced hemolysis from to and identified articles containing reports of patients. baseline characteristics of the patients are shown in table . when available, blood group, dat, hb drop, and outcome are indicated. all reports showed positive dat, except for a case of yin et al. ( ) ; in this case, dat was performed days after ivig administration and the dat negativity might have been due to a rapid removal of sensitized rbcs. in the majority of patients, the outcome was positive: out of patients recovered with or without packed rbc transfusions; three patients died after ha, with the hemolytic episode representing a precipitating factor of a severe underlying condition. elution experiments were performed and the search for blood group antibodies revealed anti-a and anti-b specificity in the majority of cases; anti-d specificity was assessed in four reports, often associated with other specificities ( , , ) . a search for other specificities such as anti-band or anti-gal was not performed. only one report detected anti-c specificity in three patients; in one of them associated with anti-d irregular antibodies ( ) . although studies were restricted to blood group antibodies, this finding demonstrated that polyvalent igg preparations might contain clinically significant non-blood group antibodies, which are not part of the lot-release criteria in that their titration is not yet required by the european pharmacopeia. antibodies in ha, such as anti-c, may have unexpected hemolytic consequences ( ) ( ) ( ) ( ) ( ) . beside passive transfer of alloantibodies, igg administration also has been demonstrated to lead to unspecific enhanced erythrocyte sequestration, in particular, in patients with underlying inflammatory disorders ( , ) . in , the canadian ivig hemolysis pharmacovigilance group elaborated criteria to define an "ivig-induced hemolysis" ( ) . they included a reduction of hb levels ≥ g within days after ig administration, with frontiers in immunology | primary immunodeficiencies appearance of a positive dat and, at least, two of the following criteria: increase in the reticulocyte count, elevation of ldh and unconjugated bilirubin serum levels, low haptoglobin, hemoglobinuria, hemoglobinemia, presence of significant spherocytosis, in the absence of alternative causes of anemia. the passive transfer of igg alloantibodies through igg concentrates is difficult to explain as polyvalent igg is prepared from plasma of thousands of donors. since immunization to rbc alloantigens can occur because of past transfusions or pregnancy, the hypothetical numbers of alloimmunized plasma donors should be rather low. recently, other mechanisms underlying alloimmunization related to molecular mimicry have been demonstrated ( ) . the mechanism of high-dose ivig-induced ha is complex and it might vary from patient to patient. ivig cause hemolysis due to: (i) diseaseassociated pre-coating of rbcs; (ii) igg with hemolysis triggered by passive transfer of igg binding to blood group antigens; (iii) transfer of high levels of alloantibodies to rbc pre-coated at a low level only; or (iv) transfer of clinically tolerable levels of isoagglutinins plus transfer of additional rbc-reacting physiological autoantibodies. indeed, hemolytic reactions could not be related exclusively to transfer of alloantibodies. hence, antibodies other than histo-blood group alloantibodies (pre-)coated to rbcs might contribute to hemolysis in igg recipients need to be identified. in addition, hemolytic episodes may possibly be precipitated by some sort of complexed/denatured igg that co-purify with other igg in the product ( , , , ) . recently, a two hit mechanism for ivig-induced hemolysis has been proposed: the passive transfer of alloantibodies through ivig representing the first hit and the underlying inflammatory state representing the second hit ( ) . nowadays all commercial ig products have to undergo anti-a and anti-b testing and regulatory requirement ask for respective igg antibody titers of ≤ : at % solution strength (w/v) ( , ) . nevertheless, hemolysis might occur even in recipients of igg products that meet these specifications ( ) . consequently, it has been suggested that igg recipients should be monitored for clinical signs and symptoms of hemolysis ( ) . with the detection of the immunomodulatory potential of igg concentrates, their clinical use has continuously increased ( ) . to cover the need, at a first glance, an increase of the volume of plasma fractionated seems to be the most convenient option. however, this might economically not be viable because fractionation of plasma products is interconnected ( ) and before increasing output of one product (e.g., ivig), the market absorbance of the other products as well (e.g., albumin) must be ascertained. on a longer-run, a more viable option is to improve recovery. considering recovery, the cold-ethanol fractionation apparently has reached its limits. as of today, four manufactures have invested into a"modern"fractioning technique on the basis of ion-exchange chromatography. ion-exchange chromatography allows elevated recovery at high purity. as of today, five ivigs, one scig, and one anti-d concentrate are fractionated by ion-exchange chromatography. pharmacovigilance has shown that all chromatographically fractionated ivig and scig, more or less prominently, show a tendency for elevated frequencies of hemolytic aes. anti-a and anti-b alloantibody titers are now lot-release criteria (see above) as they constitute the major risk parameter for hemolytic reactions mediated by igg concentrates. to overcome the threat of end up on the dark side of the moon, two manufacturers have taken measures to reduce anti-a and anti-b titers in their igg products. one measure chosen was adsorption of the alloantibodies by affinity chromatography ( ) . reported reduction in both alloantibodies was significant and levels were similar to those in cold-ethanol fractionated immunoglobulins ( ) . the other measure chosen was reduction in anti-a using an automated indirect agglutination test for donor screening and exclusion of high-titer donations (approximately . %) from plasma pooling and fractionation ( ) . this measure reduced anti-a in the igg concentrate by one titer step. to ensure staying on the safe and sunny side, the manufacturer has announced the introduction of an alloanti-a and alloanti-b immune-affinity chromatography step into the manufacturing process ( ) . preliminary results indicate depletion in anti-a and anti-b by > % in investigational lots. subsequently, we want to discuss possible consequences of (extensive) removal of antibodies reacting with histo-blood group antigens a and b. three facts have initiated our thinking about possible consequences of removal of histo-blood group a and b reacting www.frontiersin.org antibodies from igg concentrates. (i) in collaboration with hans u. lutz, formerly biochemistry eth zurich, we have observed the non-intended removal of natural anti-c autoantibodies regulating complement activation by large-scale immune-affinity adsorption of iga from an igg concentrate ( ) . anti-c antibodies belong to the family of "nabs" and have a particular role in homeostasis: they control activation of complement, among others, in the frame of nab-mediated opsono-phagocytosis of altered or senescent cells, including rbcs ( ) ( ) ( ) . thus, the intention to target one particular antibody by affinity chromatography might reduce that antibody specificity but at the same time affect other specificities as well. (ii) it should be kept in mind that the blood groups a and b are in fact "histo-blood group" antigens, i.e., they are also found on white blood cells, t lymphocytes, and proteins and also can be found in soluble form ( ) . alloantibodies reacting with histo-blood group antigens a and b thus have much broader tissue recognition than rbcs only. in addition, alloanti-a and alloanti-b belong to the population of nabs recognizing non-self and most likely participate in primary host defense ( ) . (iii) in contrast to cold-ethanol fractionation, where low titers of alloanti-a and alloanti-b are achieved on basis of their isoelectric points (ieps), the (extensive) immune-affinity removal might affect a much wider iep range, thereby removing broadly reacting antibodies and impairing some desirable functions of the igg concentrate. thus, the struggle for staying on the sunny side of the moon might have consequences for the antibody repertoire in an igg concentrate. antibodies reacting with terminal di-, tri-, and tetrasaccharides belong to the large family of human anti-glycan nabs. histo-blood group a and b epitopes in terminal position are tetrasaccharides. alloantibodies to these tetra-saccharides are found in the plasma of healthy individuals depending on the blood group they have. a considerable body of research into the nature of these nabs has been performed so far, all using for isolation the corresponding terminal di-or tri-saccharides ( ) ( ) ( ) . recently, the repertoire and epitope specificity of such immunoglobulins was addressed in depth by including the tetra-saccharide as well ( , ) . it proved that serum of healthy individuals contain respectable amounts of di-or tri-saccharide-reacting nabs. these nabs proved to be pseudo-anti-a and pseudo-anti-b nabs as they are not reacting with the tetra-saccharide of histo-blood groups a and b. in contrast, alloanti-a and -b antibodies able to react with tetra-saccharides are reacting with the corresponding terminal di-and tri-saccharides. reasoning about the biological role of these "high-titer and population conservative" anti-di-and anti-tri-saccharide nabs and the consequence of their potential removal by immunoaffinity is outlined below. a population of the anti-glycan nabs are the anti-αgal nabs which recognize galα - gal and galα - (fucα - )gal epitopes. anti-αgal nabs have been described being xenoreactive, recognizing bacterial galα - gal ( ) and having tissue homeostatic function. the daily removal of altered/senescent cells of the body is~ . removal is mainly mediated by apoptosis (no inflammation, no necrosis). rbcs, when they do not encounter a pathological condition, over their life span of - days remain intact although they shrink, do not undergo apoptosis but progressively become senescent, mainly due to cumulative oxidative stress. removal of intact rbcs with a daily turnover of~ × , corresponding to~ g cell mass, is effectuated by increased exposure of otherwise cryptic structures such as spectrin, band , or αgal epitopes. these exposed structures are recognized by low affinity, high avidity, c -bearing nabs, which promote the efficient removal of intact senescent rbcs ( , , ) . immunoaffinity adsorption by tri-saccharides columns of di-and tri-saccharide reacting nabs from igg concentrates can eliminate anti-histoblood a and b alloantibodies while it also eliminates αgal and this might have a janus effect. the face directed to the sun tells that adsorbing αgal nabs reacting with altered and senescent self on rbc might prevent an increase in the igg load of rbcs over the threshold level of relevant hemolysis in individuals at risk. the face directed to the dark indicate that adsorption of tissue homeostatic antibodies might deprive an igg concentrate of potentially beneficial antibodies. although they are nabs, tri-saccharide reacting antibodies can be induced by feeding bacteria bearing the corresponding carbohydrate epitopes ( ) . these inducible nabs are considered to participate in primary host defense. other antibodies possibly involved in primary host defense are the anti-αgal nabs. they show a broad specificity and can react with a number of related αgal-terminated oligosaccharides, including those on bacteria ( ) . thus, the immunoadsorption of di-and trisaccharide reacting nabs might diminish the potential of an igg concentrate to mediate primary host defense. therefore, when choosing affinity resins for immunoadsorption, there might be some aspects worth to consider. in summary, the principles of avoiding co-fractionation through cold-ethanol fractionation ( ) versus immune-affinity removal of histo-blood group alloantibodies can have an impact on the presence of homeostatic and first-line defense antibodies. according to present knowledge, only resins coated with the corresponding tetra-saccharides can ascertain the selective removal of histo-blood group alloantibodies presumably involved in ha. resins coated with the corresponding di-and tri-saccharides also remove blood group alloantibodies, however not selectively. such resins in addition might remove a broad range of nabs present in igg concentrates at relative high amounts. in the literature, the use of tri-saccharide-coated resin was reported ( ) ( ) ( ) . we have found no information available in the public domain indicating which type of resin is/will be used for reduction of the histo-blood group alloantibodies in large-scale fractionation of igg. furthermore, we suggest that the effect of reduction of anti-a and ant-b reacting antibodies by immune-affinity on the antibody repertoire of igg concentrates can only be assessed by, e.g., using pathogens/commensals, which share the saccharide epitopes, that have been used to coat the affinity resins or alternatively by exposing senescent rbcs stripped off the iggs coated in vivo. finally, techniques are required, which allow detection of low affinity, high avidity nabs. ivig administration-related aes, including thrombosis, have been extensively described ( ) . thrombotic aes are severe aes and patients with risk factors require a special care. reported average incidence of ivig-induced thrombosis ranges from to % ( ) . recognized risk factors for ivig-induced thrombosis frontiers in immunology | primary immunodeficiencies include male gender; age > ; diabetes; renal insufficiency, dyslipidemia; hypertension; immobility; coronary disease; pre-existing vascular disease, family history of early thromboembolic disease; atrial fibrillation, high-dose and high-speed ivig infusions. ivig-induced thrombosis is reported both as venous events such as thrombosis stroke, pulmonary embolism (pe), deep venous thrombosis (dvt), and arterial ischemia events such as myocardial infarct and stroke. the mechanisms leading to ivigassociated thrombosis are still not completely clear; three main mechanisms have been proposed, emphasizing the role of an increased blood viscosity causing a hypercoagulable state ( ) , the role of anticardiolipin antibodies passively transferred through ivig ( ) , and the role of factor xia or other biologically highly active factors passively transferred via igg concentrates, such as pka. avoiding activated coagulation factors in igg concentrates starts with appropriate anticoagulation of donated blood/plasma, i.e., careful mixing of anticoagulant and sample over the whole donation process. alterations in an established manufacturing process neglecting appropriate controls can also lead to increased risk of transmission of activated coagulation factors. high mw proteins passively transferred by ivig are probably contributing to this phenomenon ( ) . in patients with other risk factors, such as vascular disease, the increase in blood viscosity can precipitate thromboembolic events. as elderly individuals are prone for such aes, we like to point to the possibility of elevated altered/senescent self-reacting with infused homeostatic nabs being a possible factor facilitating thrombotic events as well. a relationship between ivig administration and cerebral vasospasm has also been suggested by sztajzel et al. ( ) ; blood viscosity is a determinant for oxygen delivery to the tissues, and changes in viscosity can lead to a reduction in cerebral or myocardial perfusion. we systematically reviewed case reports related to iviginduced thrombosis from to (figure ) . literature search identified articles containing reports concerning patients ( , , , . when data were available, diagnosis, risk factors, the number of ivig infusion prior to thrombosis event, and outcome have been indicated. baseline characteristics of the patients are shown in table . high-dose ivig induced thromboembolic events in patients at low to medium ivig doses. marie et al. ( ) observed that the frequency and type of arterial events was inversely related to the time elapsed from ivig infusion; almost % ( versus reports) of arterial ischemic events occurred within h following ivig, while about % of venous thrombosis occurred after more than h. no correlation between number of infusions and occurrence of ae was observed. the main risk factors observed in this review were hypertension ( cases, % of prevalence), previous vascular disease ( %), and dyslipidemia ( %). average mortality for thrombotic events was % (arterial ischemia % versus venous thrombosis %, pe representing the main venous fatal event). predicting iviginduced thrombosis is difficult. risk factors should be assessed for each patient including instrumental exams when needed. doppler ultrasound can be useful as early diagnostic tool for thrombosis or to detect the presence of abnormal blood flow especially after prolonged immobility. ivig should be administered at low ir to reduce the risk. the administration of antiplatelet or anticoagulant prophylaxis was suggested in patients with several risk factors ( ) . however, thrombotic events have been reported even after several previous uncomplicated courses of treatment. www.frontiersin.org ( ), arrhythmia ( ) first ( ), several ( ) arterial ( ), venous ( ) days recovery ( ), death ( ) al-riyami et al. ( ) second ( ), third ( ), several ( ) arterial ( ), venous ( ) h ( ), days ( ), weeks ( ) recovery ( ) stamboulis et al. ( ) several ( ) arterial ( ) h ( ), days ( ) death ( ) clinical and immunological characteristics of patients described in case reports. numbers in parenthesis indicate the number of patients with the given condition. in such cases, patients should be examined for signs and symptoms of thrombosis during each courses of ivig. immunoglobulin g concentrates are widely acknowledged to offer a safe, high-dose, long-term therapy option for a variety of diseases. aes occur rarely and mainly are mild to moderate. deviations from this rule of thumb are addressed by authorities and the plasma fractionation industry to achieve corrections. above, we have reviewed two types of ae which have shown elevated frequency in the near past. we tried to give some insights which might help in reducing frequencies of aes bed side. authors are deeply grateful to hans-hartmut peter, freiburg, germany, for his careful reading of the manuscript, the valuable comments, and the correction of english. epidemic hepatitis b caused by commercial human immunoglobulin intravenous administration of human γ-globulin adverse reactions following administration of human gamma-globulin igg antibodies to iga in two patients with hypogammaglobulinemia treated with commercial gammaglobulin contact-activated factors: contaminants of immunoglobulin preparations with coagulant and vasoactive properties fatal thrombotic events during treatment of autoimmune thrombocytopenia with intravenous immunoglobulin in elderly patients effect of high-dose intravenous immunoglobulin therapy on blood rheology pathogen safety of immunoglobulin preparations population screening for variant creutzfeldt-jakob disease using a novel blood test: diagnostic accuracy and feasibility study intravenous immunoglobulin: exploiting the potential of natural antibodies inherent specificities in natural antibodies: a key to immune defense against pathogen invasion the natural human igg anti-f(ab') antibody recognizes a conformational igg hinge epitope stimulation of complement amplification by f(ab') -containing immune complexes and naturally occurring anti-hinge antibodies -possible role in systemic inflammation natural autoantibodies to fcγ receptors in intravenous immunoglobulins multi-faceted role of naturally occurring autoantibodies in fighting pathogens immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura high-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood concurrent presence of agonistic and antagonistic anti-cd autoantibodies in intravenous ig preparations a differential concentration-dependent effect of ivig on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms verträglichkeit und virussicherheit von intravenösen immunglobulinen occurrence of hemolytic reactions (hrs) on the same day as immune globulin (ig) product administrations during cerebral venous and sinus thrombosis associated with subcutaneous immunoglobulin injection and oral contraceptive use multiple immunoglobulin intolerance without antibody's anti-immunoglobulin a: a case report common variable immunodeficiency: a patient with anaphylaxis to intravenous and subcutaneous immunoglobulin prospective study on cvid patients with adverse reactions to intravenous or subcutaneous igg administration severe adverse reaction to subcutaneous immunoglobulin therapy in a patient with common variable immunodeficiency evaluation of the relationship between injection site reaction rate and scig doses in patients with primary immunodeficiencies immunthemotherapy: a guide to immunoglobulin prophylaxis and therapy prophylactic infusions with an unmodified intravenous immunoglobullin product causing few side-effects in patients with antibody deficiency syndromes the role of anti-iga antibodies in causing adverse reactions to gamma globulin infusion in immunodeficient patients: a comprehensive review of the literature prevalence of immunoglobulin a deficiency (igad) in shanghai blood donors and efforts to establish a rare blood bank of igad in shanghai hammarström l, persson maa, smith cie. anti-iga in selective iga deficiency -in vitro effects and ig subclass pattern of human anti-iga immunoglobulin prophylaxis in patients with antibody deficiency syndromes and anti-iga antibodies follow-up of anti-iga antibodies in primary immunodeficient patients treated with gamma-globulin anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia: detection of ige antibodies to iga management of immunoglobulin a deficiency: lessons from haemovigilance screening of canadian blood services donors for severe immunoglobulin a deficiency large scale detection of iga deficient blood donors unexpected reactions of the anti-iga antibody particle gel immunoassay allergic transfusion reactions from blood components donated by iga-deficient donors with and without anti-iga: a comparative retrospective study anti-iga antibodies in common variable immunodeficiency (cvid): diagnostic workup and therapeutic strategy induction of unresponsiveness against iga in iga-deficient patients on subcutaneous immunoglobulin infusion therapy release of cytokines, soluble cytokine receptors, and interleukin- receptor antagonist after intravenous immunoglobulin administration in vivo neutropenia as a complication of intravenous immunoglobulin (ivig) therapy in children with immune thrombocytopenic purpura: common and non-alarming igg dimers in liquid intravenous immunoglobulin preparations laboratory parameters measured during infusion of immunoglobulin preparations for intravenous use and related tolerability welltolerated liquid intravenous immunoglobulin g preparations (ivgg) have a low immunoglobulin g dimer (igg-dimer) content an animal model for the detection of hypotensive side effects of immunoglobulin preparations key role of macrophages in hypotensive side effects of immunoglobulin preparations. studies in an animal model vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages -studies in a rat model hypotension with intravenous immunoglobulin therapy: importance of ph and dimer formation therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin g dimers: studies in experimental immune thrombocytopenia tumor necrosis factor and intravenous gammaglobulins in common variable immunodeficiency ivig adverse reactions: potential role of cytokines and vasoactive substances acute hemolysis after intravenous immunoglobulin amid host factors of abo-mismatched bone marrow transplantation, inflammation, and activated mononuclear phagocytes intravenous immunoglobulin induces interferon-γ and interleukin- in vivo human igg can form covalent dimers variable regionconnected, dimeric fraction of intravenous immunoglobulin enriched in natural autoantibodies ivig -mechanisms of action igg dimers in multidonor-derived immunoglobulins: aspects of generation and function immunoglobulin g dimer: an idiotype-anti-idiotype complex a view of the human idiotypic repertoire -electron microscopic and immunologic analyses of spontaneous idiotype-anti-idiotype dimers in pooled human igg polyreactive antibodies in multidonorderived immunoglobulin g: theory and conclusions drawn from experiments comparative analysis of antigen specificities in the monomeric and dimeric fractions of intravenous immunoglobulin monomerization of dimeric igg of intravenous immunoglobulin (ivig) increases the antibody reactivity against intracellular antigens antibodies in the dimer fraction of ivig have the capacity to bind beta amyloid an analysis of anti-fas and anti-siglec- autoantibodies in monomeric and dimeric fractions of ivig dimeric ivig contains natural anti-siglec- autoantibodies and their antiidiotypes intravenous immunoglobulininduced hemolytic anemia in a patient with juvenile dermatomyositis intravenous immunoglobulin-induced haemolysis: a case report and review of the literature massive intravascular haemolysis after high dose intravenous immunoglobulin therapy maternal hemolysis after intravenous immunoglobulin treatment in fetal and neonatal alloimmune thrombocytopenia hemolytic anemia following intravenous immunoglobulin therapy in patients treated for kawasaki disease: a report of cases ivig -a hemolytic culprit haemolysis after treatment with intravenous immunoglobulin due to anti-a hemoglobinuria and acute kidney injury requiring hemodialysis following intravenous immunoglobulin infusion a pediatric case series of acute hemolysis after administration of intravenous immunoglobulin acute hemolysis after high-dose intravenous immunoglobulin therapy in highly hla sensitized patients hemolytic transfusion reactions after administration of intravenous immune (gamma) globulin: a case series analysis hemolytic anemia following intravenous immunoglobulin administration acute hemolysis in a patient with cytomegalovirus pneumonitis treated with intravenous immunoglobulin (ivig) a prospective study of the immediate and delayed adverse events following intravenous immunoglobulin infusions adverse effect of polyvalent immunoglobulin in the treatment of guillain-barré syndrome hemolysis after administration of high-dose immunoglobulin in a patient with myocarditis haemolytic anaemia associated with high dose intravenous immunoglobulin therapy in a child with guillain-barré syndrome insisting on intravenous polyvalent immunoglobulin therapy in polymyositis in spite of the occurrence of sever hemolytic anemia -poursuite du traitement d'une polymyosite par les immunoglobulines intraveineuses polyvalentes malgré la survenue d'une anémie hémolytique sévère severe hemolytic anemia following high-dose intravenous immunoglobulin administration in a patient with kawasaki disease hemolytic anemia associated with intravenous immunoglobulin hemolysis during immunoglobulin therapy -hemolisis durante el tratamiento con inmunoglobulinas hemolytic anemia following highdose intravenous immunoglobulin administration acute hemolysis due to passively transfused high-titer anti-b causing spontaneous in vitro agglutination hemolysis after high-dose intravenous ig immune hemolysis, disseminated intravascular coagulation, and serum sickness after large doses of ivig for kawasaki disease acute intravascular haemolysis associated with high dose immunoglobulin after bone marrow transplantation for acute myelogenous leukemia hemolysis after intravenous immune globulin therapy: relation to igg subclasses of red cell antibody autoimmune hemolytic anemia in kawasaki disease: a case report haemolysis induced by intravenously-administered immunoglobulin massive intravascular hemolysis associated with intravenous immunoglobulin in bone marrow transplant recipients hemolytic anemia following intravenous gamma globulin administration hemolysis following intravenous immune globulin therapy transient haemoglobin drop following high dose intravenous immunoglobulin in vivo administration of intravenous immunoglobulin (ivig) can lead to enhanced erythrocyte sequestration hemolysis in patients with antibody deficiencies on immunoglobulin replacement treatment batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-rh d activity hematologic toxicities associated with intravenous immunoglobulin therapy anti-a and anti-b activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method international collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-a and anti-b in normal intravenous immunoglobulin products european directorate for the quality of medicines and healthcare. anti-a and anti-b haemagglutinins european directorate for the quality of medicines and healthcare. test for anti-d antibodies in human immunoglobulin immune complex-like moieties in immunoglobulin for intravenous use (i.v.ig) bind complement and enhance phagocytosis of human erythrocytes regulation of primary alloantibody response through antecedent exposure to a microbial tcell epitope a follow-up study of adult patients with idiopathic thrombocytopenic purpura treated with high-dose immunoglobulins and anti-d immunoglobulins hemolysis upon intravenous immunoglobulin transfusion report of the fda meeting on strategies to address hemolytic complications of immune globulin infusions kreuth ig working group. european consensus proposal for immunoglobulin therapies the art of balanced production in vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations patient safety through an ivig mastered manufacturing process. posters isoagglutinin reduction in human immunoglobulin products by donor screening igg product development isoagglutinin reduction measures. oral presentation naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates. lutz, h. u. naturally occurring antibodies (nabs) naturally occurring anti-band- antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes high doses of immunoglobulin g attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of c b in c bn-igg complexes intravenously applied igg stimulates complement attenuation in a complementdependent autoimmune disease at the amplifying c convertase level histoblood group antigens as allo-and autoantigens blood group isoantibody stimulation in man by feeding blood group-active bacteria normal human serum contains natural antibodies reactive with autologous ab blood group antigens a unique natural human igg antibody with anti-alpha-galactosyl specificity normal human serum contains high levels of anti-galα - glcnac antibodies natural anti-a and anti-b of the ab system: allo-and autoantibodies have different epitope specificity repertoire of human natural anti-glycan immunoglobulins. do we have autoantibodies? interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora innate immune and non-immune mediators of erythrocyte clearance comparison of serum anti-band and anti-gal antibody binding to density-separated human red blood cells specificity of human antibodies against galα - gal carbohydrate epitope and distinction from natural antibodies reacting with galα - gal or galα - gal anti-a and anti-b haemagglutinin trend analysis during manufacturing process of ivig. presentation at workshop "strategies to address hemolytic complications of immune globulin infusions in vitro evaluation of the efficacy and biocompatibility of new, synthetic ab immunoabsorbents high molecular weight blood group a trisaccharide-polyacrylamide glycoconjugates as synthetic blood group a antigens for anti-a antibody removal devices specific antibody filter (saf) binding capacity enhancement to remove anti-a antibodies intravenous immunoglobulin: adverse effects and safe administration thromboembolic complications of intravenous immunoglobulin therapy in patients with neuropathy: a two-year study high-dose intravenous immunoglobulin and serum viscosity: risk of precipitating thromboembolic events antired blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations soluble hla class i and class ii concentrations in commercial immunoglobulin preparations high-dose intravenous immunoglobulin treatment and cerebral vasospasm: a possible mechanism of ischemic encephalopathy? high dose intravenous immunoglobulin may be complicated by myocardial infarction fatal case of bilateral internal jugular vein thrombosis following ivig infusion in an adolescent girl treated for itp cerebral sinus thrombosis following iv immunoglobulin therapy of immune thrombocytopenia purpura graft rupture after high-dose intravenous immunoglobulin therapy in a renal transplant patient cerebral sinus thrombosis in a patient with humoral immunodeficiency on intravenous immunoglobulin therapy: a case report a case of deep vein thrombosis and pulmonary thromboembolism after intravenous immunoglobulin therapy acute stroke with high-dose intravenous immune globulin venous and arterial thrombosis following administration of intravenous immunoglobulins intravenous immunoglobulin-associated vena cava thrombosis intravenous immunoglobulin-associated arterial and venous thrombosis; report of a series and review of the literature thromboembolic events as an emerging adverse effect during high-dose intravenous immunoglobulin therapy in elderly patients: a case report and discussion of the relevant literature diffuse venous thromboemboli associated with ivig therapy in the treatment of streptococcal toxic shock syndrome: case report and review deep vein thrombosis after intravenous immunoglobulins associated with methylprednisolone deep venous thrombosis after high-dose intravenous immunoglobulin in the treatment of pemphigus vulgaris thromboembolic complications of intravenous immunoglobulin treatment acute myocardial infarction following intravenous immunoglobulin therapy for chronic inflammatory demyelinating polyneuropathy in association with a monoclonal immunoglobulin g paraprotein stroke and deep venous thrombosis complicating intravenous immunoglobulin infusions thrombosis complicating high dose intravenous immunoglobulin: report of three cases and review of the literature acute thromboembolic events associated with intravenous immunoglobulin infusion in antibody-deficient patients transverse sinus thrombosis and ivig treatment: a case report and discussion of risk-benefit assessment for immunoglobulin treatment thrombotic complications after intravenous immunoglobulin therapy in two patients pulmonary embolism after intravenous immunoglobulin adverse effects of intravenous immunoglobulin therapy in patients with autoimmune diseases acute myocardial infarction associated with high dose intravenous immunoglobulin infusion for autoimmune disorders. a study of four cases deep venous thrombosis of the arm after intravenous immunoglobulin infusion: case report and literature review of intravenous immunoglobulin-related thrombotic complications cerebral infarction complicating intravenous immunoglobulin therapy in a patient with miller fisher syndrome central retinal vein occlusion complicating treatment with intravenous immunoglobulin acute myocardial infarction during treatment with intravenous immunoglobulin for idiopathic thrombocytopenic purpura (itp) myocardial infarction as a complication of immunoglobulin therapy iatrogenic central retinal vein occlusion and hyperviscosity associated with high-dose intravenous immunoglobulin administration the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -e zjxjox authors: lee, cheryl yi-pin; lin, raymond t. p.; renia, laurent; ng, lisa f. p. title: serological approaches for covid- : epidemiologic perspective on surveillance and control date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e zjxjox since december , the novel coronavirus, sars-cov- , has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. early detection of sars-cov- is one of the crucial interventions to control virus spread and dissemination. molecular assays have been the gold standard to directly detect for the presence of viral genetic material in infected individuals. however, insufficient viral rna at the point of detection may lead to false negative results. as such, it is important to also employ immune-based assays to determine one's exposure to sars-cov- , as well as to assist in the surveillance of individuals with prior exposure to sars-cov- . within a span of months, extensive studies have been done to develop serological systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during sars-cov- infection. the vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. this review aims to provide a concise yet extensive collation of current immunoassays for sars-cov- , while discussing the strengths, limitations and applications of antibody detection in sars-cov- research and control. the ongoing pandemic, which originates from a newly emerged coronavirus, sars-cov- , was discovered in the city of wuhan in china's hubei province in december ( ) . to date, due to rapid transmission globally, there are more than two million laboratory-confirmed human infection cases, with a few hundred thousand deaths across countries and territories (https://www.who.int/emergencies/diseases/novel-coronavirus- / situation-reports/). this unprecedented crisis led to a worldwide effort to rapidly characterize the immunobiology of sars-cov- , while mitigating further spread of this deadly pathogen. sars-cov- is a single stranded, positive sense rna virus that belongs to the coronaviridae family of the betacoronavirus genus ( ) . it has a genome size of ∼ kilobases that encodes for multiple structural proteins comprising the spike (s), the envelope (e), the membrane (m), and the nucleocapsid (n), as well as non-structural proteins ( ) (figure ) . infection by sars-cov- causes an acute respiratory disease termed the coronavirus disease . the clinical manifestations of covid- form a spectrum, from being asymptomatic to fever with mild respiratory illness, to acute respiratory distress syndrome, and death from respiratory failure or associated complications ( ) ( ) ( ) . as the reported incubation period varies among different patient cohorts, it is often difficult to ascertain the actual day of onset, and infected subjects who are asymptomatic or pre-symptomatic may go undetected ( ) ( ) ( ) . early detection of sars-cov- infection is one of the crucial interventions to control virus transmission. with the discovery of the genome organization of sars-cov- viral rna, which is adapted from genbank accession number: mn , is characterized by sequence alignment against two representative members of the betacoronavirus genus. the entire genome sequence is ∼ kilobases (kb) long. the virus, numerous diagnostic assays using quantitative reverse transcriptase pcr (qrt-pcr) were developed ( ) . qrt-pcr is the reference standard for diagnosing infections with high sensitivity and accuracy in the acute phase of illness. sars-cov- viral rna has been detected in both throat and nasal swabs of infected individuals by qrt-pcr, which becomes almost undetectable by days post-illness onset (pio) (or symptom onset) ( , ) (figure ) . apart from being costly and time consuming to perform, false negative results may arise due to improper handling of nucleic acid samples, inadequate and variable sampling resulting in insufficient viral genetic material at the point of detection (after days pio), or biological variation on when viral rna is detectable by qrt-pcr ( , ) . with the limitations of qrt-pcr, immunoassays may offer another figure | schematic illustration on the window period of detection for either viral rna or antibodies in sars-cov- -infected individuals. presence of sars-cov- viral rna (boxed in pink) in throat or nasal swab of patients are typically undetectable by day post illness onset (pio) ( , ) . sars-cov- -specific antibodies (boxed in blue): igm is detectable as early as days pio, and peaks between and weeks pio ( , ) . igm response was still detectable after more than month pio ( ) . both iga and igg are present as early as days pio, and peaks after weeks pio in serum samples ( , , , ) . there are currently no reports on the presence of these sars-cov- -specific antibodies in the later phase pio, as indicated by dotted lines. this depicts the importance of serological studies to identify individuals with current or prior exposure to sars-cov- that went undetected, by testing for either igm, igg, or iga antibodies against sars-cov- . illustration was created using biorender. avenue to reduce undiagnosed cases, with the advantage that rapid test formats may deliver results in a relatively shorter time and lower cost ( ). immunoassays are another diagnostic approach that can provide information on both active viral infections and past exposures (figure ). to date, many commercial companies and research institutes have developed serological assays to detect sars-cov- antibodies from patient serum or plasma samples ( , ) . closely related to another pathogen, severe acute respiratory syndrome coronavirus (sars-cov), these assays mainly target immunogenic coronavirus proteins: s protein, which is the most exposed viral protein, and n protein, which is abundantly expressed during infection ( , , ) . in addition, the receptor-binding domain (rbd), which is located along the s protein, is also a target of interest to detect the presence of sars-cov- -specific antibodies ( , ) . in recent pre-prints deposited in medxriv and bioxriv, it was shown that both anti-sars-cov- -igm and igg levels increase gradually along with infection phases, with igm being detected as early as days pio, which peaks between two to three weeks pio ( , ) . one study has reported that sars-cov- -specific igm is still present in the serum after month pio ( ) . sars-cov- -specific igg antibodies, on the other hand, can be present as early as days pio, and peak after days pio ( , ) (figure ) . these observations are similar to what was previously reported during a sars-cov infection ( ) . however, interestingly, one study demonstrated that longitudinal profiling of both antibodies in a population of covid- patients showed no specific chronological order in terms of igm and igg seroconversion ( ) , which was also observed in patients infected with sars-cov and another human coronavirus, middle east respiratory syndrome coronavirus (mers-cov) ( , ) . in addition, there seems to be no correlation between seroconversion rates with age, gender or time of hospitalization ( ) . these findings on sars-cov- -specific antibodies seroconversion against the s viral protein suggest the importance to test for both igm and igg antibodies to confirm a positive infection. expectedly, similar to what was reported for sars-cov and mers-cov, both igm and igg levels seems to be correlated with disease severity, with a higher level of both antibodies present in patients with more severe sars-cov- infection ( , , , ( ) ( ) ( ) . in contrast to other flu-like infections such as influenza, instead of igg , igg appears to be the dominant igg subtype during sars-cov- infection ( , , ) . as a majority of the human population has prior exposure to endemic human coronavirus infections including alphacoronaviruses ( e and nl ), and other betacoronaviruses (oc and hku ) ( ) , it is crucial to validate the specificity and sensitivity of current immunoassays against sars-cov- to avoid false positive outcomes. within the s protein antigen, cross-reactivity was observed when samples were tested against sars-cov s and s subunit proteins, and to a smaller extent, with mers-cov s protein ( table ) . interestingly, there was no cross-reactivity with the s subunit of mers-cov ( ) . the high level of crossreactivity between sars-cov and sars-cov- can be attributed to the high degree of genetic homology ( , , ) . furthermore, detailed analysis revealed a highly conserved s subunit domain across coronaviruses, which may explain for the cross-reactivity observed with only the s protein of mers-cov, and not with the s subunit ( , ) . these data suggest that using an s subunit-based immunoassay may be more specific than the entire s antigen for diagnosing sars-cov- infections. another immunogenic target, the rbd, which lies along the s protein is usually the target of many neutralizing antibodies against sars-cov ( ) . a substantial level of cross-reactivity by sars-cov rbd-induced antibodies to sars-cov- rbd was described ( table ) ( ) . of clinical relevance, these antibodies were also able to cross-neutralize sars-cov- pseudovirus infection, signifying the potential of an immunotherapy-based treatment ( ) . while one non-peer reviewed study has shown that rbd-based serological assays are more sensitive than s subunit-based assays in identifying antibodies in mild covid- patients ( ), other non-peer-reviewed studies have described a lower degree of antibody response to the rbd as compared to full-length s protein, plausibly reflecting the larger number of epitopes present on the larger s antigen ( , ). due to a high level of similarity of % between sars-cov and sars-cov- n proteins, the n antigen of sars-cov was also used for serological detection of sars-cov- -specific antibodies ( table ) ( ) . these n-based assays were reported to be more sensitive than s subunit-based tests ( ) . the use of sars-cov antigens to diagnose sars-cov- infections may be reliable, given that sars-cov has not circulated in the human population since ( ). in addition, an earlier report has demonstrated waning of sars-cov-specific antibodies, therefore being undetectable in % of patient serum samples after years ( ) . since respiratory diseases are the hallmark of coronavirus infections, which activate mucosal immunity, several studies have exploited the detection of iga to diagnose sars-cov- infection in patients (table ) ( , ) . although a strong iga response was also detected in covid- patients where peak seroconversion was achieved by two weeks pio (figure ) , igabased immunoassay has been hypothesized to be less specific than igg-based elisa due to cross-reactivity with serum samples from patients infected by other coronaviruses ( ) . with the availability of immunoassays utilizing various coronavirus structural proteins, the use of more than one different antigen-based serological approach may be essential to establish a true positive sars-cov- infection. in addition, frontiers in immunology | www.frontiersin.org the use of saliva samples and other bodily fluid swabs as a less invasive alternative, which have been done for other viral infections including hiv and measles, should also be explored for serological testing of sars-cov- infections ( , ). apart from using immunoassays for the early detection of sars-cov- infected individuals, it is also critical to determine the regions where sars-cov- -specific antibodies bind to help guide vaccine designs. using sars-cov-derived b-cell epitopes that have been experimentally identified from positive b-cell assays ( ), out of linear b-cell epitopes have an identical match with sars-cov- protein sequences without any mutations ( ) . notably, majority of these matches were located at both the s and n viral antigens, with only from the m protein, and none in the e protein ( ). on the other hand, conformational b-cell epitopes identified from the same database were located on the s antigen. however, unlike the linear epitopes, none of these mapped identically to the sars-cov- protein ( ). further mapping the residues of linear b-cell epitopes onto available sars-cov s protein structure revealed several regions on the s subunit that may allow cross-neutralization of both sars-cov and sars-cov- ( , ) . in contrast, conformational b-cell epitopes mapped onto the s subunit, resulting in very few identical residues within sars-cov and sars-cov- ( ). these findings indicate that sars-cov-specific antibodies targeting these discontinuous regions may not be able to cross-react with sars-cov- ( , ) . as these regions are computationally predicted, serological studies using patient samples are necessary to validate the importance of these regions for serology and in controlling sars-cov- infection. it also remains imperative to identify other sars-cov antibodies that may recognize the conformational epitopes of sars-cov- s protein, which can greatly reduce the amount of time needed to develop novel neutralizing antibodies. the findings derived from serological assays can provide valuable information that would help to support the diagnosis, treatment, and prevention of sars-cov- infections. characterization of antibody profiles suggested that any suspected individuals with undetectable antibody levels against sars-cov- after days pio may be a true negative case, since both anti-sars-cov- igm or igg seroconversion should have already occurred ( , ) . however, these findings may be limited to the relatively small sample size (< patients) and may require further validation with a larger cohort. in order to reinforce diagnosis, it would be advisable to perform multiple assays against different viral antigen. in addition, the information of antibody seroconversion is crucial in determining the optimal timepoints to collect serum or plasma samples for immunoassay screening, as well as obtaining peripheral blood b cells for the generation of therapeutic monoclonal antibodies ( ) . currently, in order to rapidly generate neutralizing monoclonal antibodies against sars-cov- , repurposing of existing sars-cov-specific antibodies was demonstrated. to date, two human sars-cov-specific antibodies, cr and d , have been shown to recognize sars-cov- ( , ) . cr recognizes an epitope along the rbd of sars-cov- , which differs largely at the c-terminus residues to the rbd of sars-cov ( ) . unfortunately, this variation in sequence impacted the ability of cr to crossneutralize sars-cov- . monoclonal antibody d , on the other hand, targets the rbd along the s subunit of both sars-cov and sars-cov- with similar affinities, thereby enabling cross-neutralization against sars-cov- infection ( ) . while combinatory therapy has exhibited a stronger neutralization capability against sars-cov infection ( ), a cocktail antibody approach for sars-cov- could be explored. surprisingly, reports on antibodies against the coronavirus e protein are scarce, possibly due to it being the smallest protein. however, the e antigen is involved in viral assembly, release of virions, as well as virus pathogenesis ( ) . it was demonstrated that recombinant coronaviruses lacking the e protein displayed significantly reduced viral titers, impaired viral maturation and produced avirulent virus progenies, suggesting a similar importance of e protein during sars-cov- infection ( , ) . thus, it would be worthwhile to identify or generate neutralizing antibodies that are specific against the viral e protein. during the course of an epidemic, one of the main challenges is the identification of asymptomatic infection. since these individuals do not present any distinguishable symptoms, they could be the major source of transmission ( ) . immunoassays may be able to detect mildly infected cases ( ) , which is important to ascertain the extent of community spread. while it is fast, robust and easy to perform, there are several limitations to serological assays. one of the major setbacks of immunoassays is the inability to detect the presence of infection during the early stage of disease, as antibodies take several days to be generated after exposure to foreign material ( ) . as such, a recent infection may provide false negative results during serological testing. thus, the use of rt-pcr may be more suitable to diagnose an early acute sars-cov- infection. furthermore, due to the unique genetic makeup of each individual, there would be an inherent variability of the antibody response ( ) . this could possibly explain the difference in antibody profiles elicited among individuals infected with sars-cov- ( ). cross-reactivity could potentially be a limitation of immunoassays as it severely impacts the specificity and sensitivity of the test. although the phylogenetically closest coronavirus, sars-cov, has not been reported to be circulating in the human population since ( ), other endemic human coronaviruses may still pose a problem to accurately diagnose patients with true sars-cov- infection. while a recent study has demonstrated negligible cross-reactivity from human coronavirus, nl , to sars-cov- ( ), validation with other human coronaviruses remains to be investigated. in addition, prior findings on the s protein sequence and neutralization antigenicity of other coronaviruses suggest that antibodies neutralizing clinical human coronavirus isolates may not have the same degree of cross-reactivity with laboratory strains of human coronaviruses, thereby affecting the sensitivity of immunoassays ( ) ( ) ( ) . given the rapid increase in the number of confirmed covid- cases coupled with the shortage in test kits to meet rising demands, decentralized point-of-care tests (poct) may be another alternative to facilitate sars-cov- diagnosis. such tests include lateral flow assay (lfa), which is a paper-based platform for the detection and quantification of analytes in complex mixtures ( ) . to design lfa for sars-cov- detection, an antibody specific to the viral antigen, or a viral antigen that is detectable by patient serum or plasma samples can be immobilized on a nitrocellulose membrane. detection of binding between the analyte and capture antibody by a detector antibody will give rise to a colored line, closely resembling home pregnancy kits ( ) . poct is advantageous as it is usually designed to be rapid, sensitive, highly accessible, and easily performed, requiring only a small amount of sample ( ) . meanwhile, several hundreds of candidate pocts are being evaluated for their applicability toward identifying sars-cov- -infected individuals ( ) . however, pocts can't replace rt-pcr and it is crucial that these developing tests are rigorously assessed prior to use. it is important to note that wrong use and interpretation could lead to disastrous public health consequences ( ) . rapid development of diagnostic tools and immune-based assays are important early interventions against the ongoing sars-cov- pandemic. the availability of serological assays that target a diverse range of viral antigen has no doubt assisted in the accurate diagnosis of covid- patients. essentially, data generated through serological studies can greatly aid in supplementing the results from qrt-pcr, as well as contribute to seroepidemiology, which has been shown to help in the design of virus elimination programs ( ) . moving forward, this extensive collation of the current immunoassays against sars-cov- will provide insights toward monoclonal antibodies discovery and characterization for the development of a sars-cov- vaccine. ln and lr conceived the presented idea. cl wrote the manuscript and prepared the figures. ln, lr, and rl revised the manuscript. all authors approved this manuscript for publication. this work was supported by core research grants provided to singapore immunology network by the biomedical research council (bmrc), and by the a * ccelerate gap-funded project (accl/ -gap -r h-h). new sars-like virus in china triggers alarm novel coronavirus -ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies 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( ) seroepidemiology: an underused tool for designing and monitoring vaccination programmes in low-and middle-income countries diagrams are created with biorender. the authors wish to thank drs. siew-wai fong and yi-hao chan for critical comments of this manuscript. key: cord- - u otbf authors: vainionpää, r.; waris, m.; leinikki, p. title: diagnostic techniques: serological and molecular approaches date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: u otbf virus laboratory diagnostics has an increasingly important role in modern patient care. virological methods are needed to investigate the etiology of acute viral infection or the reactivation of a latent infection, as well as to follow virus load in antiviral treatments. serological assays are also used for screening of blood products for the risk of certain chronic infections, evaluation of the immune status, and need for prophylactic treatments in connection with organ transplantations. for diagnostic purposes the following approaches can be used: demonstration of presence of infectious virus or its structural components directly from a patient's specimens or investigation of specific antibody response in serum specimens. amplification techniques, most commonly polymerase chain reaction (pcr) is currently the workhorse of nucleic acid testing for the detection and quantitation of virus genomes. virus isolation is used to demonstrate infectious virus in a patient's specimens, whereas virus antigens are investigated by antigen detection assays. serological diagnosis is based on either the demonstration of the presence of virus-specific igm antibodies or a significant increase in the levels and/or avidity of specific igg antibodies. immunoassays are the most commonly used serological assays. point-of-care tests (poc tests), for antigens, antibodies, and also nucleic acids are also becoming more and more common in diagnostic use. in order to reach the best diagnostic efficiency for each patient it is important to select the most suitable method using the right sample collected at the right time. glossary eia enzyme immunoassays are methods used to estimate virus-specific igg and igm antibodies or virus antigens by enzyme-labeled conjugates. pcr by the polymerase chain reaction (pcr) and with specific primers, dna sequences can be multiplied. rt-pcr for rna, nucleic acid has to be transcribed with reverse transcriptase (rt) enzyme to complementary dna prior to pcr. specific virus diagnostics can be used to determine the etiology of acute viral infection or the reactivation of a latent infection. two approaches can be used: demonstration of a specific antibody response or of the presence of the virus itself. nucleic acid testing has become the main approach for the demonstration of the presence of virus while cultivation is used by fewer specialized laboratories and antigen detection methods have moved to the point of care. serological methods are used for measuring the antibody response caused by an active infection. in this article, we briefly describe the principles of the most important serological methods and molecular applications that are used to provide information about the viral etiology of the clinical condition presumed to be caused by a viral infection. the diagram of the course of acute virus infection (figure ) indicates the optimal methods for viral diagnosis. following transmission, the virus starts to multiply and after an incubation period clinical symptoms appear with simultaneous shedding of infectious virus. the presence of infectious virus or viral structural components can be investigated directly from various clinical specimens either by nucleic acid detection assays, virus isolation, or antigen detection assays. irrespective of the method of direct virus detection, specimens collected at the site of symptoms give the most conclusive diagnosis. virus-specific antibodies appear somewhat later (from some days to weeks, called a window period). when the virus-specific antibody production reaches the level of detection, at first immunoglobulin m(igm) antibodies and some days later immunoglobulin g(igg) antibodies appear, and the amount of infectious virus starts to decrease. if this is the first encounter with this particular virus, that is, a primary immune response, igg antibody levels can stay at a relatively low level, whereas in a later contact with the same antigen, that is, in secondary response, igg levels increase rapidly and reach high levels while igm response may not be detectable at all. antibodies are usually investigated from serum samples taken at acute and convalescent phase of the infection. in selected cases other materials such as cerebrospinal fluid and other body fluids can also be analyzed. in order to reach the best diagnosis for each patient, it is important to select the most suitable method using the right sample collected at the right time. during most primary infections igm antibody levels peak at - days after the onset of illness and then start to decline, disappearing after some weeks or months. an igm response is usually not detected in reactivated infections or reinfections. the production of igg antibodies starts a few days after igm response and these antibodies often persist throughout life. serological diagnosis is usually based on either the demonstration of the presence of specific igm antibodies or a significant increase in the levels of specific igg antibodies between two consecutive samples taken - weeks apart. the antigen for the test can be either viable or inactivated virus or some of its components prepared by virological or molecular methods. isotype-specific markers or physical separation are used to demonstrate the isotype of the reacting antibody. in some cases, even igg subclass specificities are determined although they have limited value in diagnostic work. during the early phase of primary infection the specific avidity of igg antibodies is usually low but it increases during the maturation of the response. diagnostic applications of the measurement of the avidity of igg antibodies against specific antigens have been developed to help distinguish serological responses due to acute infections from those of chronic or past infections. serological assays are useful for many purposes. in primary infections they often provide information about the etiology even after the acute stage when infectious virus or its components can no longer be demonstrated in the samples. they are widely used for screening of blood products for the risk of certain chronic infections, evaluation of the immune status, and need for prophylactic treatments in connection with certain organ transplantations. serology may also be used to confirm an acute infection in cases when the virus can be asymptomatically present. they are also widely used for epidemiological studies, determination of vaccine-induced immunity, and other similar public health purposes. serological assays have their limitations. in some infections the antibody response is not strong enough or the limited specificity of the antigens used in the assay does not allow unambiguous interpretation of the results. in infections of newborns the presence of maternal antibodies may render the demonstration of the response in the baby impossible. in immunocompromised patients the serological response is often too weak to allow the demonstration of specific responses. in these cases other virological methods should be considered. other clinical specimens than sera can be used for antibody assays. igm and igg antibody determinations from cerebrospinal fluid are used for diagnosis of virus infections in the central nervous system although new molecular methods are increasingly replacing them. recently, increasing attention has been given to the use of noninvasive sample materials such as saliva or urine. they are becoming important for public health purposes but their value for diagnosing individual patients is still limited. antibodies that decrease the infectious capacity of the virus are called neutralizing antibodies. they are produced during acute infection and often persist during the entire lifetime. they are also useful as an indication of immunity. both igm and igg antibodies participate in the neutralization. in the assay, known amounts of infectious virus are mixed with the serum sample and incubated for a short period after which the residual infectivity is measured using cell cultures or test animals. this infectivity is then compared with the infectivity of the original virus and the neutralizing capacity is calculated from this result. today, neutralizing antibody assays are often done by plaque reduction assays with better accuracy but with somewhat more complex technical requirements. neutralizing antibody assay is specific and sensitive, but time-consuming and laborious, and therefore it is not widely used in routine diagnostic services. many viruses bind to hemagglutinin molecules found at the surface of red blood cells of various animal species and this can cause aggregation of red cells in suitable conditions. prevention of this aggregation, called hemagglutination inhibition, by specific antiviral antibodies in the patient's serum has been widely used for diagnostic purposes. the test, known as hemagglutination inhibition test, has important diagnostic and public health applications in certain infections, most notably in influenza where antibodies measured by this test show additional specificity compared to other tests and therefore provide more detailed information about the immunity and past infections of individuals. however, for the diagnosis of individual patients, the assay is no longer widely used and is replaced by more modern immunoassays. in the test, a virus preparation with a predetermined hemagglutinating capacity is mixed with the serum sample and after proper incubation the residual hemagglutination capacity is measured. both igm and igg antibodies are able to inhibit hemagglutination. the complement fixation test (cft) is a classical laboratory diagnostic test, which is still used for determination of virus antibodies in patient sera or cerebrospinal fluid samples during an acute infection. the test mainly measures igg antibodies. the test is based on the capacity of complement, a group of heat-labile proteins present in the plasma of most warm-blooded animals to bind to antigen-antibody complexes. when the complexes are present on the surface of red blood cells, complement causes their lysis which can be visualized by a suitable experimental setup. in the actual test, the complement in the patient's serum is first destroyed by heating; the serum is then mixed with appropriate viral antigen and after incubation; when the antigen-antibody complexes are formed, exogenous complement (usually from fresh guinea pig serum) is added. this complement then binds to the complexes and having been 'fixed,' it is then no longer able to cause lysis of added indicator red cells. usually, sheep red cells coated with antisheep red cell antibodies are used as indicator to measure the presence of any residual complement. the effect is measured by a suitable test protocol. serial dilutions of the patient serum are used and the highest dilution where the serum can still prevent complement activity in the indicator system is taken as the cft titer of the sample. the tests are usually carried out on microtiter plates and the results are observed by eye. cft is still used for diagnosis of acute virus infection. it measures certain types of antibodies which occur only during the acute phase of the infection. therefore, cft is not suitable for investigation of immune status. the assay procedure is quite complex, because the test is dependent on several biological variables, which have to be standardized by pretesting. the method is less sensitive than many other immunoassays. in addition, the method is very labor intensive and is not amenable to automation. the use of cft in virus diagnostics is increasingly replaced by modern immunoassays. in immunoassays, antibodies binding to specific immobilized antigens can directly be observed using bound antigens and proper indicators such as labeled anti-immunoglobulin antibodies. the antigens can be immobilized to plastic microtiter plates, glass slides, filter papers or any similar material. different immunoassays are nowadays widely used to measure virus-specific igm and igg antibodies. the most recent formats of immunoassays make it possible to detect simultaneously both antigens and antibodies decreasing significantly the window period between infection and immune response. numerous commercial kits with high specificity and sensitivity are available. automation has made immunoassay techniques more rapid, accurate, and easier to perform. in the basic format of solid-phase immunoassays, virus-infected cells, cell lysates, purified or semipurified, recombinant viral antigens or synthetic peptides are immobilized to a solid phase, usually plastic microtiter wells or glass slides. patient's serum is incubated with the antigen and the bound antibody, after washing steps, is visualized using labeled anti-immunoglobulin antibodies ('conjugate') ( figure (a) ). if the label used is an enzyme, the test is called enzyme immunoassay (eia) or enzymelinked immunosorbent assay (elisa) and the bound antibody is detected by an enzyme-dependent color reaction. if a fluorescent label is used, the method is called immunofluorescent test (ift). the enzyme labels most commonly used are horseradish peroxidase (hrp) and alkaline phosphatase (ap). in hrp-eia the color-forming system consists of ortho-phenyldiamine (opd) as a chromogen and hydrogen peroxidase (h o ) as a substrate. if the hrp-conjugate is bound to antibody-antigen complexes, the colorless chromogen becomes yellow and color intensity is measured with a photometer at a wavelength of - nm. the intensity of the color is proportional to the amount of bound conjugate and to the amount of specific antibodies in a patient serum sample. if the serum contains no specific antibodies, the conjugate is not bound and no color reaction occurs. by using either anti-igg or anti-igm conjugates it is possible to determine separately immunoglobulin subclasses. the specificity and sensitivity of these immunoassays are generally high. the most common source of potential misinterpretation is false positive igm reaction due to the presence of rheumatoid factor, itself igm molecule, reacting with virus-specific igg. the specificity can be improved by using an additional incubation step where igm antibodies are first enriched ('captured') in the sample by using anti-igm immunoglobulin. alternatively igg can be adsorbed or blocked by serum pretreatment. immunofluorescent tests were used in the past for measuring virus-specific antibodies, but are now replaced by eia techniques. the principle of the method is similar to eias. in ift, infected cells are placed on a glass slide and bound antibodies are detected by fluorescein-labeled anti-immunoglobulin antibodies. the glass slides are examined under a fluorescence microscope. the method is specific and sensitive, but quite labor intensive, and reading the test demands considerable experience. in some infections (e.g., that caused by human immunodeficiency virus (hiv)), antibodies against certain components of the virus are more informative than other less-specific antibodies and they are detected by immunoblotting assays. different virus antigens, prepared by gel diffusion or other techniques, are absorbed as discrete bands on a solid strip of cellulose or similar material and the strip is incubated with the patient's serum. antibodies present in the serum bind to specific antigens and are detected using an hrp-conjugate and nitroblue tetrazolium as the precipitating color chromogen. the color reaction is observed and compared to positive and negative control samples assayed on separate strips. immunoblotting assays are commonly used to confirm highly sensitive eia screening assay results for antibodies against hiv, hepatitis c virus (hcv) and human t-cell lymphoma virus (htlv). a technique known as lateral-flow technology has also been used to identify antibodies or antigens. these tests involve application of serum or other samples directly on a strip of suitable material such as cellulose, where the antibodies are diffused laterally and eventually reach a site in the strip where appropriate antigen has been applied and chemically fixed. specific antibodies become bound to the site while nonreacting antibodies diffuse out from the area. the presence of antibodies is visualized using labeled conjugates. although such tests are not quantitative, they are valuable for infections where the presence of specific antibodies is indicative, such as hiv infection. performance of the test is often very simple and the result is available in a few minutes or a few hours, making such tests suitable for bed-side screening. in more advanced tests, several different antibodies can be detected by a single assay and the test conditions can be modified further so that antigens can also be detected. many such tests have become commercially available in recent years. for some applications, coated latex particles have replaced strips with fixed antigen as the solid phase. binding of specific antibodies can be visualized with chromogenic or otherwise labeled indicator antibodies or a positive reaction can be detected by agglutination of the latex particles. the presence of viral antigens in clinical specimens, such as nasopharyngeal aspirates, fecal specimens, vesicle fluids, tissue specimens, as well as serum samples can be demonstrated by antigen detection assays. in immunofluorescence tests, cells from a clinical specimen are fixed on a glass slide and viral antigens present in the cells are detected by fluorescein-labeled virus-specific antibodies. less reader-dependent results can be obtained using enzyme or other immunoassays. solubilized antigens in clinical specimens are first captured using specific monoclonal antibodies bound to a solid phase, and are then detected with virus-specific detector antibodies (figure (b) ). the functionality of monoclonal antibodies (mabs) with high specific binding affinity is better preserved when labeled with a small molecule as compared to a bulky enzyme molecule. in eia, biotinylated mabs are used with streptavidin-enzyme conjugate. in time-resolved fluoroimmunoassay (tr-fia), detector mabs are labeled with an europium chelate. antigen detection methods are especially recommended in the case of virus reactivation, for example, for herpes simplex and varicella zoster virus diagnosis where the serological response can be very weak. antigen detection assays are also widely used in respiratory tract infections like influenza and respiratory syncytial virus infections. a simple test for the demonstration of rotavirus and adenovirus antigens in children with gastroenteritis is also available. direct demonstration of viral nucleic acids in clinical samples has become the technique with the widest repertoire of diagnostic virus targets. using the polymerase chain reaction (pcr) with specific primers, viral sequences can be rapidly multiplied and identified. these techniques have largely replaced classical virus isolation. they are rapid to perform and in many cases more sensitive than virus isolation or antigen detection methods making earlier diagnosis possible. they have proved particularly valuable for the diagnosis of emerging viruses and viruses that cannot be cultivated such as papillomaviruses, parvoviruses, hepatitis viruses, and rhinovirus species c. semiquantitative and quantitative applications have been developed allowing monitoring of viral load during antiviral treatment. these tests cannot distinguish between viable and replication-incompetent virus, warranting caution in the interpretation of the results in certain cases. while sensitivity to cross-over contamination throughout the diagnostic pathway cannot be undermined, use of real-time pcr technology has diminished these problems in clinical laboratory settings. the specificity of these tests is based on the extent of pair-matching sequences between the viral nucleic acids and the primers. extremely high sensitivity is typical for pcr methods; - copies of viral nucleic acid can be detected in about one to few hours. pcr methods are available for both rna and dna viruses. for rna viruses viral nucleic acid has to be transcribed with reverse transcriptase (rt) enzyme to complementary dna in a combined assay called rt-pcr. viral nucleic acid is extracted from the sample material and amplified in three successive steps. the double-stranded dna is first heat-denaturated and separated into single strands. the specific target fragment of dna strand is then amplified (figure ) by pairs of target-specific oligonucleotide primers, each of which anneal to one strand of unfold double-stranded dna. each annealed primer acts as an origin for heat-stable polymerase enzyme and a complementary strand is synthesized via sequential addition of deoxyribonucleotides. in real-time pcr, annealing and extension steps are often combined to a single step at c. these cycles are repeated about times, each cycle resulting in an exponentially increasing numbers of copies. after the amplification is completed, the products can be detected by several methods. agarose gel electrophoresis combined with ethidium bromide staining of the products is a classical method (figure ) . the size of the amplified product is compared to control amplicons and other standards in the same gel. various hybridization assays, based on labeled complementary oligonucleotides (probes), are also used to improve the sensitivity and specificity of the detection. for applications with a large number of targets, such as respiratory virus detection or papillomavirus typing, conventional pcr with post-pcr hybridization is still a popular solution. products of highly multiplexed pcr assays can be identified by probe hybridization on microarrays using scanning fluorometer or on fluorescent microbeads using flow-cytometric bead counting. the amplified fragments can also be sequenced giving additional information about the virus. comparison of the sequences with known virus sequences allows identification of species, strains, or subtypes that may be important for public health or medical purposes. sequencing after rt-pcr is also the current method-of-choice for investigating the emergence of antiviral drug resistance among hiv-infected patients. real-time pcr instruments monitor accumulation of amplicons by measuring the fluorescence continuously in each cycle of the reaction. fluorescence is generated by a dsdna dye or a fluorescent probe system included into the reaction mix. the earlier the amplification product becomes detectable over the background, the higher is the amount of virus in the sample ( figure ) . a dsdna dye (e.g. sybr green i) reacts with any dsdna formed during amplification, but amplicon specificity can be confirmed by melting curve analysis. it also allows testing for more than one virus from the same sample ( figure ). there are many alternative probe chemistries for real-time pcr, the most common including dual label probes. since probes are usually more selective for their target sequences than primers, probe based real-time pcr methods offer highest specificities with fastest reaction times. multichannel instruments allow the use of probes with different exitation and emission spectra for the simultaneous detection of several different analytes in a single tube. the pcr assays are extremely sensitive and can therefore be influenced by inhibitors of the polymerase enzyme that are sometimes present in clinical samples. internal controls can be included into reaction mixtures. nucleases present in samples or in figure rt-pcr with real-time detection (a) and with melting curve analysis (b) for the detection of respiratory syncytial virus (rsv), rhinovirus, and enterovirus in respiratory secretions (c). c t is a threshold cycle number, t m is a melting temperature, and ntc is a nontemplate control. unpublished results by waris m, tevaluoto t, and Ö sterback r. reagents can also cause false negative results by degrading viral nucleic acids. furthermore, amplicons may also cause product carryover and false positive results. extremely high care has to be applied in handling the clinical specimens, the reagents, as well as the reaction products. the key to a succesful pcr method is the careful design of the primers and probes. they have to be specific for the target and follow certain structural rules to produce efficient amplification. the high specificity of the pcr, especially probe-based real-time pcr, makes the technique sensitive for mutations, sense or non-sense, in the target sequences of highly variable viruses. that must be taken into the consideration not only in design of the assays, but also in continuous evaluation of them for changes in sensitivity caused by new mutations affecting critical primer or probe binding sites. one of the great advantages of the pcr technology is its potential to detect new emerging viruses. by using primers from related viruses or so-called generic primers important information regarding the new virus can be obtained for further development of more specific tests. a recent example is the middle-east respiratory syndrome (mers) coronavirus, for which specific diagnostic tests became available soon after the taxonomic position of the virus became known. the technology also allows safe handling and transport of virus samples, since extraction buffers added to the samples inactivate virus infectivity. poc tests are becoming increasingly common in clinical practice. in proper use, they offer a cost-efficient diagnostic guidance for quick clinical decision making. their performance is rarely as good as that of corresponding laboratory tests; use of them should be quality-controlled, and a back-up should be arranged with a virology lab. most of them are based on easy-to-use lateral-flow or latex particle technology and are able to give the result in a few minutes. poc tests are nowadays available for antibody screening of an increasing number of virus infections (hiv, hepatitis c virus (hcv), varicella-zoster virus (vzv), cytomegalovirus (cmv), epstein-barr virus (ebv)). with the breakthrough of nucleic acid testing, antigen detection methods are becoming obsolete in high-end virus laboratories, but their development have continued for poc testing. most commercially available tests for antigen detection are lateral flow immunochromatographic assays, and they are typically targeting influenza viruses, respiratory syncytial virus, adenoviruses, rotavirus, norovirus and hepatitis b virus. multianalyte testing for common respiratory virus antigens is possible with a commersial multianalyte respiratory infection poc test platform including a small bench-top instrument suitable for polyclinical use. also compact all-in-one pcr systems for the detection of respiratory pathogens are now available. driven by public health, scientific and commercial interests, new diagnostic tests for the laboratory diagnosis of viral infections are continuously being developed. the development of new molecular detection methods continues on two lines. on the one hand, it includes compact, fully integrated automates requiring minimal training to run and suitable for poc use. on the other hand, separate instruments for specimen handling, nucleic acid extraction, pcr set-up, and amplification are integrated into high throughput flow systems, which allow efficient use of both commercial and laboratory designed assays. use of multianalyte methods is becoming a practical reality and they might significantly change diagnostics of infectious diseases in future. multiplex pcr in a single tube or miniature format still have the challenge of reaching the sensitivity of simpler pcr targeting only one or few analytes. microarray technology for pcr product detection competes with sequencing, but still provide a rapid alternative for identification of a large number of virus types. next generation sequencing technology will be the primary way of identification of new or emerging viruses. simple microarrays, readable without bioinformatics, would reduce the cost of serological screening and allow the use of control measures to improved quality of the results. outside the specific virus diagnostics, measures of virus induced interferon response is an area of potential development. next-generation sequencing technologies in diagnostic virology avidity of igg in serodiagnosis of infectious diseases principle and practice of clinical virology clinical and economical impact of multiplex respiratory virus assays microarray-based detection and genotyping of viral pathogens detection and monitoring of virus infections by real-time pcr point-of-care diagnostics for global health key: cord- -d b fjl authors: bernard, serge; shirai, june; lantier, isabelle; bottreau, elisabeth; aynaud, jean marie title: lactogenic immunity to transmissible gastroenteritis (tge) of swine induced by the attenuated nouzilly strain of tge virus: passive protection of piglets and detection of serum and milk antibody classes by elisa date: - - journal: veterinary immunology and immunopathology doi: . / - ( ) - sha: doc_id: cord_uid: d b fjl abstract piglets of eight sows vaccinated by different routes with the attenuated tge mutant coronavirus, nouzilly (n) strain, and piglets from two field seropositive sows were challenged with a virulent tge strain. on the day of challenge and days after challenge, milk and serum samples from sows were analysed for their level of neutralizing antibodies, total immunoglobulin classes and tge antibody classes by an elisa. no direct relationship was seen between the level of protection of the litters and the titres of the different antibody classes on the day of challenge. however, an inverse correlation was seen days after challenge between protection and the level of tge antibodies. transmissible gastroenteritis (tge) causes acute and highly contagious viral enteritis in pigs, and is considered to be a major cause of death in newborn piglets. after natural infection or experimental oral infection of pregnant sows with a virulent strain of tge virus, lactogenic immunity is highly protective for piglets, and neutralizing antibodies in milk are mainly associated with the iga fraction (bohl, ) . in contrast, in sows immunized with cell culture-attenuated tge strains, lactogenic immunity is poorly protective and neutralizing antibodies in milk are associated with the igg fraction (sail and bohl, ) . in a previous investigation (aynaud et al., ) , an attenuated tge mutant virus, nouzilly strain (n strain), resistant to acidity and digestive gastric enzymes, was used for oral immunization of seronegative pregnant sows. the post-vaccinal lactogenic immune response gave good protection to suckling piglets against virus challenge. however, the passive protection rate was not related to the level of neutralizing activity in milk and serum of sows after challenge exposure (shira'/et al., ) . in the present investigation, our objective was to analyse the immune response of pregnant sows vaccinated with the n strain of tge, using an elisa technique. the levels of igg, igm, and iga were evaluated in serum and milk of sows on the day of, and days after virulent challenge of the piglets. cell culture rp.d. is a pig kidney cell line which has been described elsewhere (laude et al., ) . the swine testis (st) cell line (mcclurkin and norman, ) was kindly supplied by dr. e.h. bohl (wooster, ohio, u.s.a. ) . both cell lines were cultured in minimal essential medium (mem) containing iu/ml penicillin and #g/ml streptomycin sulphate, and were supplemented with % fetal calf serum. the virus, serum and milk whey used for the antibody titration were diluted in serum-free mem containing twice the normal antibiotic concentration. purdue-ll is a high-passage tge strain, cultured in rp.d. cells. the virus suspension was harvested at the first sign of a cytopathic effect (about h post-infection) and stored at - ° c. this strain was used for the virus neutralization (vn) antibody test. the nouzilly strain (n) is a high-passage strain obtained in our laboratory by serial cycles ( ) of survivor selection in gastric juice of adult pigs (aynaud et al., ; nguyen et al., ) . this attenuated strain was used for vaccination of pregnant sows against tge. virus suspensions were prepared in the same way as the purdue-ll strain. the virulent challenge gep-ii strain of tge was isolated by p. vannier (personal gift) from a field outbreak in france. a virulent virus stock (about ldso/ml) was prepared from the contents of the small intestine of colostrum-deprived newborn piglets h post-inoculation with the gep-ii strain. this strain was free of rotavirus antigens, and was filtered ( . ttm) before being used for the oral virulent challenge of the newborn piglets. tge virus neutralizing antibodies were titrated by a micro-method in st cells (aynaud et al., ) . briefly, doubling dilutions of the serum and milk were made in -well microplates, and tissue culture-infectious dosess (tcidso) of the purdue-ll strain were added to each well. the mixtures of samples and virus were incubated for min at ° c in % co , % air. onehundred/tl of fresh st cell suspension ( /ml) in % fetal calf serum supplemented mem was then added to each well. tge antibody-positive serum was used as a reference. results were read after days of incubation, the titre of the sample was calculated as the arithmetic mean of triplicates of the lowest dilution which protected the cells from the cytolytic effect of the virus. eight tge seronegative (neutralizing antibody activity) large white and mei shan pregnant sows were bred in our institute for the experiment. two field seropositive pregnant sows were also used (no. and ). eight sows received pfu of the n strain, to days before parturition. vaccine doses were given per os in ml of macilvaine buffer ( . m, ph ) to six fasting sows, and intramuscularly in ml of pbs to two sows. booster administration was carried out with the same dose of n strain virus, to days before farrowing, by different routes of administration (oral, intramuscular and conjunctival) as shown in table . the animals which had been experimentally inoculated with tge virus were housed in isolated units. one ml of ldso of gep-ii strain was orally-administered to all the piglets in a litter at to days old. clinical signs and the mortality rate of the piglets were noted every day up until days after challenge. blood and milk samples were collected from each sow on the day and days after challenge. milk samples were obtained manually after milk let-down was induced by intravenous injection of oxytocin. whey was prepared by centrifugation at ×g for min. an aliquot was collected from the middle portion between the cream layer and the deposited casein and debris. serum and milk whey were stored at - ° c until use. total amounts of igg, igm and iga in the serum and milk were measured by a competitive, immunocaptive, elisa technique as described previously . briefly, the samples were diluted in pbs con-taining . % tween and % gelatin and mixed with chromatographicallypurified peroxidase-conjugated (grade i, boehringer, w. germany) porcine igg, igm or iga (nakane and pierce, ) on -well microplates (dynatech microtiter). the mixture was transferred to microplates (nunc immunoplates - ) coated with rabbit anti-porcine immunoglobulin and incubated for h at ° c. dilution of a known quantity of purified immunoglobulin of the various classes, treated as described above were used to produce reference curves between ng and ~g/well. the optical density at nm was quantified after h of contact with , ' -azino-di-( -ethyl-benzthiazoline sulfonate (abts) (boehringer, w. germany) and i-i . the titres of tge virus specific igg, igm and iga antibodies in serum and milk were evaluated by an elisa technique. a mixture of monoclonal antibodies (delmas et al., ; laude et al., ) specific for structural proteins of tge virus was used for coating plates as described previously . after washing, the plates were saturated overnight with % gelatin in pbs. virus material from supernatants of st cells infected with purdue- strain of tge virus ( pfu) was incubated in pbs, containing . % tween and % gelatin, in the precoated plates for h at ° c. control plates were set up with supernatant from uninfected st cell cultures. the diluted samples, containing unknown antibodies, were incubated in the same buffer in the microplates ( zl/well) for h at ° c. the samples were then incubated with conjugated immune complexes (pig igg or igm or iga-peroxidase labelled/specific anti-pig igg, igm, iga) (bernard and lantier, ) for h at °c. the enzymatic reaction was performed with abts under the same conditions as described above. results were expressed as the difference in optical density of samples between infected and uninfected st cell supernatant. the relationships between the titres of the different antibody classes, and neutralizing antibodies in milk and serum collected on the day or days after challenge exposure were estimated by the non-parametric spearman rank-test. all of the piglets challenged with our virulent strain of tge virus have shown clinical signs of diarrhea. this parameter was therefore not considered to be selective and we chose the mortality rate of the piglets, up to days after challenge, to estimate the passive protection rate. the passive protection rate of piglets and the neutralizing antibody activity levels in serum and milk of immune sows are presented in table . neutralizing antibody activities were always higher in serum than in milk. after challenge exposure of piglets, a strong increase in neutralizing antibody activity was observed in serum and milk of immune sows with the exception of sow no. . this observation suggested a secondary immune response caused by virulent tge virus excreted by challenged piglets. whatever the conditions of immunization, there was no correlation between the neutralizing activity level in serum or milk and the passive protection rate of piglets (p> . ). the mean quantities of immunoglobulin classes in serum and milk on the day and days after challenge of piglets are presented in table . no difference was seen between the two samples at the times in the levels of igg, igm or iga in serum or milk. the individual variation of the level of milk immunoglobulins was very high. a correlation existed between the level of each class of immunoglobulin in the milk and serum (spearman test, p < . , results not shown). the only parameter which correlated with protection was the increase in the serum igm titre. the antibody class responses to tge virus of immune sows are shown in fig. (serum) and in fig. (milk wheys) . on the day of challenge, no direct correlation was seen between the passive protection rate and the level of any class of tge antibody in serum or milk. statistical analysis (table ) , showed no other associations except a linkage between the level of the igg, iga and igm antibodies in the serum. no correlation was seen with protection. ten days after challenge, neutralizing activity (table ) and tge antibody classes detected by elisa were greatly increased in the serum (fig. ) and milk (fig. ) of the sows (except sow no. ). the inverse relationship between protection rate and the antibody titre at this time was confirmed by statistical analysis (table ). further analysis of statistical correlation between the level of antibody activity in serum and milk is given in table . no relationship was seen between the different antibody titres in samples taken on the day and days after challenge, neither in milk nor in serum. however, on the day of challenge, a linkage was seen between the serum neutralizing antibody level and the milk igg tge antibodies. ten days after challenge, the pattern of the relationship between the activities of the antibody class in serum and milk, was compatible with an anamnestic response in the serum of sows and of diffusion into the milk (table ). table relationship between passive protection rate and titre of specific anti-tge antibody classes ( igg, iga, igm) in serum and milk, on the day and days after challenge the efficacy of the vaccinal n strain was estimated against standardized challenge of ldso of gep-ii virulent virus (aynaud et al., ) . according to our previous experiments, we estimated that in this age range ( to days old) the use of only one seronegative susceptible control was sufficient to show the validity of the challenge. unfortunately, this field virulent strain does not grow in tissue culture, so it is not possible to analyse the neutralizing antibodies against this virus. however, we have analysed the neutralizing activity of many monoclonal (delmas et al., ) and polyclonal antibodies against many different strains of the virus (personal data), and we have never found a fundamental difference between the neutralizing activities. for convenience we choose the purdue-ll virus to analyse our samples. the most interesting result of the preliminary report about the immune response of sows vaccinated with n strain of tge (shira'i et al., ) was the absence of correlation between the passive protection rate of the litter and the neutralizing antibody titre in serum or milk of sows on the day of challenge. in the present work, using eight other sows, we confirmed the absence of linkage between protection and neutralizing antibody level on the day of challenge. we also demonstrated a statistically significant inverse relationship between the neutralizing antibody titre in milk days after challenge and protection of the litters (table ). ten days after challenge, it is clear that an inverse relationship exists between the protection and the titre of all the antibody classes in serum and milk ( fig. and , table ). this secondary response was modulated by the quantity of virus excreted by the piglets and the immune status of the digestive tract. absence of, or a low secondary immune response of sows days after challenge could be a good a posteriori marker of lactogenic passive protection. in a previous report (shira '/et al., ) , we analysed the antibody classes which were responsible for the neutralizing activities in serum and milk days after challenge by chromatographic separation. the low titres in the sample collected on the day of virulent challenge obliged us to search for a more sensitive technique. we used therefore an appropriate elisa to allow us to study the antibody class distribution on the day of challenge. the pattern of the igg, iga and igm tge antibody classes did not show any relationship with protection. with high virulent tge virus, antibody of the iga class was the main immunoglobulin involved in the passive protection of the suckling piglets (bohl and saif, ; saif and bohl, ; saif and bohl, ) . however, the protection mediated by iga is also dependent on the oral route of vaccination with virulent virus since intramuscular injection does not elicit iga synthesis (abou-youssef and ristic, ; kodama et al., ; woods, ) . with cell culture-attenuated tge virus neutralizing antibody, but not iga antibody, was detectable in the serum of sows which had been either orally or intramuscularly inoculated (kodama et al., ; woods, ) . with the attenuated n strain it seems that iga antibody activity is not sufficient to explain the protection of the litters. however, purified igg tge antibody gives broadly the same passive protection as igm and iga to artificiallyfed piglets (stone et al., ) and bovine hyperimmune colostrum whey, rich in igg , considerably reduced the mortality of passively-fed piglets following tge challenge (stepanek et al., ) . it is very difficult to compare an in vitro antibody detection test with the in vivo protection rate due to attendant complex multiparametric physiological phenomena. characteristics of the n strain such as resistance to acidity and to digestive enzymes (aynaud et al., ) and its high content of structural antigen (nguyen et al., ) is sufficient to explain the in vitro immunogenicity but not to explain the in vivo protection induced by this virus. according to bohl (bohl et al., a,b; bohl and saif, ) the routes of immunization of the sow, the virulence of the virus, the time of immunization and the processing of the viral epitope would be parameters that affect the pattern of the immune response. the mastery of successful oral vaccination will be achieved only with a greater understanding of all these parameters together with many others. protective effect of immunoglobulins in serum and milk of sows exposed to tge virus transmissible gastroenteritis (tge) of swine: survivor selection of tge virus mutants in stomach juice of adult pigs a new conjugate for the elisa quantitation of porcine iga iga purification from sow milk detection of transmissible gastroenteritis coronavirus antigens by a sandwich elisa technique transmissible gastroenteritis passive immunity in tge of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes immunology of transmissible gastroenteritis antibody response in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus antigenic structure of transmissible gastroenteritis virus. ii. domains in the peplomer glycoprotein characterization of immunoglobulin a antibody in serum of swine inoculated with transmissible gastroenteritis virus serum immunoglobulin a antibody response in swine infected with transmissible gastroenteritis virus, as determined by indirect immunoperoxidase antibody test in vitro properties of low and high passaged strains of transmissible gastroenteritis coronavirus of swine antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virus proteins studies on transmissible gastroenteritis of swine. ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis enzyme labeled antibody preparation and application for the localization of antigens etude compar~e de souches de coronavirus de la gastroent~rite transmissible: conditions de la r~plication virale et de la synth~se des antig~nes structuraux role of secretory iga in passive immunity of swine to enteric viral infections passive immunity against enteric viral infections lactogenic immunity to transmissible gastroenteritis of swine induced by attenuated nouzilly strain of tge virus: neutralizing antibody classes and protection preparation of hyperimmune cow colostrum whey and its use in the protection of piglets against transmissible gastroenteritis efficacy isolated colostral iga, igg and igm (a) to protect neonatal pigs against the coronavirus of transmissible gastroenteritis efficacy of vaccination of sows with serologically related coronaviruses for control of tge in nursing pigs we are grateful for the technical assistance of ph. bernardet, r. delaunay and d. musset, and for the correction of the manuscript by anita rees and julia woodman. key: cord- -bdoavpsb authors: nemoto, manabu; kanno, toru; bannai, hiroshi; tsujimura, koji; yamanaka, takashi; kokado, hiroshi title: antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine date: - - journal: j vet med sci doi: . /jvms. - sha: doc_id: cord_uid: bdoavpsb a vaccine for equine coronavirus (ecov) is so far unavailable. bovine coronavirus (bcov) is antigenically related to ecov; it is therefore possible that bcov vaccine will induce antibodies against ecov in horses. this study investigated antibody response to ecov in horses inoculated with bcov vaccine. virus neutralization tests showed that antibody titers against ecov increased in all six horses tested at days post inoculation, although the antibody titers were lower against ecov than against bcov. this study showed that bcov vaccine provides horses with antibodies against ecov to some extent. it is unclear whether antibodies provided by bcov vaccine are effective against ecov, and therefore ecov challenge studies are needed to evaluate efficacy of the vaccine in the future. doi: . /jvms. first isolated in the united states in [ , ] . two-fold serial dilutions of serum were mixed with an equal volume of viral suspensions containing two hundred % tissue culture infective doses per . ml and incubated for min at °c. then, . ml of each mixture was applied to hrt- g cells on -well plates and incubated for to days. virus-neutralizing antibody titers were expressed as the reciprocal of the highest serum dilution that inhibited viral cytopathic effects. statistical analysis was carried out using ekuseru-toukei (ssri, tokyo, japan). logarithmic transformations of the reciprocal antibody titers were made to stabilize variances. antibody titers after logarithmic transformation were analyzed by one-way anova with dunnett's multiple comparison post hoc test using the antibody titers at dpi as control. a p-value of < . was considered statistically significant. the virus-neutralizing antibody titers of horses inoculated with or ml of the bcov vaccine are shown in table . in horses inoculated with ml of vaccine, the geometric mean antibody titers against bcov at , , , , and dpi were , , , , and , respectively, and the geometric mean antibody titers against ecov were , , , , and (table ) . compared with the antibody titers at dpi, the antibody titers against both bcov and ecov significantly increased at , , and dpi. in horses inoculated with ml of vaccine, the geometric mean titers against bcov were , , , , and , respectively, and the geometric mean titers against ecov were , , , , and ( table ). the antibody titers against bcov significantly increased at , , , and dpi, and the antibody titers against ecov significantly increased at , , and dpi in comparison with the antibody titers at dpi. this study showed that in all horses inoculated with the bcov vaccine antibody titers against ecov increased from dpi, although the antibody titers against ecov were lower than those against bcov. maximum antibody titers against ecov in each horse ranged from to . an experimental inoculation study conducted earlier also showed that neutralizing antibody titers against ecov strain nc , which is the same strain as used in this study, were to in three horses at days after their inoculation with ecov-positive feces [ ] . however, in horses naturally infected by ecovs in the and outbreaks in japan [ , ] , the geometric means of neutralizing antibody titers were . ( horses) and . ( horses), respectively. thus, the antibody titers of horses inoculated with the bcov vaccine were similar to the titers of the experimentally infected horses but were lower than the titers of horses naturally infected in actual outbreaks. an experimental challenge study using cattle inoculated with inactivated strain no. /h showed that inoculated cattle possessing neutralizing antibody titers of more than showed no clinical signs after challenge with a virulent bcov, whereas in contrast, inoculated cattle possessing neutralizing antibody titers of less than developed watery diarrhea and fever [ ] . needless to say, the animal species, strain of challenge virus, and method of virus neutralization test in that study are different from our present study. nevertheless, the antibody titers of all vaccinated horses in the present study were no more than , and we therefore consider that the bcov vaccine will have limited efficacy against ecov infection in horses. to clarify this, ecov challenge studies in horses inoculated with the bcov vaccine will be needed to evaluate the efficacy the vaccine. the three horses inoculated with ml of the vaccine did not exhibit any adverse reaction during this study. in contrast, two out of the three horses inoculated with ml of the vaccine exhibited swelling at the inoculation site after the second vaccination. none of the horses developed a fever after the vaccinations. administration of more than ml of the vaccine to horses would likely increase the risk of adverse reactions. as described above, a significant increase in antibody titers against ecov was observed from dpi irrespective of whether or ml was administered. additionally, the differences in antibody titers against ecov at each dpi from dpi were less than twofold between horses inoculated with and ml. these results suggest that inoculation of ml is suitable for horses as well as for cattle. although horse no. had no detectable antibodies against ecov before vaccination, the horse already had antibodies against bcov (table ) . that horse was born and had been kept at a farm that reared cows before coming to our facility. in saudi arabia, dromedary camel coronavirus hku was detected in apparently healthy horses kept at facilities that reared camels, sheep, goats, and chickens [ ] . hku , which is closely related to bcov, is endemic in camels of the middle east [ ] and the hku -positive horses frequently came into contact with camels and other animals [ ] . hku may have been transmitted from infected camels to those horses. horse no. in the present study may have also been in contact with infected cows, and bcov may have been transmitted from infected cows to the horse. however, because there is no epidemiological information, it is unknown whether there were in fact bcov-infected cows at the farm or whether horse no. had shown any clinical signs. this study showed that a bcov vaccine provides horses with antibodies against ecov to some extent. it is unclear whether the antibodies provided by the bcov vaccine are sufficient to be effective against ecov, and therefore ecov challenge studies in horses are needed to evaluate the efficacy of the vaccine in the future. virus taxonomy: ninth report of the international committee on taxonomy of viruses disease associated with equine coronavirus infection and high case fatality rate necrotizing enteritis and hyperammonemic encephalopathy associated with equine coronavirus infection in equids characterization of a coronavirus isolated from a diarrheic foal coronavirus infections in horses in saudi arabia and oman antigenic variation among recent japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups prevalence of disease with inference of equine coronavirus infection among horses stabled in a draft-horse racecourse complete genome analysis of equine coronavirus isolated in japan experimental inoculation of equine coronavirus into japanese draft horses epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain equine coronavirus: an emerging enteric virus of adult horses emerging outbreaks associated with equine coronavirus in adult horses field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows protection studies on winter dysentery caused by bovine coronavirus in cattle using antigens prepared from infected cell lysates novel betacoronavirus in dromedaries of the middle east genomic characterization of equine coronavirus acknowledgments. the equine coronavirus strain nc was kindly provided by dr. j. s. guy key: cord- -rya w sh authors: kang, xiaoping; li, yuchang; fan, li; lin, fang; wei, jingjing; zhu, xiaolei; hu, yi; li, jing; chang, guohui; zhu, qingyu; liu, hong; yang, yinhui title: development of an elisa-array for simultaneous detection of five encephalitis viruses date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: rya w sh japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), and eastern equine encephalitis virus (eeev) can cause symptoms of encephalitis. establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. currently, there are still no multiple antigen detection methods available clinically. an elisa-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. an elisa-array method for the simultaneous detection of five encephalitis viruses was developed in this study. seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the elisa-array. the elisa-array assay is based on a "sandwich" elisa format and consists of viral antibodies printed directly on -well microtiter plates, allowing for direct detection of viruses. the developed elisa-array proved to have similar specificity and higher sensitivity compared with the conventional elisas. this method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. the results demonstrated that the developed elisa-array is sensitive and easy to use, which would have potential for clinical use. japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), eastern equine encephalitis virus (eeev), sindbis virus(sv), and dengue virus(dv) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [ ] . establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. currently, elisa and ifa are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [ , ] . there are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [ ] [ ] [ ] . however, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost. antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [ ] [ ] [ ] [ ] . liew [ ] validated one multiplex elisa for the detection of antigens; anderson [ ] used microarray elisa for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that elisa-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays. however, the application of elisa-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. in this study, we developed an elisa-based array for the simultaneous detection of five encephalitis viruses. seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an elisa-array assay. the assay was validated using cultured viruses and inoculated chicken eggs with patient sera. the results demonstrated that this method combined the advantage of elisa and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus. monoclonal antibodies were prepared from hybridoma cell lines constructed by prof. zhu et al. purification was conducted by immunoaffinity chromatography on protein g affinity sepharose [ ] . specific monoclonal antibodies ( d against jev, b against tbev, f against sv, b against serotype dv, f against serotype dv, e against eeev, and a against flavivirus) were selected for this study. all of the antibodies were raised according to standard procedures. using d , b , f , b , f , and e as capture antibodies, detection antibodies ( a , f , and e ) were coupled to biotin-nhs ester(pierce, germany) at °c for h according to the manufacturer's instructions. unincorporated biotin was removed by desalt spin column (pierce). immunologic reactions were reported by streptavidin-hrp (cwbio, beijing, china) and super signal elisa femto maximum sensitive substrate. purified goat-anti mouse antibody was used as a positive control. jev and dv were cultured in c / cells; sv, tbev, and eeev were cultured in bhk- cells. the culture of tbev and eeev was conducted in biosafety level facility, however, jev, dv and sv were conducted in biosafety level facility. viral titers were determined by the % tissue culture infectious dose (tcid ) method. all the cultures were inactivated by . % β-propionolactone at °c overnight, then °c for h to decompose β-propionolactone. antibodies were spotted using a biodot machine (bd ;california, usa) on elisa plates ( nl/dot). the plates were blocked with % bsa-pbs in °c for h, followed by washing times with pbs containing . % tween- for min each. then, the plates were dried, sealed, and stored at °c before use [ ] . when spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the elisa-array assay. the optimization was evaluated by dot morphology and signal intensity. the tested spotting buffers included × phosphate buffer saline (pbs), pbs + % glycerol, and × pbs + % glycerol+ . % triton-x . a range of monoclonal antibody concentrations ( . , . , . , . , and . mg/ml) were compared. following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, hpr-labeled avidin, and substrate, followed by signal evaluation. antigen binding was performed in pbs(containing . % tween- and % fcs) at °c for h, followed by washing times( × pbs containing . % tween- ). incubation of elisa plates with biotinylated detecting antibody cocktails was performed in pbs (containing . % tween- and % fcs) at °c for h. after washing, specific binding of the detecting antibodies was reported by streptavidin-hrp and stained with super signal elisa femto maximum sensitive substrate (thermo scientific, rockford, usa) [ , , ] . visualization of the plate was performed in ae cool ccd image analyzer(beijing bgi gbi biotech company., ltd, china). the signal intensity and background of each spot was read out and recorded with "monster"software. the positive signals were defined as a signal value > and a signal value (sample)/signal value (negative) > . the identical antibodies used in the elisa-array format were also tested in a conventional elisa format to determine the difference in sensitivity and specificity of the two methods. the conventional elisas were performed at the same time as the elisa-array assays to ensure similar reaction conditions. the conventional elisas were performed in an identical maner to the elisa-array, except that antibodies were coated at a concentration of μg/ml in pbs (ph . ), and substrate tmb was used instead of super signal elisa femto maximum sensitive substrate [ , ] . three serum samples were collected from patients with nervous system symptoms and histories of tick bites. the serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. days later, the liquid was collected and divided into two portions (one for inactivation and one for rna extraction). the rna and inactivated samples were stored at - °c before use. rna was extracted from the inoculated chicken eggs using a rneasy mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's instructions. all rna extraction procedures were conducted at bsl- facilities. the primers and probes were used as previously described [ ] . the real-time rt-pcr was conducted with a quti-teck q-rt-pcr kit (qiagen inc,). the reaction consisted of μl of × reaction buffer ( . μl reverse transcription enzyme, and nmol/l primers and probes). rna and deionized water were added to a final volume of μl. pcr was performed with a lightcycler . (roche, switzerland) [ ] . optimization of the elisa-array assay the spotted array layout is depicted in figure and the efficacy of three different spotting buffers on the quality of the printed elisa-arrays were investigated by spot morphology observation and signal intensity comparison. the spotting concentration of the capture antibodies varied from . to . mg/ml (each was serially diluted -fold). the efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. figure illustrates the array layout and figure demonstrates the result of the three spotting buffers and spot concentration of antibody b by tbe virus culture detection. cross reaction detection was also conducted by applying jev, yf, and dv cultures. spot morphology observation (figures a, b , and c) demonstrated that spotting buffer containing pbs with % glycerol produced tailed spot morphology; buffers containing pbs alone and pbs with % glycerol + . % triton-x gave good spot morphology (round and full). buffers containing pbs with % glycerol and pbs with % glycerol+ . % triton-x produced higher signal intensities than pbs alone. thus, pbs with % glycerol+ . % triton-x was adopted as the optimized spotting buffer for subsequent experiments. simultaneously, the spot concentration evaluation suggested that . mg/ml was optimal. at this concentration, the signal intensity was higher and the cross-reaction did not appear (figure d ). consequently, spotting concentration optimization of other capture antibodies ( d , f , e , and b ) demonstrated that . mg/ml was also suitable(data not shown). the optimized elisa array layout is shown in figure , which was applied in the following experiments. successful detection of viral pathogens requires a test with high sensitivity and specificity. to evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. by testing serially-diluted viral cultures, including dv- , dv- , jev, tbe, sv, and eeev, the sensitivity of elisaarray and the identical conventional elisa were compared ( table ). the detection limit of the two methods was compared and demonstrated. the cross-reactivity test was conducted using bhk- and vero cell lysate, yellow fever virus (yfv) cultures ( × tcid /ml, west nile virus(wnv) cultures( × tcid /ml), and western equine encephalitis virus( × tcid /ml). the results demonstrated that neither the elisa-array nor traditional elisa displayed cross-reactivity. equal volumes of cultured tbev, jev, dv- , dv- , sv, and eeev were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. a cocktail of biotin conjugated antibody ( a , e , and f ) was used in all tests. the results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (figures and ) . chicken eggs inoculated with infected human serum were used for validation of the elisa-array assay. all samples showed high reaction signals with capture antibody b , which was specific for tbev ( figure b ). the elisa-array assay suggested that the three patients were all infected with tbev. to verify the results tested by elisa-array, rna extracted from chicken eggs was applied to a real time-rt-pcr assay using primers and probes targeting tbev. the results were also positive (figure a) . the consensus detection results confirmed that the elisaarray assay was reliable. to be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [ , , ] . multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. thus, an elisa-array, which combines the advantages of elisa and protein array, meets the above requirements. it has been reported that an elisa-array has been used in the diagnosis of cancer and auto-allergic disease [ , ] ; however, no study has reported the detection of viral pathogens. in this study, we developed a multiplex elisa-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens. the production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [ ] . cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. first, we prepared and screened monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. then, the antibodies were screened by an elisa-array with a double-antibody sandwich elisa format. the antibodies which produced cross-reactivity and low-positive signals were excluded. finally, six antibodies were selected as capture antibodies. another monoclonal antibody, a , which could specifically react with all viruses in the genus flavivirus was used for detecting antibody against dv, jev, and tbev. for the detection of eeev and sv, although the detecting and trapping antibodies were the same ( f and e , respectively), the antibodies produced excellent positive signals. the epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. as one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay. currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. in the elisa-array assay, this problem exists as well. because of the limitation of available antibodies, this assay could only detect pathogens. in the future, with increasing numbers of suitable antibodies, especially specific antibodies against flavivirus, this elisaarray might be able to test more pathogens and be of greater potential use. to make the assay more amenable to multiple virus detection, the assay protocol was optimized. in addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. heat inactivation was performed by heating the viral cultures at °c for h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. thus, β-propiolactone treatment was chosen as the virus-inactivation method. a conventional elisa is a standard method in many diagnostic laboratories. we compared the elisa-array with a conventional elisa and confirmed that the advantage of the elisa-array was evident with comparable specificity and higher sensitivity than elisa. the time required for the elisa-array is significantly less than for conventional elisa ( h vs. a minimum of h, respectively). furthermore, less igg is required for printing than for coating elisa plates. coating of a single well in microtiter plate requires μl of a μg/ml antibody solution, which is equivalent to ng of igg. for the elisa-array, only nl of a μg/ml antibody solution is required for each spot, which is equivalent to . ng of igg. with the characteristics of ease of use, sensitivity, specificity, and accuracy, the elisa-array assay would be widely accepted for clinical use. direct broad-range detection of alphaviruses in mosquito extracts multiplexed protein measurement: technologies and applications of protein and antibody arrays multiplexed sandwich assays in microarray format detection of adamantane-resistant influenza on a microarray a novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis advances in viral disease diagnostic and molecular epidemicological technologies assessment of some tools for the characterization of the human osteoarthritic cartilage proteome high-throughput microarray-based enzyme-linked immunosorbent assay (elisa) development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis multiplexed sandwich assays in microarray format validating a custom multiplex elisa against individual commercial immunoassays using clinical samples application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer human neutralizing sars-cov specific fab molecules generated by phage display simultaneous multianalyte elisa performed on a microarray platform a multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis c virus infection and typing of whole blood a simple and rapid protein array based method for simultaneous detection of biowarfare agent microarray immunoassay for the detection of grapevine and tree fruit viruses development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna a duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses high diagnostic accuracy of antigen microarray for sensitive detection of hepatitis c virus infection the role of biosensors in the detection of emerging infectious diseases submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was supported by the national natural science foundation of china (grant no. ) and national key research special foundation of china (grant no. zx - ). authors' contributions xk: designed the study, performed the laboratory testing, analyzed the test results, and co-wrote and edited the manuscript. h l and yl performed the virus cultures. lf performed laboratory testing. xc, fl, and gc performed real time-pcr. qz and yy organized the overall project and helped edit the manuscript. all of the authors read and approved the final manuscript. state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology, beijing , china the authors declare that they have no competing interests. key: cord- -yvsyo l authors: berger, a.; preiser, w. title: sars date: - - journal: encyclopedia of environmental health doi: . /b - - - - . - sha: doc_id: cord_uid: yvsyo l severe acute respiratory syndrome (sars) emerged in southern china in late . it first spread within guangdong province and then to other parts of china. via air travelers, it quickly reached various countries around the globe, causing several major hospital outbreaks. within weeks, the causative agent, a previously unknown coronavirus (sars-cov), was identified, thanks to an unprecedented international effort led by the world health organization (who). its origin was quickly traced to wild animals traded locally for culinary purposes. masked palm civet and some other species seem to have acted as intermediate hosts. since then, sars-like coronaviruses were found in different bat species in china and elsewhere, and bats are now regarded as the wildlife reservoir for sars-cov. fortunately, the sars outbreak could be contained within months. until july , it had caused cases, with deaths. once adequate measures such as isolating patients and quarantining their contacts were strictly adhered to, further transmission between human beings could be interrupted. sars is an example of how rapidly an infectious agent can spread in the modern world. at the same time, it should serve as a showcase of how international cooperation and modern science can help to combat the spread of infectious diseases. in an unprecedented move, who initiated a collaborative multicenter research project to identify the causative agent of sars. avian influenza was quickly ruled out. several laboratories diagnosed active infections with chlamydia or paramyxoviruses in somebut far from allpatients suffering from sars. however, there was good evidence that neither of these were plausible etiological agents for the novel infectious disease. the network comprised laboratories with access to sars patient samples and those with particular expertise on emerging or respiratory viruses. for several weeks, a spirit of cooperation rather than competition prevailed. samples were exchanged and results and findings shared continuously via daily telephone conferences and a password-protected website. within weeks, groups in hong kong, germany, canada, and the united states of america found a hitherto unknown member of the coronavirus family in sars patients. this was achieved through independent studies but certainly benefited greatly from the real-time sharing of interim results. although different approaches were employed, including cell culture, electron microscopy, and molecular techniques, sequencing data showed all laboratories had identified the same coronavirus, previously unknown to human and veterinary virology ( figure ) . it was shown that sars patients seroconverted against this virus in the course of their illness; healthy, unexposed control individuals lacked antibody reactivity. however, it remained to be proven that this novel coronavirus was indeed the etiological agent for sars rather than an 'innocent bystander' newly discovered by thorough studies. after experimental infection of macaques with the newly isolated agent was shown to cause a sars-like illness, and subsequent reisolation of the agent, all of koch's postulates had been fulfilled. on april , who officially announced that the provisionally termed sars-associated coronavirus (sars-cov) was the causative agent of sars. based on this breakthrough, tests for the detection of viral sequences and specific antibodies were quickly developed and made available to affected countries. in addition, numerous scientists embarked on programmes to develop vaccines and drugs or antibodies for prophylactic or therapeutic use. within a few months, the sars outbreak was brought under control. on july , who declared that the last chain of person-toperson transmission had been interrupted. measures including source isolation of patientswho only became infectious after onset of clinical symptomsstrict infection control in health care facilities, timely identification and quarantining of exposed contacts, and perhaps also measures to increase social distance, such as travel warnings and screening of travelers, had led to this remarkable and remarkably rapid success. thorough and consistent implementation of these measures eventually brought an end to the sars outbreak even in the worst affected areas. in the meantime, however, several areasdifferent chinese provinces other than guangdong, most prominently the capital, beijing, but also toronto in canada, and taiwanpaid a high price for not implementing adequate countermeasures in a timely fashion. typically, a so-called 'superspreader,' that is, a highly contagious sars patient, would seek treatment at a poorly prepared facility, and by the time the danger was realized, scores of staff and patients had become infected and themselves become sources of spread. interestingly, despite the rapid identification of the agent and laboratory tests becoming available almost immediately, these formidable achievements did not contribute much to the containment of the outbreak. instead, it was the prudent and thorough use of 'old-fashioned' measures such as isolation and quarantine that proved to be the key to success. identification of suspected cases was based on clinical and epidemiological criteria: high fever (> c) plus symptoms of respiratory tract infection plus an exposure history, the details of which depended on each location's sars status at the time. an additional positive sars-cov test result or radiological or pathological evidence of pneumonia or respiratory distress syndrome would make it a probable case. the final case count from november until july is , with deaths. since mid- , sars has reappeared on four occasions. three involved laboratory-acquired infections, which demonstrates the dangers of breaching biosafety procedures and the risks of subsequent further spread in the community by secondary transmission outside of the laboratory. the fourth sars outbreak was due to reintroduction from the reservoir. to minimize the risk of reemergence, who has issued guidelines for the surveillance of possible sars cases. risk categories to guide adequate national surveillance strategies to guard against the possible (re-)emergence of sars are emergence from wildlife or other animal reservoirs, emergence or introduction from laboratories or via international travel, or low risk of sars-cov emergence or introduction. who also urges all countries to conduct an inventory of all laboratories working with or storing sars-cov and to ensure strict enforcement of biosafety procedures. before sars, only two coronaviruses (hcov- e and hcov-oc ) were known to infect humans. because they both cause only relatively minor disease and are difficult to propagate in the laboratory, they received relatively little attention by diagnostic and research laboratories. in contrast, several coronaviruses were recognized as animal pathogens, infecting pigs, dogs, rabbits, cats, mice, rats, turkeys and chicken. some of these had for some time played important roles in veterinary medicine. it was obvious that the majority of human sars cases were acquired through transmission from other sars sufferers (which proved key to its eventual control). however, it seemed highly unlikely that this was a previously existing disease entity that was only then being recognized; its propensity to cause nosocomial outbreaks, often in hospital settings, would not have been overlooked for long. the very first sars cases were identified retrospectively in various localities in guangdong province. the first of these early cases occurred in foshan in november , and several more over the following weeks in nearby places. interest soon focused on these early index cases: was there any characteristic these patients had in common that could point to a source of their sars-cov infection? there was indeed; many of these early index patients either worked in kitchens or at markets or lived nearby where they were constantly exposed to a multitude of species of domestic and wild animals that were being traded, kept in captivity, and finally slaughtered and prepared for consumption. this provided the impetus for studying animals being sold at guangdong markets. infection with coronaviruses closely related to sars-cov was identified in different species, most commonly in the masked palm civet (paguma larvata) but also in the chinese ferret badger (melogale moschata) and the raccoon dog (nyctereutes procyonoides). a further, small sars outbreak occurred again in guangdong in late /early ; molecular analysis of virus isolates from human cases and animals sampled at the same place and time confirmed that this was zoonotically acquired from paguma larvata. however, further investigations revealed that this widely consumed species is most likely not the animal reservoir, as most populations studied were uninfected. later research identified a multitude of coronaviruses in different species of bats in asia and elsewhere, among them what is probably the progenitor of sars-cov. studies by different groups demonstrate that sars was the result of spillover from a wildlife reservoirmost likely batsinto an intermediate host (or hosts; most importantly paguma larvata) and from there to the human population. rapid viral evolution was demonstrated and most likely was the pivotal factor that allowed sars-cov to rapidly adapt to nonreservoir species. table shows the distribution of several different coronaviruses in humans and animals and their classification into different groups. disease caused by sars-cov may present with rather nonspecific clinical signs and symptoms. the differential diagnosis is therefore wide and may include various common respiratory pathogens, including influenza, parainfluenza, and respiratory syncytial viruses (rsv) the laboratory diagnosis of sars remains a challenge; in fact, despite the rapid identification of sars-cov as the etiological agent, testing contributed little to the successful control of the outbreak. insufficient test specificity on occasions caused false-positive results, leading to considerable confusion. in many viral diseases, virus shedding is greatest during the early symptomatic phase, that is, around, and immediately following the onset of symptoms. unfortunately, virus excretion is comparatively low during the initial phase of sars. it peaks in respiratory specimens and in stools at around day after the onset of the clinical illness. in addition, there are currently no laboratory tests available to reliably diagnose sars in the first few days of illness. the highest test sensitivity is achieved with bronchoalveolar lavage fluid or lung biopsy tissue at the onset of illness. because of the invasiveness of such procedures and the associated risk of transmission, nasopharyngeal aspirates and throat washings, taken with respiratory precautions and preserved in viral transport medium, remain the most important diagnostic specimens. most commonly, viral genome detection, usually by reverse transcriptase-polymerase chain reaction (rt-pcr), is used diagnostically. nucleic acid amplification tests have been designed targeting the orf b or nucleoprotein genes; it has not been clearly proven in clinical studies that they are superior. real-time rt-pcr of nasopharyngeal aspirates is the most sensitive and rapid method. furthermore, the determination of viral load in nasopharyngeal specimens or serum has been shown to be of clinical value, as it is an important prognostic factor. to avoid false-positive results, who stipulates that the following should be tested: at least two different clinical specimen types (e.g., nasopharyngeal aspirate and stool), or the same type of clinical specimen but collected on at least two occasions during the course of the illness (e.g., sequential nasopharyngeal aspirates), or the same original clinical sample but two different assays or by repeating the same assay but using a new rna extract for each test. sars-cov is cultivable on vero and caco cells not only from respiratory materials but also from fecal samples ( figure ). once isolated, the virus must be identified as sars-cov using further tests. cell culture is a very demanding test, but currently (with the exception of animal trials) the only means to show the existence of a live virus. antigen detection in respiratory and fecal specimens using enzyme immunoassay (eia) is generally less sensitive than rt-pcr. however, antigen detection in serum specimens with monoclonal antibodies or monospecific polyclonal antibody against the viral n protein was found to be a sensitive and specific test for the diagnosis of sars. as serum antibody levels start to rise from day after onset of illness, the sensitivity of the serum antigen assay progressively decreased to % at day . antibody testing allows the indirect diagnosis of sars-cov infection and is unsuitable during the acute illness. positive antibody test results indicate previous infection with sars-cov. seroconversion from negative to positive or a fourfold rise in the antibody titer from acute to convalescent serum indicates a recent infection. a negative antibody test result later than days after the onset of illness is likely to indicate that no infection with sars-cov has taken place. virus-specific serum igg, igm, and iga antibodies against sars-cov appear at around the same time, between days and after the onset of symptoms. antibody testing is therefore generally not useful during the first week of illness. for antibody testing, the indirect immunofluorescent antibody test is more commonly performed than the neutralizing antibody test in cell cultures that again requires a biosafety level laboratory (figure ) . a recombinant nucleocapsid eia may be used as a rapid screening test and possesses a higher sensitivity, with detection as early as day after onset of illness. single low-titer positive antibody results can be due to cross-reactivity with other human coronaviruses and require confirmation by more specific western blotting or neutralization assays. who regards seroconversion or an at least fourfold rise in antibody titer between an acute and a convalescent serum as proof of infection. the updated who guidelines for the global surveillance of sars of october replace all previous who guidance on sars surveillance and response. one key recommendation is that independent of the test used, who strongly recommends that during the interepidemic period all countries seek verification of laboratory-confirmed cases of sars ('preliminary positive' cases), preferably by an external laboratory that is part of the who sars international reference and verification laboratory network (figure ) . human sars-cov are no longer circulating in the human population, nevertheless the virus is still present in the animal reservoir and virological laboratories and can reemerge anytime. there is no single test that can be used to diagnose sars with a reasonable degree of accuracy. diagnosis, therefore, continues to rely on the clinical examination, supported by case definitions that include the risk assessment according to the updated who guidelines (tables and ). the initial symptoms of sars are nonspecific, complicating the differential diagnosis. the mean incubation period is days with the range of - days although there are isolated reports of longer incubation periods. unlike influenza virus, where the patients are most infectious in the first days of illness, transmission from symptomatic sars patients usually occurred on or after the fifth day of onset of disease, which is in line with the rising viral load in nasopharyngeal secretions that peaked at around day . there have been no reports of transmission occurring before the onset of symptoms. the typical clinical presentation of sars is that of viral pneumonia with rapid respiratory deterioration. patients initially develop influenza-like prodromal symptoms. fever, malaise, myalgia, headache, rigors, and nonproductive cough are the major presenting symptoms, whereas rhinorrhea and sore throat are less frequently seen. clinical deterioration, often accompanied by watery diarrhea, commonly occurs week after the onset of illness. although history of fever is the most frequently reported symptom, it may be absent on initial measurement. severe cases develop rapidly progressing respiratory distress and oxygen desaturation with approximately % requiring intensive care. chest radiographs typically show ground-glass opacities and focal consolidations, especially in the periphery and subpleural regions of the lower zones. progressive involvement of both lungs is not uncommon. features during the later stages have sometimes included spontaneous pneumothorax, pneumomediastinum, subpleural fibrosis and cystic changes. nosocomial transmission of sars-cov has been a striking feature of the sars outbreak. the majority of the cases have occurred in adults. children are less commonly affected than adults and usually have a milder illness. the overall mortality rate was approximately %. age and the presence of comorbidities are poor prognostic indicators. although the sars outbreak was still ongoing during the first half of , significant funding was made available for sars-related research. immunomodulators (i.e., corticosteroids, intravenous immunoglobulins, thymosin, and anti-tnf) were empirically used for the treatment of sars during the initial epidemic. the correlation between viral load and clinical outcome suggests that suppression of viral replication by effective antiviral drugs should be the key to preventing morbidity and mortality. numerous potential antiviral agents have been identified using different approaches. in vitro susceptibility test results demonstrate that ifn-alpha and ifnbeta have some potential activity. ribavirin has good activity when tested in human caco- cells despite its lack of activity in vero cells. the viral proteases are important targets for the development of antiviral drugs. protease inhibitors like nelfinavir, glycyrrhizin, chloroquine, and many others as well as many herbal formulations have been found to possess some antiviral activity against sars-cov in vitro. in addition, the use of nitric oxide (s-nitro-n-acetylpenicillamine) inhalation as an experimental form of rescue therapy for sars appeared to have inhibitory activity against sars-cov. screening of chemical libraries has identified several inhibitors of the viral protease and helicase. identification of angiotensin-converting enzyme (ace ) as an obligatory cellular receptor for table risk categories for the emergence of sars emergence of sars-cov-like viruses from wildlife or other animal reservoirs -countries/areas identified as source(s) of the epidemic in - in southern china or areas with an increased likelihood of animal-to-human transmission of sars-cov-like viruses from wildlife or other animal reservoirs. emergence or introduction of sars-cov from laboratories or international travel -countries/areas at potentially higher risk of sars-cov emergence or introduction due to the presence of laboratories in which sars-cov or sars-covlike viruses are being studied or in which clinical specimens infected with sars-cov are being processed or stored. or -countries/areas with entry of large numbers of persons from areas in which wildlife or other animal reservoirs of sars-cov-like viruses are found. low risk of sars-cov emergence or introduction -countries/areas that never reported cases or reported only imported cases during the - epidemic, and that do not conduct research using live sars-cov-like viruses or store clinical samples from sars cases. sars-cov contributed to understanding of the sars-cov entry process, and helped to characterize two targets of antiviral therapeutics: the sars-cov spike protein and ace . however, most of the chemicals or approaches have not been evaluated in human or animal models. various approaches toward producing a vaccine against sars have been pursued, including the use of inactivated sars-cov, plasmid dna, and adenovirus vectors. one obvious problem any vaccine would face is whom it should be given to, in the absence of sars-cov transmission. however, waiting until a renewed outbreak occurs before commencing vaccination means that precious weeks would be lost until individuals at risk become immune. the administration of preformed antibodies (obtained from human or animal donors or, more recently, produced in vitro) is effective in preventing a number of different infections, such as hepatitis b, varicella, and rsv. similarly, it could be shown that neutralizing antibodies against sars-cov can protect experimental animals from infection. using screening of a large naive antibody library by antibody phage display technology, neutralizing antibodies were identified and produced that were protective in vitro. there was no evidence of enhancement of sars-cov infection by subneutralizing concentrations of these antibodies, and immune escape mutants were not generated. in theory, this could be an ideal tool: if sufficient stocks of such human monoclonal antibodies could be procured, they would be ready for use when and wherever sars reemerges. one could then passively immunize anyone in contact with the source or patients, affording immediate protection. in summary, sars was a novel severe infectious disease that presumably originated in guangdong in southern china. large-scale wildlife trade and consumption favored the emergence of this zoonosis from a hitherto unrecognized animal reservoir. after its zoonotic origins, the new agent quickly spread within the human population, being transmitted mostly via the respiratory route through close human-to-human contact. sars was rapidly disseminated via the metropolis hong kong through international air travel. its important potential for nosocomial transmission in the community and in hospitals was soon recognized and allowed for appropriate measures to be instituted in most places. there was an unprecedented rapid gain of knowledge through global networking and international collaboration, led by who. unfortunately it took some major outbreaks, some of them affecting industrialized countries with modern health care systems, until the first sars epidemic was controlled. thus in the end rapid success was achieved through 'traditional' sanitary measures, mainly rapid identification of suspect cases and isolation of the diseased. fortunately, sars-cov hasat least so farnot established itself permanently in the human population. its relatively low transmissibility (with the exception of 'superspreaders') led to a low basic reproduction number r ; together with the fact that infected individuals only become infectious themselves once they have developed clinical disease, this made 'traditional' public health measures very effective. is the fact that the first pandemic of the twenty-first century was quickly contained reason to take comfort in the knowledge that the history of humankind has reached a phase in which biomedical sciences and information technology are able to deal with such threats? probably not. first, the rapid success was remarkable. unfortunately, though, this does not reflect preparedness. in , donald burke proposed the following criteria for identifying virus families with a high pandemic risk: ( ) those that had caused pandemics in human populations in the recent past; ( ) those with the proven ability to cause larger epizootics; and ( ) those with an intrinsic propensity to rapidly undergo evolution on the basis of high mutation rate or genome organization favoring recombination ('intrinsic evolvability'). interestingly, even before the sars outbreak (which obviously fulfills the first criterion), the coronavirus family should clearly have been regarded as high risk: starting in the late s, the porcine epidemic diarrhea coronavirus (pedv) caused a severe swine epizootic in europe and asia (second criterion). the high evolvability of the coronavirus family had also been demonstrated: the porcine respiratory coronavirus (prcv) evolved through a deletion mutation in the s gene from the transmissible gastroenteritis virus (tgev); the mutant has a different tissue tropism and is less virulent. second, infectious disease emergence from zoonotic sources was a well-recognized threat long before sars. nevertheless, it took anotherfortunately minor -sars outbreak before decisive steps were taken to control at least the trade in the directly implicated paguma larvata in southern china. starting shortly after sars had been controlled, another agent with pandemic potential has caused an ongoing epizootic of unprecedented proportions: highly pathogenic influenza a (h n ) virus. transmission to humans occurs almost exclusively through close contact with infectedand sickpoultry. despite the possibility that this avian influenza virus will become fully 'humanized' and trigger the next influenza pandemic, even activities recognized as high risk, such as 'wet markets,' have mostly not been curtailed. finally, the lessons from sarsparticularly the experiences of beijing, toronto, and taiwan that suffered devastating sars outbreaks during the later phase of the outbreakemphasize over and over again the enormous importance of open and timely communication. at times, this may mean admitting to problems that (at least initially) reflect negatively on the country or territory concerned. in the medium to long term, however, such openness is the only way to prevent consequences that would be much worse. although this was widely accepted in the wake of the sars experience and has had a strong influence on the new international health regulations that entered into force in june , the information policies of some countries during the h n epizootic unfortunately do not attest to this at all. notwithstanding its grave consequences in humanitarian, political, and economic terms, sars should serve as an example what can be achieved through international cooperation, modern science, and rigorous use of 'traditional' approaches. even if prudent precautions were suddenly adopted, sars will certainly not have been the last highly pathogenic novel infectious agent that crosses the species barrier into the human population. although bats must be very high on the list of 'culprits' for emerging viral diseases, having been identified as reservoir hosts for a number of emerging viruses, there is no reason why other groups of animals should not figure as prominently in future. the mechanisms behind emergence have been studied. these may be linked to the agent, the new host species (i.e., human beings and in certain cases also the intermediate hosts) or to the connection between those. many of the factors and determinants of disease emergence are related to human activities. although this has accompanied humankind throughout history, and in fact may have helped shape human history, the speed and the extent of human-induced changes has accelerated markedly in recent times. although in the case of sars people were lucky in the end, this may be quite different next time round. it will be important to improve understanding of emergence to minimize the risks of what is a natural phenomenon but much aggravated by human behavior. severe acute respiratory syndrome (sars)Àparadigm of an emerging viral infection the evolvability of emerging viruses development of antiviral therapy for severe acute respiratory syndrome severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses viral zoonosesÀa threat under control? human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets bats, civets and the emergence of sars wet marketsÀa continuing source of severe acute respiratory syndrome and influenza? relevant websites www.cdc.gov/eid. emerging infectious diseases: (search for sars) health protection agency (uk) information and guidance on sars released by the world health organization (who) international office of epizootics key: cord- - zjqflhz authors: sØrensen, morten drÆby; sØrensen, brian; gonzalez‐dosal, regina; melchjorsen, connie jenning; weibel, jens; wang, jing; jun, chen wie; huanming, yang; kristensen, peter title: severe acute respiratory syndrome (sars): development of diagnostics and antivirals date: - - journal: ann n y acad sci doi: . /annals. . sha: doc_id: cord_uid: zjqflhz abstract: the previously unknown coronavirus that caused severe acute respiratory syndrome (sars‐cov) affected more than , persons worldwide and was responsible for more than deaths during the first outbreak in – . for reasons unknown, the sars virus is less severe and the clinical progression a great deal milder in children younger than years of age. in contrast, the mortality rate can exceed % for persons at or above the age of . as part of the sino‐european project on sars diagnostics and antivirals (sepsda), an immune phage‐display library is being created from convalescent patients in a phagemid system for the selection of single‐chain fragment variables (scfv) antibodies recognizing the sars‐cov. in february , the new and previously unknown deadly coronavirus causing severe acute respiratory syndrome (sars-cov) was brought to the attention of the world health organization (who) by dr. carlo urbani and his colleagues. the sars virus originated in the province of guangdong in southern china in november where it initially was thought to cause atypical pneumonia. however, within a short time the virus spread to hong kong, singapore, vietnam, canada, the united states, taiwan, and several european countries. concerted efforts of the scientific community led to a very rapid identification of a novel coronavirus as the etiological agent of sars and the full genome sequencing of the virus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sars-cov genome is ∼ kb in length and contains potential open reading frames (orfs). , according to the who, the sars-cov affected more than , individuals worldwide and was responsible for over deaths during the first outbreak in - . for reasons unknown the sars virus is less severe and the clinical progression a great deal milder in children younger than years of age. in contrast, the mortality rate was highest among patients > years and can exceed % for persons at or above the age of years. in hong kong, where people died from sars, the mortality rate for children (age - years) was %. on the other hand, . % of the cases were in persons older than years, most of whom showed a history of chronic disease (http://www.hku.hk/ctc/sars hk ). at present no experimental evidence can explain the observed age distribution. however, it should be noted that a recently discovered coronavirus strain, nl , exhibits a markedly different age distribution with regard to clinical symptoms. the coronaviruses are a group of viruses that have a crown-like (coronal) appearance. the sars-cov are positive-strand rna viruses and the virion consists of a nucleocapsid core encapsulated by the three envelope glycoproteins: spike (s), membrane (m), and envelope (e) proteins that are common to all members of the genus. the rna is packaged by the nucleocapsid (n) protein into a helical nucleocapsid. the sars virus n protein has only a % identity with the other known coronaviruses and has been suggested to be a major immunogen. the s protein is known to be a major target of the cellular immune response and plays an important role in the initial roles of infection. continuous work is being performed to find an early diagnosis and therapy of sars. several different approaches have been taken. diagnosis has been performed by serologic testing using indirect fluorescent antibody or enzymelinked immunosorbent assays (elisa) specific for sars-cov antibody. , detection of the sars-cov itself has been done using clinical specimens of serum, nasal secretions, and stool. this was done through viral isolation and electron microscopy, viral culture, or reverse transcription polymerase chain reaction (rt-pcr) to test for viral rna. , neutralizing antibodies (nabs) have been found against the sars virus. one of the targets of these nabs is the s glycoprotein, especially toward the metallopeptidase angiotensin-converting enzyme (ace ) binding domain [s( - )]. nabs have been selected against the ace domain. , within the sepsda consortium significant progress has been made, with regard to both a structural understanding of the sars-cov - and the development of putative therapies. to further increase the understanding of viral biology and to help devise novel therapies, an effort to harvest the protective immunity generated by convalescent patients has been initiated. antibodies against the sars proteins can be obtained in different ways. the most commonly used are hybridomas to make monoclonal antibodies and immunization followed by collection of antiserum. selections using phage display have been performed, selecting scfv antibodies from semi-synthetic (nonimmune) libraries - and with immune library. the first time a fusion protein was displayed on the surface of filamentous bacteriophages was in by g.p. smith, who showed that foreign dna fragments could be inserted in the middle of gene iii to create a fusion protein. the phage particle displays a protein or peptide on the surface and carries the gene for the displayed protein or peptide inside the particle, giving a linkage between phenotype and genotype. this allows for the selection of phage displaying a protein or peptide with affinity for a given target, and at the same time the gene encoding the protein or peptide is co-selected. in this way, it is possible to screen millions of different displayed proteins or peptides. in a single-chain antibody fragment (scfv) was displayed on the surface of filamentous bacteriophage for the first time by mccafferty et al. in came the first publications displaying libraries of fragment antigen-binding (fab) fragments on giiip , and on gviiip. subsequently, better and larger libraries have been constructed and used for selection of antibodies against numerous different antigens. the creation of large phage-display libraries gives the potential of isolating human antibodies against most antigens, making it possible to bypass both hybridoma technology and immunization. in addition, because no immune system is involved in the selection of antibodies by phage display, it is possible to select antibodies against toxic compounds, lethal pathogens, and highly conserved antigens. in general two kinds of antibody libraries can be created from donors: immune and naïve (nonimmune) libraries. the immune libraries are made from immunoglobulin (ig) variable region (v) genes derived from immunized donors. an immune library is biased toward a specific antigen, which leads to the selection of higher-affinity antibodies (compared to naïve libraries of the same size). the naïve libraries are made from ig v genes derived from nonimmunized donors or from synthetic v-genes. the naïve library is unbiased and can therefore select antibodies against virtually any antigen. creation of immune phage-display libraries for immunized donors has shown a particular efficiency in selecting neutralizing antibodies (nabs) against different viruses, for example, rabies, varicella-zoster, hepatitis a and e, measles, and respiratory syncytial virus. the use of phage display seems ideal for the selection of antibodies against the sars-cov. creating an immune library, based on peripheral blood lymphocytes from convalescent patients in a phagemid system, would provide the possibility of developing an early diagnosis and therapy of sars. nab has been selected, but an early and fast diagnosis is still important, should another outbreak occur. against s domain at n-terminal residues to of sars-associated coronavirus s protein. severe acute respiratory syndrome (sars): multi-country outbreak pneumonia causes panic in guangdong province identification of a novel coronavirus in patients with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus mechanisms and enzymes involved in sars coronavirus genome expression severe acute respiratory syndrome among children severe acute respiratory syndrome epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains linkage of recognition and replication functions by assembling combinatorial antibody fab libraries along phage surfaces by-passing immunization: human antibodies from v-gene libraries displayed on phage making antibody fragments using phage display libraries mating antibody phage display with proteomics the human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries neutralizing human antibodies to varicella-zoster virus (vzv) derived from a vzv patient recombinant antibody library neutralizing human monoclonal antibodies to hepatitis a virus recovered by phage display monoclonal antibodies that neutralize hev recognize an antigenic site at the carboxyterminus of an orf protein vaccine neutralizing human fab fragments against measles virus recovered by phage display efficient generation of respiratory syncytial virus (rsv)-neutralizing human moabs via human peripheral blood lymphocyte (hu-pbl)-scid mice and scfv phage display libraries key: cord- -lxa pvt authors: pelfrene, e.; mura, m.; cavaleiro sanches, a.; cavaleri, m. title: monoclonal antibodies as anti-infective products: a promising future? date: - - journal: clinical microbiology and infection doi: . /j.cmi. . . sha: doc_id: cord_uid: lxa pvt abstract background the paucity of licensed monoclonal antibodies (mabs) in the infectious diseases arena strongly contrasts with the ready availability of these therapeutics for use in other conditions. aims this narrative review aims to assess the potential of monoclonal antibody-based interventions for infectious diseases. sources a review of the literature via the medline database was performed and complemented by published official documents on licensed anti-infective mabs. in addition, ongoing trials were identified through a search of the clinical trial registration platform clinicaltrials.gov. content we identified the few infections for which mabs have been added to the therapeutic armamentarium and stressed their potential in representing a readily available protection tool against biothreats and newly emerging and reemerging infectious agents. in reviewing the historical context and main features of mabs, we assert a potentially wider applicability and cite relevant examples of ongoing therapeutic developments. factors hindering successful introduction of mabs on a larger scale are outlined and thoughts are offered on how to possibly address some of these limitations. implications mabs may represent important tools in treating or preventing infections occurring with reasonably sufficient prevalence to justify demand and for which existing alternatives are not deemed fully adequate. future initiatives need to address the prohibitive costs encountered in the development process. the feasibility of more large-scale administration of alternative modalities merits further exploration. in order to ensure optimal prospect of regulatory success, an early dialogue with competent authorities is encouraged. in , bezlotoxumab became the second monoclonal antibody authorized for use throughout the european economic area directed against an infectious agent. it targets clostridium difficile toxin b, and as such aims to prevent recurrences of the infection in adults at high risk of repeated bouts [ ] . this follows a much earlier eu regulatory approval of palivizumab (in ), which is administered to prevent serious lower respiratory tract disease requiring hospitalization caused by respiratory syncytial virus (rsv) in children at high risk for the disease [ ] . licensing of these two monoclonal antibodies (mabs), nearly two decades apart, illustrates the gap to date in clinical development of this therapeutic tool against infections. this is consistent with the experience of other regulatory authorities, such as the us food and drug administration (fda), which granted similarly phrased labels in the united states for both products [ , ] . additionally, the fda recently licensed ibalizumab as a rescue therapy in heavily treatmentexperienced adults with multidrug-resistant hiv- infection and also previously approved raxibacumab (in ) and obiltoxaximab (in ), both intended for treatment of inhalational anthrax (in combination with appropriate antibacterial medicines) and for prophylaxis when alternative therapies are not available or are not appropriate [ e ] . the latter two products were assessed by fda according to its 'animal rule,' largely relying on efficacy data generated in a relevant animal model [ e ] . in general, the use of mabs is viewed as an attractive biodefense measure, countering deliberate threats [ ] . the technology platform may also offer a rapid protection against newly emerging and reemerging infectious agents when no other treatment or prophylaxis is available. witness to this, for example, is the ongoing research in mabs to treat patients with middle east respiratory syndrome [ ] . the experimental use of a tobacco plantederived mixture of mabs (zmapp) during an ebola virus disease outbreak in western africa provides an additional example in this respect [ , ] . interestingly, the paucity of currently licensed mabs in the infectious diseases arena strongly contrasts with the ready availability of these therapeutics for use in other conditions, mainly oncology and chronic inflammatory or rheumatic afflictions. indeed, for these disease areas, mabs globally generate a sizeable yearly income [ ] . this narrative review revisits the potential applicability of mabs in targeting infections and outlines their prospects for successful therapeutic development. we undertook a search on medline complete regarding trials and reviews for antibody-based therapeutics in infectious diseases. the terms used in different combinations were as follows: 'therapeutic antibodies,' 'monoclonal' and 'infectious diseases,' as well as pathogen-specific terms. the results were limited to englishlanguage reports and surveyed documents published from january to march . our methodology was further complemented by published official documents on anti-infective mabs licensed to date by eu and us regulatory authorities. some of the trials were identified through a search of the clinical trial registration platform clinicaltrials.gov. historically, immune sera were used to treat or prevent infectious diseases before the availability of vaccines and antibiotics, with polyclonal sera (hyperimmune sera) still being used against conditions such as rabies, diphtheria, tetanus and botulism [ ] . these sera are typically administered when rapid protection is required, following known exposure in an unvaccinated individual (or vaccination is nonexistent) or when previously acquired immunity has waned. as with hyperimmune sera, mabs can be deployed irrespective of preexisting immune protection status and equally may benefit the immunocompromised host [ , ] . however, they avoid some of the disadvantages attributed to sera derived immunoglobulins, such as potential (exceedingly low) transmission of infectious hazards and batch variability [ ] . over the years, monoclonal antibody engineering has evolved. the first licensed monoclonal antibody, okt , indicated to prevent acute kidney transplant rejection, used a murine hybridoma technique [ , ] . early commercial successes with mabs were limited, however, by the availability of suitable myeloma cell lines. also, occurrence of adverse immune reactions, by provoking a human anti-mouse antibody response, is a recognized setback for use of the technique. to limit such unwanted effects whilst also enhancing effector function, chimeric (mouseehuman) antibodies first replaced murine hybridomas and more recently humanized (i.e. antibody structure containing less than % nonhuman sequences) and fully human mabs have been developed [ , ] . new expression tools became available, such as phage display libraries, transgenic mice, chinese hamster ovary (cho)-based biomanufacturing and highly scalable plant technology, as well as the possibility to produce mabs using human b cells isolated from subjects in convalescence or after vaccination [ e ]. thus, current methods have made production more efficient, and importantly, the use of human and humanized mabs poses fewer safety concerns as opposed to older techniques and serum therapy [ , ] . with exquisite specificity being the hallmark, mabs generally harbour a low potential for off-target adverse reactions [ ] , although it makes them vulnerable to escape mutants, potentially rendering this therapeutic measure ineffective [ ] . hence, this drawback justifies the development of mabs cocktails, with the antibody constituents binding to different epitopes [ , ] . also, alternative antibody formats have been engineered, simultaneously addressing different targets involved in pathophysiologic processes. as such, bispecific antibodies aim to ensure enhanced potency and breadth of protection [ e ]. other developments include antigen-binding (fab) fragments, single chain variable fragments (scfv) and pairs linked in different ways, as to create formats combining optimal size, half-life, activity and safety [ , ] , as well as novel delivery systems such as antibodyeantimicrobial conjugates and radioimmunoconjugates [ , ] . effector functions can be modified: fc domain changes may enhance the affinity to fc receptors on phagocytic cells, nk cells and b cells, and hence enforce antibody-dependent killing, whilst mutations leading to increased binding affinity for neonatal fc receptor (fcrn) contribute to an extended half-life of the antibody [ ] . in principle, mabs could target a wide range of biologic agents, encompassing bacterial and viral pathogens, fungi and associated toxins, with their action exerted either directly (e.g. preventing cell entry or neutralizing toxins) or via indirect mechanisms (e.g. modulating inflammatory responses or promoting opsonic phagocytosis) [ , , ] . to date, licensed antibacterial mabs target exotoxins, with the antigeneantibody complexes primarily removed via the reticuloendothelial system [ , , , ] . instead, mabs may bind to structural cell surface components (proteins and exopolysaccharides), with subsequent direct bactericidal clearance or immune system dependent cytotoxicity (antibody or complement dependent) [ ] . in this regard, the pharmacodynamic mode of action varies amongst the candidate products currently under development [ ] . notwithstanding previous disappointment in translational research [ ] , activities which target staphylococcus aureus and pseudomonas aeruginosa are the most developed so far (b. françois et al., 'safety and tolerability of a single administration of ar- , a human monoclonal antibody, in patients with severe pneumonia caused by staphylococcus aureus: first in man trial,' abstract , paper presented at european congress of clinical microbiology and infectious diseases ) [ , ] . indeed, in an era of increased awareness and intensified effort to mitigate the antimicrobial resistance threat, mabs may offer a welcome addition to the therapeutic toolkit [ ] . although unlikely to compete directly with conventional antimicrobials in treating serious bacterial infections, mabs might act synergistically if both measures are administered concurrently. to date, promising results to that effect have been shown in animal models [ , ] . further on, antibody administration is expected to provide less selective pressure for cross-resistance and may preserve the gut microflora [ , ] dan important consideration in view of our expanding knowledge on the role of the human gut microbiome in both health and disease [ ] . nevertheless, the success for a wide introduction of mabs in treating established bacterial infections will be largely dependent on ready and specific pathogen identification. this necessitates further investment and research into validated point-ofcare rapid diagnostics (e.g. pcr-based and fluorescence in-situ hybridization technologies), allowing prompt targeted therapy [ ] . also, because the effect of a single dose can last for one to a few months, the use of recombinant antibodies as prophylaxis could theoretically allow for a decrease in conventional antimicrobial usage. hence, in recent efforts, clinical development programmes intend to demonstrate that mabs against bacterial pathogens will protect at risk patients (e.g. prevention of s. aureus ventilatorassociated pneumonia in colonized, mechanically ventilated patients in the intensive care unit) (clinicaltrials.gov nct , nct ) [ ] . mabs may provide a broad neutralizing, potent activity against highly conserved viral epitopes. alternatively, the antibodies can target receptors and coreceptors located on host cells [ ] . with research still in infancy, mabs which recognize virally infected cells and induce antibody-dependent cytotoxicity may offer an additional mechanism of action [ ] . hitherto, mabs have been licensed as hiv rescue therapy in adults and as an rsv infection preventive in high-risk children [ , ] . whilst ibalizumab targets the conformational epitope of the cd t-cell receptor, blocking cell entry by hiv- , other candidates under development, either also target host antigens or alternatively directly bind hiv- antigens [ e ]. research further includes the promise of bispecific and trispecific broadly neutralizing antibodies [ , ] . with regard to rsv, palivizumab interferes with virus attachment and fusion by binding rsv f protein [ ] . presently rsv research is focused on extending the half-life of rsv mabs, aiming to protect infants throughout their first rsv season with a single dose administration [ ] . other currently studied viral targets include influenza, ebola and zika [ ] . recent viral outbreaks have brought a heightened awareness to prepare against emerging infections [ ] . for such threats, mabs could potentially be indicated as treatment of infected individuals and could as well provide targeted prophylaxis for individual protection and transmission interruption (possibly changing the dynamics of a nascent epidemic) [ ] . for example, within the context of ongoing product development targeting influenza, for which promising results were obtained in ferret models and early clinical development, broadly neutralizing mabs might be suitable at the emergence of a pandemic outbreak, at least for specific at-risk groups such as exposed contacts and healthcare professionals [ e ]. in that scenario, they could be used to bridge the time gap before availability of strain-specific vaccines [ ] . with a potential to provide a comparably efficacious product, the replacement of currently indicated hyperimmune sera by mabs deserves to be explored, taking into account feasibility issues related to their clinical development [ ] . for example, in the face of periodic outbreaks of diphtheria in various world regions, a global short supply of equine diphtheria antitoxin provides new impetus in developing a suitable monoclonal antibody alternative [ ] . so far, though, activities have been most advanced in respect to rabies, for which at least two different products are considered [ ] . for one of these, a single human monoclonal antibody (sii-rmab), a recently completed noninferiority controlled trial concluded it as a potential safe and potent alternative to human rabies immunoglobulin-containing postexposure prophylaxis regimen [ ] . as illustrated by the ongoing efforts, anti-infective mabs constitute a thriving focus of research, but in order to become a success, multiple challenges need to be overcome. first, technical barriers remain. for instance, proper selection of bacterial targets remains fraught with uncertainties: highly conserved outer membrane proteins may be masked and hence unavailable for binding with antibodies; conversely, epitopes located on exopolysaccharides are typically not conserved (i.e. different serotypes exist), posing limitations to the effectiveness of a targeting single monoclonal antibody [ ] . moreover, bacterial defence mechanisms developed in response to host immunoglobulins (e.g. production of antibody degrading proteinases) can impede the success of antibody-based therapy. for viral infections, a single lasting treatment is hampered in the face of multiple strains, rapid evolution and selection of escape mutants [ ] . in addition, for some pathogens, viraemia often peaks before appearance of symptoms [ , ] . in these circumstances, rapid point-of-care diagnostics may be fundamental to the success of the intervention, allowing prompt therapy initiation in those most at risk of developing serious illness [ , ] . second, regulatory product approval pathways are not well optimized for emerging threats. indeed, a small number of patients and unpredictable outbreak dynamics may impede the conduct of confirmatory controlled clinical trials. also, human challenge studies can provide supportive evidence of protection for only a limited number of conditions [ , ] . in the absence of adequate risk mitigation, such an approach is not expected to be feasible for most of the emerging infections [ , ] . alternatively, however, for those threats lacking sustained human-to-human transmission, assessment could be based mainly on data obtained from animal challenge studies and on pharmacokinetic/ safety evaluation of the product in healthy volunteers, on the condition that animal models can be established that are sufficiently representative of human disease [ , ] . the availability of protocols to test the effectiveness and safety of the therapeutic in the field at the time of an outbreak would then probably constitute a condition to the licensing. third, business models and potential markets do vary depending on the disease target, but on the whole, investment and product development are hindered by lack of clarity on how to position the use of mabs amongst preventive and therapeutic options [ ] . uncertainties remaindfor instance, in selecting appropriate trial endpoints and delineating populations best suited to the intervention [ ] . additionally, development is constrained by high investment expenses amidst unsure future returns. appropriate pricing is considered a prerequisite for market success, and hence increased streamlining, optimization and innovative processes (e.g. quality-by-design) may exert downward pressure on costs. moreover, research into new delivery methods should be expanded and intensified, ensuring convenient administration to a large number of people. this may entail the development of novel intramuscular and inhalation formulations. in this respect, dna plasmid-delivered mabs produced by muscle cells in vivo are singled out as a potential way to circumvent some of the limitations in immunoglobulin g administration [ ] . next-generation mabs might offer further opportunities, even allowing a single product to target multiple pathogens [ , ] . from a regulatory viewpoint, tools are available in the european union to ensure a swift evaluation process and early access to the market, including specific scientific guidance or dedicated advice to companies (table ) [ ] . in this regard, obtaining and complying with european medicines agency's scientific advice has shown to be predictive of regulatory success [ ] . concerning emerging infections, broad stakeholder engagement (with national and international public health agencies, private sector, funding organizations and regulatory authorities) has been advocated. it is argued that this may entail the creation of an independent global agency, as to develop and implement a coherent strategy for global biopreparedness [ ] . although research and clinical development of mabs has intensified, it remains to be proven if a new dawn is on the horizon for this product class to become commonplace in the fight against an array of infections. inroads have been made so far, as illustrated by the few licensed mabs against infectious agents or associated toxins. however, for a broader anti-infective potential to be fully exploited, scientific, regulatory and commercial barriers to development need to be addressed. amongst other initiatives, an early dialogue with regulatory authorities is encouraged to ensure streamlined development and to enable early access for patients. orphan drug: medicine for diagnosis, prevention or treatment of a lifethreatening or chronically debilitating condition that is rare or where medicine is unlikely to generate sufficient profit to justify research and development costs. incentives apply (e.g. fee reductions). prime priority scheme: support for development of medicines of major interest that target unmet medical need continuous support and early interactions with reviewers; sa at key development milestones involving additional stakeholders such as hta. potential for combination with one or several early access tools at time of maa. early access accelerated review reduced evaluation time frame rapid assessment of medicines that are of major interest for public health, especially ones that are therapeutic innovations (unmet medical need). conditional maa earlier authorization of medicines for patients with unmet medical needs, on basis of less complete clinical data eligibility includes medicines for seriously debilitating or life-threatening diseases, emergency situations, orphan drugs. comprehensive data are expected to be generated after authorization within agreed time frame but risk/benefit ratio is clearly positive at time of approval. compassionate use benefits seriously ill patients who cannot be treated satisfactorily or cannot enrol in ongoing clinical trials pertains to unauthorized medicinal products for chronically, seriously debilitating or life-threatening diseases, with no satisfactory treatment authorized in eu; targeted at a group of patients rather than individual; or undergoing centralized maa or clinical trials ema, european medicines agency; fda, us food and drug administration; hta, health technology assessment bodies; maa, marketing authorization application; sa, scientific advice. european medicines agency; committee for medicinal products for human use. zinplavadepar summary for the public european medicines agency; committee for medicinal products for human use. synagisdepar summary for the public (ema/ / ) drugs@fda. fda approved drug products: zinplava drugs@fda. fda approved drug products: synagis drugs@fda. fda approved drug products: trogarzo drugs@fda. fda approved drug products: raxibacumab drugs@fda. fda approved drug products: anthim product development under the animal rule: guidance for industry raxibacumab for the treatment of inhalational anthrax animalto-human dose translation of obiltoxaximab for treatment of inhalational anthrax under the us fda animal rule antibodies for biodefense treatment strategies for middle east respiratory syndrome coronavirus clinical care of two patients with ebola virus disease in the united states multi-national prevail ii study team. a randomized, controlled trial of zmapp for ebola virus infection the therapeutic monoclonal antibody market therapeutic antibodies for infectious diseases mortality and morbidity among infants at high risk for severe respiratory syncytial virus infection receiving prophylaxis with palivizumab: a systematic literature review and meta-analysis bezlotoxumab for prevention of recurrent clostridium difficile infection history of passive antibody administration for prevention and treatment of infectious diseases therapeutic use of okt monoclonal antibody for acute renal allograft rejection monoclonal antibody therapeutics: history and future efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning molecular determinants of human neutralising antibodies isolated from a patient infected with zika virus broadly cross-reactive antibodies dominate the human b cell response against pandemic h n influenza virus infection history and practice: antibodies in infectious diseases monoclonal antibody-based therapies for microbial diseases antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology analysis of respiratory syncitial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab combination therapy using chimeric monoclonal antibodies protects mice from lethal h n infection and prevents formation of escape mutants the coming of age of engineered multivalent antibodies bispecific antibodies against hiv a multifunctional bispecific antibody protects against pseudomonas aeruginosa an engineered human antibody fab fragment specific for pseudomonas aeruginosa pcrv antigen has potent antibacterial activity pharmacokinetic and pharmacodynamic considerations for the use of monoclonal antibodies in the treatment of bacterial infections novel antibodyeantibiotic conjugate eliminates intracellular s. aureus antibody therapies for the prevention and treatment of viral infections antibodies for the treatment of bacterial infections: current experience and future prospects assessment of panobacumab as adjunctive immunotherapy for the treatment of nosocomial pseudomonas aeruginosa pneumonia the future of antibiotics and resistance assessment of an antiealpha-toxin monoclonal antibody for prevention and treatment of staphylococcus aureuseinduced pneumonia pcrv antibodyeantibiotic combination improves survival in pseudomonas aeruginosaeinfected mice antibody-based therapy to combat staphylococcus aureus infections the human intestinal microbiome in health and disease better tests, better care: improved diagnostics for infectious diseases targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases hiv- entry inhibitors: recent development and clinical use phase a study of the ccr monoclonal antibody pro administered intravenously to hiv-infected adults antibodies in hiv- vaccine development and therapy trispecific broadly neutralizing hiv antibodies mediate potent shiv protection in macaques respiratory syncytial viruseneutralizing monoclonal antibodies motavizumab and palivizumab inhibit fusion a highly potent extended half-life antibody as a potential rsv vaccine surrogate for all infants monoclonal antibodies for emerging infectious diseasesdborrowing from history new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets the hemagglutinin a sstem antibody medi prevents and controls disease and limits transmission of pandemic influenza viruses passive immunization for influenza through antibody therapies, a review of the pipeline, challenges and potential applications identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication comparison of a novel human rabies monoclonal antibody immunoglobulin for postexposure prophylaxis: a phase / , randomized, single-blind, noninferiority, controlled study population modeling of influenza a/h n virus kinetics and symptom dynamics point of care strategy for rapid diagnosis of novel a/h n influenza virus efficacy and safety of treatment with an anti-m e monoclonal antibody in experimental human influenza randomized controlled trials for influenza drugs and vaccines: a review of controlled human infection studies design, recruitment and microbiological considerations in human challenge studies ethical criteria for human challenge studies in infectious diseases guideline on procedures for the granting of a marketing authorisation under exceptional circumstances an engineered bispecific dna-encoded igg antibody protects against pseudomonas aeruginosa in a pneumonia challenge model crossneutralization of four paramyxoviruses by a human monoclonal antibody exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a monoclonal antibody scientific advice and protocol assistance factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the european medicines agency emerging infectious diseases: a proactive approach all authors report no conflicts of interest relevant to this article. the views expressed in this article are the personal views of the authors and ought not be understood or quoted as being made on behalf of or reflecting the position of the european medicines agency or one of its committees or working parties. key: cord- - fd bu authors: parma, y.r.; chacana, p.a.; rogé, a.; kahl, a.; cangelosi, a.; geoghegan, p.; lucchesi, p.m.a.; fernández-miyakawa, m.e. title: antibodies anti-shiga toxin b subunit from chicken egg yolk: isolation, purification and neutralization efficacy date: - - journal: toxicon doi: . /j.toxicon. . . sha: doc_id: cord_uid: fd bu shiga toxins (stx and stx ) are the main virulence factors of enterohemorrhagic escherichia coli (ehec), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. the aim of this study was to evaluate the antibodies against stx obtained from egg yolks of laying hens immunized with a recombinant stx b subunit. a high specific response in serum was observed days after the first immunization and igy antibodies were extracted from day th and purified from egg yolk. a concentration of . mg of total igy/ml of egg yolk was obtained, of which % were antigen specific. the ability of anti-stx b igy to recognize stx b and stx either in solid-phase or in solution were evaluated and compared with anti-stx b rabbit antibodies by western blotting and elisa. the protective efficacy of igy against stx was determined by in vitro and in vivo experiments. the results show that igy was able to recognize stx b and stx in denatured conditions, attached to a solid-phase and free in solution. the anti-stx b igy could effectively block the biological activity of stx on vero cells and protect mice from stx challenge. the data suggest that immunization of hens with stx b could be a strategy to obtain at low cost a relatively high concentration of anti-stx egg yolk igy, able to neutralize stx lethal activity. igy technology could be an useful tool for research, diagnosis and therapy of ehec infection. shiga toxins (stx and stx ) are the main virulence factors of enterohemorrhagic escherichia coli (ehec), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. the aim of this study was to evaluate the antibodies against stx obtained from egg yolks of laying hens immunized with a recombinant stx b subunit. a high specific response in serum was observed days after the first immunization and igy antibodies were extracted from day th and purified from egg yolk. a concentration of . mg of total igy/ml of egg yolk was obtained, of which % were antigen specific. the ability of anti-stx b igy to recognize stx b and stx either in solid-phase or in solution were evaluated and compared with anti-stx b rabbit antibodies by western blotting and elisa. the protective efficacy of igy against stx was determined by in vitro and in vivo experiments. the results show that igy was able to recognize stx b and stx in denatured conditions, attached to a solid-phase and free in solution. the anti-stx b igy could effectively block the biological activity of stx on vero cells and protect mice from stx challenge. the data suggest that immunization of hens with stx b could be a strategy to obtain at low cost a relatively high concentration of anti-stx egg yolk igy, able to neutralize stx lethal activity. igy technology could be an useful tool for research, diagnosis and therapy of ehec infection. Ó elsevier ltd. all rights reserved. enterohemorrhagic escherichia coli (ehec), a subset of shiga toxin producing e. coli (stec), are important human foodborne pathogens (kaper et al., ) . transmission is most frequently associated with the consumption of contaminated food or unpasteurized dairy products and infections can also be acquired through person-to person contact (griffin, ) . the clinical manifestations of ehec infections range from watery diarrhea, or hemorrhagic colitis (hc), to the most severe outcome, the life threatening hemolytic uremic syndrome (hus) (nataro and kaper, ) . hus occurs in about - % of individuals, primarily very young and elderly subjects (rivas et al., ) . although ehec infections are a serious problem in developed countries, argentina has the highest incidence of hus in the world, with approximately new cases observed each year in children under years of age (rivas et al., ) . stec also referred to as verotoxin producing e. coli (vtec), produce cytotoxins structurally related to those produced by shigella dysenteriae type . shiga toxins (stx and stx ) are the main virulence factors involved in the pathogenesis of hus and belong to the class known as ab toxins composed of one a subunit and five identical b subunits. the a subunit ( kda), possesses enzymatic rna n-glycosidase activity that hydrolyzes the n-glycoside bond of adenosine of the s rrna of s ribosomes, and hence inhibits protein synthesis; b subunits ( kda) are mainly involved in receptor binding (jeong et al., ) . stec strains produce stx , stx (or their variants), or both of these toxins. although the mechanisms of action of stxs are thought to be similar, cytotoxicity of stx may be stronger than that of stx ; the % lethal dose in mice of purified stx is ng, whereas stx has a % lethal dose of ng (tesh et al., ) . additionally, epidemiological data indicate that stx producing strains are more frequently associated to severe illnesses such as hus than stx producing strains (karmali et al., ) . due to their extreme toxicity, shiga toxins are classified in category b of bioterrorism agents by the center for disease control and prevention (cdc, ) . immunoglobulins are widely used for a variety of purposes, such as in diagnostic tests, purification columns, and passive immunotherapy (kim et al., ) . therefore, research and diagnostic community constantly demand new alternatives and procedures to produce cost-effective antibodies. the use of laying hens to produce polyclonal antibodies is an alternative to the use of mammals, such as rabbits and, since more than two decades, egg yolk antibodies (igy) are a low cost and ethical alternative (schade et al., ; schade and chacana, ) . compared with the stressful bleeding of mammals to obtain serum, igy can be easily obtained non-invasively from the egg yolk. from the economical point of view, the amount of antibodies produced by a single hen is similar to that of a large mammal such as a sheep or goats, whereas maintenance costs are much lower (schade et al., ; zhang, ) . igy from serum is actively transferred into the yolk by a receptor -mediated process (schade et al., ) and the amount of the immunoglobulin varies between and mg per egg (schade et al., ) . thus, a substantial amount of antibody can be produced from just one hen (up to g of total igy per chicken per year), of which - % is expected to be specific to the antigen of interest (mine and kovacs-nolan, ) . in contrast to mammalian igg, igy antibodies do not activate mammalian complement, do not cross-react with fc receptors, mammalian rheumatoid factor, or human anti-mouse antibodies, thus eliminating false-positive results in serological assays (schade et al., ) . also, chickens are able to develop a better response against mammalian antigens, due to the phylogenetic distance between mammals and birds (schade et al., ) . at present, there are no established specific prophylaxis or therapy strategies for ehec borne disease or the prevention of its complications (orth et al., ; tzipori et al., ) , nor early and reliable predictors of the severity of the disease. but, the rapid diagnosis of stec infection and early intervention before the onset of systemic diseases are desirable to prevent or ameliorate toxin-related complications, including hus, as a proper supportive treatment can be determined. besides, rapid laboratory diagnosis and subtyping of stec isolates leads to prompt detection of outbreaks and implementation of control measures (cdc, ) . in this work, polyclonal antibodies were raised in chicken against the stx b subunit. the antibodies were extracted from egg yolk, purified and analyzed for their binding and neutralizing capabilities against stx holotoxin. the comparative performance of anti-stx b igy was evaluated by immunoblotting, elisa and in vitro and in vivo neutralization against polyclonal antibodies obtained from immunized rabbits. the stx holotoxin, corresponding to stx -edl variant (krüger et al., ) , was obtained from stec :h strain t - isolated from cattle by parma et al. ( ) . briefly, the bacteria were grown in luria bertani (lb) broth at c with shaking at rpm until od nm reached , . the culture was treated with mitomicin c ( . mg/ml) and incubated overnight at c with shaking. after centrifugation at ,  g, min at c, the supernatant was filter-sterilized and aliquots were stored at À c. a negative control for the sandwich elisa assay was prepared performing the same protocol with e. coli dh a strain. stx b subunit was produced by a cloning method based in site-specific recombination reactions that mediate the integration and excision of phage lambda into and from the e. coli chromosome (gateway cloning technology, invitrogen). a dna fragment encoding stx b was obtained by pcr amplification using dna extracted from the reference strain e. coli edl and the following pairs of synthetic primers that include recognition sites (attb) for the recombination: ggggacaagtttgtacaaaaaagcaggcttctagaa gaaggagatatacatatgaagaagatgtttatggcggtt (attb -forward, sequence corresponding to attb is underlined). ggggaccactttgtacaagaaagctgggtggatcctta tcagtgatggtgatggtgatgccgaccttcgatgtcattattaa actgcactt (attb -reverse, sequence corresponding to attb is underlined and his-tag codons are in italics). the accuracy of the final dna construction was confirmed by dna sequencing of different clones. the plasmid obtained was called pdeststx b. for expression of recombinant stx b subunit, bl -ai Ô e. coli cells were transformed by pdeststx b. isolated colonies were then grown with shaking at rpm at c to mid exponential phase (od nm: , ) in ml of luria bertani (lb) broth supplemented with mg/ml ampicillin (sigma aldrich). the cultures were induced with l (þ) arabinose (sigma aldrich) at a final concentration of . % and incubated with shaking at c overnight. the stx b was purified by affinity chromatography under native conditions using a ni-nta column (qiagen). the eluted fractions were exchanged with pbs (ph . ) by repeated dilution and concentration by using centrifugal ultrafiltration with vivaspin ( mwco, sartorius). protein yield was determined by the bradford assay using bsa as the standard protein, purity and presence of the recombinant protein in the elution fractions were determined by sds-page and western blot, respectively. one gel was stained with coomassie blue and the other was electrotransferred onto a nitrocellulose membrane (hybond ecl, amersham pharmacia biotech). membranes were blocked overnight with % skimmed milk in pbs-tween (pbs-t) . % at c and then washed three times with pbs-t. this was followed by an incubation with : dilution of anti-his-tag antibody (invitrogen) in pbs-t and then with a : dilution of the anti-mouse ecl secondary antibody (sigma aldrich). after a washing step, the membrane was developed with the ecl western blotting detection kit (amersham pharmacia biotech), according to the manufacturer's instructions. the blot was exposed for min to an x-ray film (super rx; fuji photo). serum samples were collected from the wing vein days after each immunization. eggs were collected daily starting one week after the third immunization and stored at c until further processing. egg yolks were pooled and frozen for purification of igy antibodies. hens were housed in individual cages before the beginning of laying, maintained on / h light/dark cycle and received food and water ad libitum. two new zealand white male rabbits ( kg) were immunized subcutaneously with mg of purified recombinant stx b emulsified with fca at multiple sites in the dorsal region. subsequent booster immunizations were done with an equivalent dose of stx b emulsified with fia at days , , and . blood was collected from the marginal ear vein after the second booster. final bleeding of the anaesthetized animals was done by cardiocenthesis. animals were housed individually with ad libitum water access and commercial rabbit food was supplemented with hay and carrots once a week. prior to the first immunization, serum samples from hens and rabbits and egg yolks samples were taken and used as pre-immune negative controls. presence of specific antibodies in chicken and rabbit sera was tested in a dot blot assay, using recombinant stx b subunit as antigen. egg yolk antibodies were purified by the water dilution method (wd) (akita and nakai, ) . briefly, egg yolk content was mixed with water in a : dilution and kept at À c for at least h and thereafter thawed at c. the disrupted emulsion was centrifuged at  g, min at c. the liquid phase containing the igy was filtered through a gauze tissue and ammonium sulfate was added ( . g/ml supernatant). after centrifugation at ,  g, min at c, the pellet was resuspended with ammonium sulfate m and centrifuged again. finally, the pellet was resuspended and dialyzed against pbs (ph . ) at c. for rabbit sera antibodies, blood samples were kept overnight at room temperature, sera were separated by centrifugation and thereafter stored at À c until use. igg was precipitated with ammonium sulfate ( % saturation) and stirred overnight at c (spira et al., ) . after centrifugation at ,  g for min at c, supernatants were discarded and pellets were dissolved and dialyzed against pbs (ph . ). pre-immune serum antibodies from non-immunized chickens and rabbits were also processed. purity of igy and igg was checked by sds-page and western blot and protein concentration was determined by the bradford assay, using bsa as the standard protein. specific antibodies were purified using hitrap nhsactivated hp ml columns (ge). a coupling procedure was done using recombinant stx b ( . mg in coupling buffer) according to the manufacturer's protocol. about ml of either igy or igg antibodies isolated as described in section . , were applied onto the column. unbound material was eluted with column volumes of binding buffer ( mm tris-hcl ph . ). bound antibodies were eluted with column volumes of elution buffer ( mm glycine, . m nacl ph . ) in . ml fractions into collection tubes containing ml m tris-hcl ph . . protein concentration in the eluted fractions was determined by the bradford assay. recombinant stx b and stx holotoxin were separated by . % sds-page (under reducing conditions) and transferred onto a nitrocellulose membrane (hybond ecl, amersham pharmacia biotech). the membrane was blocked overnight at c with % skimmed milk in pbs-t . %, and incubated with a : dilution of either igy or rabbit serum in pbs-t for h at c. after washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-chicken igy ( : ) or goat anti-rabbit igg ( : ) for h at c. finally, membranes were revealed using dab/h o system (pierce). microplates (nunc maxisorp) were coated overnight at c with ng/well of recombinant stx b or stx holotoxin in carbonate/bicarbonate buffer ph . and blocked at c for h with % skimmed milk in pbs-t . %. after a washing step, igg and igy (or pre-immune samples) were -fold serially diluted in pbs-t and incubated at c for h. previously, both antibodies were properly diluted to give the same starting protein concentration determined by the bradford assay (initial concentration: . mg/ml). plates were washed and incubated with horseradish peroxidase-conjugated goat antichicken igy ( : ) or goat anti-rabbit igg ( : ) for h at c. the plates were washed with pbs-t and developed with abts/h o system (pierce) and absorbance was read at nm. antibody titers were defined as the reciprocal of the dilution of igy and igg antibodies corresponding to a od nm of , . for chicken sera, -fold serial dilutions were made and antibody titers were defined as the reciprocal of the highest dilution of anti-stx b generating a signal about -fold higher than the pre-immune serum. microplates (nunc, maxisorp) were coated overnight at c with ml of igy or igg at ng/well in carbonate/bicarbonate buffer ph . . after washing with pbs-t . %, plates were blocked with % skimmed milk in pbs-t for h at c. wild type stx holotoxin -fold serially diluted in pbs-t was added and incubated at c for h. as a negative control, dh a supernatant was used in the same dilutions as stx . plates were washed and incubated with a : dilution of horseradish peroxidaseconjugated anti-stx b igg antibodies, coupled in the lab by using ez-link plus activated peroxidase (thermo scientific). plates were incubated h at c and then developed with opd (sigma) and absorbance was read at nm. minimun concentration of stx detected by igy and igg was defined as the reciprocal of the highest dilution generating a signal about -fold higher than that of dh a supernatant. african green monkey kidney (vero) cells were plated at /well on -well plates in dmem medium containing % fetal bovine serum and incubated overnight at c under % co . cytotoxic dose % (cd ) was calculated from dose-response curves geometrically as the reciprocal of the toxin dilution causing % reduction in cellular viability. igy and igg antibodies (initial concentration: . mg/ml) (or pre-immune samples) were serially diluted -fold in dulbecco's modified eagle medium (dmem) and pre-incubated for h at c with an equal volume of stx holotoxin ( cd ) (final stx concentration: cd ). for the toxin control, serially -fold dilutions of stx in dmem without antibody were prepared. the stx -igy and stx -igg mixtures and stx alone were added to the vero cell monolayer ( ml/well) and incubated for h at c under % co . the viability of the vero cells was determined by crystal violet staining (gentry and dalrymple, ) and absorbance was read at nm.percentage of neutralization was calculated by using the following formula: od (toxin þ antibody) -od (toxin only)/od (no toxin) -od (toxin only)  . results were expressed as percent of neutralization compared with total igg or igy concentration in mg/ml. all data represent the average of triplicate assays. nih mice (anlis-malbrán) of - g, were allocated randomly over the groups. during the test period mice were housed in wire-topped plastic cages with a layer of sawdust as bedding. cages were located in a room with controlled lighting ( h/day), constant temperature ( - c) and constant relative humidity ( - %). mice toxicity of holotoxin preparation was determined. serially doubling dilutions were made in saline solution and the successive dilutions, expected to cover the -to- % mortality range, were tested in separate groups of mice each. median lethal dose (ld ) was calculated by the method of reed and muench ( ) . two-fold dilutions of igy and igg diluted in pbs (initial concentration: . mg/ml) were incubated with ld of wild type stx holotoxin for h at c. groups of four mice were injected intravenously (iv) with . ml of each immunoglobulin dilution-stx mixture. the mice were monitored over the next days and symptoms of disease and death were recorded. control mice injected with only stx holotoxin were included in each assay and the results were used to confirm the l þ / test dose of the toxin and to correct the antitoxin values obtained. control mice (non-injected or injected with saline solution) were also included. antibody titers were calculated by the spearman-karber method (markus et al., ) . all procedures involving animals were reviewed by the animal care and use committee at the national institute of agriculture technology. the average yield of purified recombinant stx b protein from independent ml culture preparations was . mg ( . mg/l). sds-page analysis of the purified stx b is shown in fig. a . one band between and kda was observed in the eluted fractions. this band likely corresponds to the recombinant stx b monomeric conformation of the protein (theoretical mw: kda) (fig. b) . densitometry analysis of the protein bands on the sds-page gels revealed > % purity in the purified protein preparations. hens had a different profile of antibody production, although in both birds the highest specific titer was found after the second immunization (day th, titer: , ). in hen n , specific titer diminished after the third (titer: , ) and fourth booster (titer: ) but it was increased with the last booster to approximately the same level of specific antibody than after the second immunization. in hen n , specific antibodies remained constant after nd and rd immunization (titer: , ) but diminished significantly after the final boosters (titer: ). highly purified total antibodies preparations were obtained as most of the other proteins present in sera and egg yolk could be removed ( fig. a and b) . the final affinity purification of specific antibodies revealed that approximately % of total igy and % of total igg were antigen specific. igy and igg antibodies isolated as described in section . were used at the same protein concentration for subsequent elisa and in vitro and in vivo neutralization assays. western immunoblots (fig. , lanes and ) and indirect elisa (fig. a) indicate that igy and igg were able to recognize both denatured and native forms of recombinant stx b protein, respectively. elisa titers were higher for igy ( , ) than for igg ( ). specific anti-stx b polyclonal antibodies obtained from chicken egg yolk and rabbit sera recognized not only the denatured and native form of b subunit, used for immunization but the antibodies were also able to recognize the denatured and native wild type stx holotoxin in western blot (fig. ) , indirect elisa (fig. ) and sandwich elisa (fig. ) . unspecific signal was observed with pre-immune igy and igg antibodies, probably due to the recognition of e. coli proteins from the supernatant of stx . specific titer of chicken igy and rabbit igg against the holotoxin were of and , respectively. in the sandwich elisa, soluble native stx holotoxin was detected by igy and igg. the holotoxin could be detected in a / dilution when igy was used as capturing antibody, whereas using igg coupled to the elisa plate it was possible to detect the holotoxin in a / dilution. the cytotoxicity of stx holotoxin in vero cells was cd /ml and the lethality in mice was ld /ml. the cytotoxicity assay based in vero cells was used to test if anti-stx b antibodies from both species were able to prevent the toxin activity in vitro. both igy and igg antibodies neutralized the cytotoxic effects on vero cells (fig. ) . the data obtained show that igg antibodies were times more efficient than igy antibodies since a concentration of . mg/ml was able to neutralize % of the toxin cytotoxicity compared with igy antibody that needed . mg/ml. vero cells that were treated with the mixture stx -pre-immune antibodies or stx alone subsequently died (data not shown). in addition, neutralizing activity was evaluated in vivo during days with native stx holotoxin that had been previously incubated with igy and igg antibodies. all animals that received only native stx ( ld ) died at the third day of the experiment, whereas mice that received . mg/ml of igy antibody survived (fig. b) . in concordance to the results observed in the in vitro assay, neutralizing capability of igg was higher than igy antibodies. this can be easily visualized with . mg/ml of igg ( fig. a ) that prevented of mice from dying at the end of the experiment compared with mice that received igy at the same concentration that all could not survive to the third day of the experiment (fig. b) . neutralizing activity was calculated by the spearman-karber method, as the antibody dilution that prevented mortality in % of the animals, resulting in : and : dilution for igy and igg antibodies, respectively. in the present report, specific egg yolk igy antibodies with binding and neutralizing capabilities against the wild type stx toxin were obtained after immunization of laying hens. the antigen that was injected consisted in the recombinant b subunit stx b, obtained with a yield of . mg/l culture, a relatively elevated production compared with the . mg/l and . mg/l reached in the procedure described by acheson et al. ( ) and marcato et al. ( ) , respectively. in this work, significant levels of specific antibodies were measured by elisa after days of initiating immunization, showing that stx b (w kda) is immunogenic for chickens. however, in some other mammal species as human (ludwig et al., ) and mice (imai et al., ) , stx b was found to be a poor immunogen. marcato et al. ( ) reported that high specific antibody titers against stx b in rabbits could only be achieved including endotoxin in the antigen preparation. so it can be hypothesized that a selective pressure to minimize a long-term immunity against the b subunit was favored in host species (johannes, ) . although a high antibody response was achieved at the beginning of immunization, elisa results from chicken sera showed that levels of specific antibodies were not constantly maintained in the immunized birds, in contrast to the results of pauly et al. ( ) . it is possible that conjugation of stx b with a carrier may be necessary (marcato et al., ) although wang et al. ( ) obtained a high titer of specific antibodies against b subunit from shiga toxin type without conjugation to a carrier and the response remained constant over one year. this suggests a possible negative effect of b subunit from shiga toxin type in laying hens. to the best of our knowledge, there is no information available regarding to this fact, though it has been reported that the stx holotoxin and the b subunit have harmful effects, including lethality, in different mammal species (huang et al., ) . early studies have established that chickens tend to be more resistant against toxins than other species (bengtson, ) . the polyclonal chicken antibodies were tested in classical immunological assays, recognizing not only the denatured toxin, but also the native holotoxin and the b monomer either in solid-phase or in solution. the stxb subunit binding capability of egg yolk igy was comparatively higher than rabbit igg. however, recognition of the b subunit in the context of the whole assembled toxin was similar for igy and igg. in general, ideal antigenic b-cell epitopes are hydrophilic, surface orientated and flexible because in most natural environments, hydrophilic regions tend to reside on the surface of proteins, while hydrophobic regions are found hidden in the interior of the protein. then, a possible justification for the difference in binding is that igy antibodies recognized epitopes in the isolated b subunit that became inaccessible in the holotoxin. in addition to the ability for binding to the native toxin, the chicken antibodies were able to neutralize crude stx preparation, both in vitro (cell culture cytotoxicity assay) and in vivo (mouse bioassay). wang et al. ( ) also evaluated the neutralizing capability of anti-stx igy antibodies by in vitro and in vivo experiments, but they tested the protective efficacy against stx . in this work, we demonstrate that hens, and also rabbits, can produce neutralizing igy antibodies against stx , which is associated to more severe course of illness (karmali et al., ) and when administered systemically it is about times more lethal to mice than stx (tesh et al., ) . also in our work, we completely neutralize the activity of shiga toxin in vitro and in vivo with an igy concentration of about times lower than that used in wang et al. ( ) . others authors have similarly generated neutralizing chicken antibodies against ricin toxin (pauly et al., ) , botulinum toxins (gomez et al., ; pauly et al., ) , clostridium difficile toxins, rabies and viper venom (motoi et al., ) . in this work, rabbit anti-stx b igg antibodies were about times more effective in stx neutralization than egg yolk antibodies (at a similar mass) in vitro and in vivo. however, to obtain mg of specific anti-stx b antibody from one hen the collection of only eggs was enough, while to reach the same amount of specific igg, the exsanguination of one rabbit was necessary. total igy yield per egg was . mg although gassmann et al. ( ) described a yield of mg igy per egg. however, there was less difference in the amount of specific antibodies per egg: . mg from this work and mg from gassmann et al. ( ) . another group (bizhanov and vyshniauskis, ) reported a recovery rate of - mg of anti-sendai virus igy per ml of egg yolk. antibiotic therapy is not recommended for food poisoning caused by enterohemorrhagic e. coli infection, because increases the risk of serious complications, such as hemolytic uremic syndrome, due to the release of shiga toxin from killed bacteria and by inducing expression of stx through replication of phages that carry stx genes (kozlov et al., ) . therefore, alternative therapeutic approaches, such as inhibiting shiga toxin activity or absorption from the intestine, are required. oral administration of igy has proved successful for treatment of a variety of gastrointestinal infections, such as bovine and human rotaviruses, bovine coronavirus, yersinia ruckeri, c. difficile, salmonella spp., edwardsiella tarda, staphylococcus and pseudomonas, (mine and kovacs-nolan, ; schade et al., ) . chicken egg yolk has previously been used as an inexpensive and effective source of igy antibodies for the passive immunization or treatment of piglets suffering enterotoxigenic f (k )þ (marquardt et al., ) and f abþ (imberechts et al., ) e. coli infections, dental caries due to streptococcus mutans in humans (smith et al., ) and porcine epidemic diarrhea virus (pedv) infection in piglets (kweon et al., ) . therefore, anti-stx igy could be an economic alternative for prophylactic and therapeutic treatment of ehec infections or stx exposure, either alone or combined with other products. furthermore, therapeutic use of igy in primates was effectively studied by leclaire et al. ( ) using neutralizing igy antibodies against the highly toxic staphylococcal enterotoxin b (seb). neutralization of stx may also be useful to decrease cattle colonization by stec (hoffman et al., ) , which is considered the main reservoir of the bacteria. since stx plays a role in both colonization and systemic disease, passive administration of anti-stx antibodies to reduce or prevent ehec infection in people has also been proposed (krystle et al., ) . this was demonstrated in mouse models with anti-stx antibodies administered before infection (donohue-rolfe et al., ) . in subsequent evaluations of the efficacy of such passive therapy, anti-stx antibodies were shown to provide protection against stec-mediated illness and death even when administered up to days after bacterial challenge (donohue-rolfe et al., ; yamagami et al., ) . besides, igy is widely used in many applications ranging from immunofluorescence, immunohistochemistry, immuno-enzyme techniques (elisa), western blotting and immunoelectrophoresis (lee et al., ; tini et al., ) . these antibodies are capable to detect multiple bacterial and parasitic organisms such as acanthamoeba spp., helicobacter pylori (shin et al., ) , microsporidia (young et al., ) , e. coli o :h (sunwoo et al., ) . in conclusion, although further studies should evaluate different adjuvants and immunization plans in chickens in order to maximize the anti-stx b igy production, this work shows that igy technology is a promising alternative to be applied in the detection of stec and the prophylaxis or treatment of hemolytic uremic syndrome. there are no conflicts of interest related to this study. expression and purification of shiga-like toxin ii b subunits immunoglobulins from egg yolk: isolation and purification studies on organisms concerned as causative factors in botulism a comparison of three methods for extracting igy from the egg yolk of hens immunized with sendai virus recommendations for diagnosis of shiga toxin-producing escherichia coli infections by clinical laboratories antibody based protection of gnotobiotic piglets infected with escherichia coli o :h against systemic complications associated with shiga toxin efficient production of chicken egg yolk antibodies against a conserved mammalian protein quantitative microtiter cytotoxicity assay for shigella toxin production of chicken antibodies against botulinic toxin a escherichia coli o :h and other enterohaemorrhagic escherichia coli bovine immune response to shiga-toxigenic escherichia coli o :h . clin shiga toxin b subunits induce vwf secretion by human endothelial cells and thrombotic microangiopathy in adamts -deficient mice production of secretory immunoglobulin a against shiga toxin-binding subunits in mice by mucosal immunization chicken egg yolk antibodies against f ab fimbriae of escherichia coli inhibit shedding of f positive e. coli by experimentally infected pigs in vitro and in vivo protective efficacies of antibodies that neutralize the rna n-glycosidase activity of shiga toxin the epithelial cell cytoskeleton and intracellular trafficking. i. shiga toxin b-subunit system: retrograde transport, intracellular vectorization, and more pathogenic escherichia coli the association between idiopathic hemolytic uremic syndrome and infection by verotoxin producing escherichia coli performance of an enzyme-linked immunosorbent assay for detection of clonorchis sinensis infestation in high-and low-risk groups the primary structure of the operons coding for shigella dysenteriae toxin and temperature phage.h shiga-like toxin verotoxins in bovine and meat verotoxin-producing escherichia coli isolates: type, number of variants, and relationship to cytotoxicity neutralizing antibodies to shiga toxin type (stx ) reduce colonization of mice by stx -expressing escherichia coli o :h immunoprophylactic effect of chicken egg yolk immunoglobulin (igy) against porcine epidemic diarrhea virus (pedv) in piglets protection against bacterial staphylococcal enterotoxin b by passive vaccination vitro studies of chicken egg yolk antibody (igy) against salmonella enteritidis and salmonella typhimurium antibody response to shiga toxins stx and stx in children with enteropathic hemolytic-uremic syndrome immunoprophylactic potencial of cloned shiga toxin b subunit recombinant shiga toxin b-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from shigatoxemia an alternative approach to the optimal design of an ld bioassay passive protective effect of egg-yolk antibodies against enterotoxigenic escherichia coli k þ infection in neonatal and earlyweaned piglets chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review production of rabies neutralizing antibody in hen's eggs using a part of the g protein expressed in escherichia coli diarrheagenic escherichia coli prevention and treatment of enterohemorrhagic escherichia coli infections in humans virulence genotypes and serotypes of verotoxigenic escherichia coli isolated from cattle and foods in argentina. importance in public health monitoring of laying capacity, immunoglobulin y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period a simple method of estimating fifty per cent endpoints chapter : diarrheagenic escherichia coli in argentina specificity of chicken (igy) versus rabbit (igg) antibodies raised against cholecystokinin octapeptide (cck- ) chicken egg yolk antibodies (igytechnology): a review of progress in production and use in research and human and veterinary medicine egg yolk compounds -livetin fractions use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of helicobacter pylori infection passive transfer of immunoglobulin y antibody to streptococcus mutans glucan binding protein b can confer protection against experimental dental caries tsh binding proteins in rat and human serum detection of escherichia coli :h using chicken immunoglobulin y comparison of the relative toxicities of shiga-like toxins type i and type ii for mice generation and application of chicken egg-yolk antibodies antibody therapy in the management of shiga toxin-induced hemolytic uremic syndrome passive protection of purified yolk immunoglobulin administered against shiga toxin in mouse models efficacy of postinfection treatment with anti-shiga toxin (stx) humanized monoclonal antibody tma- in mice lethally challenged with stx-producing escherichia coli chicken-derived igy recognizes developing and mature stages of loma salmonae (microsporidia) in pacific salmon, oncorhynchus spp the use of gene specific-igy antibodies for drug target discovery the study received financial support from anpcyt pict / and pict . key: cord- - qw t s authors: naylor, mj; monckton, rp; lehrbach, pr; deane, em title: canine coronavirus in australian dogs date: - - journal: aust vet j doi: . /j. - . .tb .x sha: doc_id: cord_uid: qw t s objective to estimate the frequency of serum antibodies (igg and igm) to canine coronavirus (ccv) in the australian dog population and evaluate the role of ccv as a causative agent of gastroenteritis. design a serological survey of antibodies to ccv among different dog populations. procedure the development and characterisation of an indirect elisa for the detection of antibodies (igg and igm) to ccv was undertaken. sera collected from both diarrhoeal and non‐diarrhoeal dogs from various populations throughout australia were tested for these antibodies to ccv. results serum samples ( ) collected from to were tested for the presence of igg antibodies to ccv. samples were divided into two categories on the basis of the number of dogs housed together. the groups were either an open population containing dogs housed as groups of three or less, or kennel populations. sera from . % of the open population and . % of kennelled dogs were positive for ccv antibodies. the prevalence of antibodies varied from zero to % in kennelled dogs. about % of dogs positive for igg antibodies to ccv were also positive for igm antibodies to ccv, indicating recent ccv infection. of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting(n = ), % were positive in the igm elisa and . % in the igg elisa for antibodies to ccv. in comparison, for those dogs presented without any history of gastroenteritis only % were positive for igm and % positive for igg. conclusion serological evidence indicates that infection with ccv in dogs is widespread throughout the australian mainland. the prevalence of antibodies varies greatly among different populations, with an average of . % positive in kennelled populations and . % in the open population. c cv was first recovered from the faeces of dogs during an epidemic of diarrhoeal disease in germany. enteritis was subsequently induced in neonatal dogs by oral inoculation of canine coronavirus (strain - ) isolated from the original outbreak. enteritis accompanied by diarrhoea was characterised by atrophy and fusion of intestinal villi. the infection was not fatal and was self-limiting. subsequently ccv has been isolated from other epidemics of canine diarrhoea and has a worldwide distribution. [ ] [ ] [ ] the extent to which ccv is important as a gastroenteric pathogen in the australian dog population is presently unknown, although ccv and cvlp have been previously reported in australia. [ ] [ ] [ ] in these reports, electron microscopy of faecal samples was used as the detection method with a relatively small number of samples. in this study we determine the prevalence of serum igg and igm antibodies to ccv from a larger number of dogs sampled from throughout australia. ccv tn- strain and crfk cell line were obtained from fort dodge laboratories, iowa, usa. crfk cells were propagated in cm cell-culture flasks at o c (corning, australia) in growth medium containing emem (trace biosciences, australia), mm l-glutamine, . % lah and % fbs not heat inactivated (csl ltd, parkville). maintenance medium for maintaining cells without division consisted of emem, mm l-glutamine, . % lactalbumin hydrolysate and % fbs not heat inactivated. positive control serum (pooled sera from dogs inoculated with ccv tn- ) and negative control serum (pooled sera from specific pathogen free dogs) were obtained from fort dodge laboratories, iowa, usa. these serum samples were further verified by serum neutralisation assay as described by mochizuki et al. elisa antigen preparation roller bottles of cm surface area (corning, australia) with crfk cells at % confluency were infected with ccv (tn- ) at a multiplicity of infection of . . after incubation for h at o c, cells were harvested by scraping with a cell scraper and resuspended in ml of pbs. cells were washed three times by centrifugation at g for min and resuspended in ml of pbs containing . % sodium deoxycholate and incubated for min on ice. open population -of the dogs tested, igg antibodies to ccv were detected in ( . %), (table ) . from this group, clinical histories were obtained for dogs. twentyeight of these dogs had signs of gastroenteritis such as diarrhoea, vomiting and weight loss and all dogs were negative for gastroenteric pathogens such as cpv as determined by independent pathology laboratories. twenty-four of the were positive for ccv antibody (igg), with elisa od results equivalent to those obtained from experimentally infected dogs (unpublished data). antibodies (igg) to ccv were detected in of the remaining for which histories were obtained but which did not have diarrhoea or any other signs of gastroenteric infection at the time of presentation. kennel populations -of the group-housed dogs, igg antibodies to ccv were detected in ( . %). ten of the different kennel populations tested contained ccv antibodypositive dogs. the range of od values determined by indirect elisa for serum igg antibodies to ccv varied from . (the cut-off was ultracentrifuged for min at , g ( o c) and the supernatant used as antigen for the elisa. control antigen was prepared in the same manner from uninfected cells. indirect elisas for the detection of serum igg or igm antibodies to ccv were developed on the basis of previously published assay methods. , control and ccv tn- antigens were diluted separately to µg per . ml in carbonate-bicarbonate buffer and incubated overnight ( °c) in -well flat-bottomed immunoplates (nunc, denmark) with . ml per well of diluted antigen. the solution was decanted from the plates and . ml per well of . % skim milk in pbs-blocking agent was added. after incubation for h at o c, plates were washed once with pbs containing . % tween . positive and negative control sera and test sera were serially diluted in pbs containing . % tween with % foetal bovine serum (csl biosciences, australia), inactivated at o c for h and added at a volume of µl per well. dilutions were incubated for a further hour at room temperature before the plates were decanted and washed three times with pbs containing . % tween . fifty µl per well of a : dilution of either hrp-conjugated rabbit anti-dog igg (sigma, australia) or hrp-conjugated goat anti-dog igm (nordic, the netherlands) was added and the plates incubated for h at room temperature. following a further three washes, µl per well of . mg per ml tetramethylbenzidine microwell peroxidase substrate (kirkegarrd & perry laboratory, usa) was added and incubated for min. one hundred µl of . m phosphoric acid was added to stop the reaction and the absorbance was read at nm. each serum dilution was performed in duplicate, with the absorbance value of the control antigen deducted from the ccv tn- antigen absorbance value to give the final value. an absorbance value greater than . was considered a positive reaction. this value was at least two times higher than background values from negative control sera (unpublished data). serum samples (n = ) were collected from dogs in two different housing groups. one group contained dogs housed in groups of no more than three animals (open population) while the other group contained dogs from commercial breeders, rescue shelters and remote settlement colonies where dogs were housed as a group of or more animals (kennel populations) ( table ) . open population -in this group, serum samples were collected from veterinary pathology laboratories, universities and by field sampling from dogs submitted for general pathology, surgery or survey purposes. these dogs were domestic pets from both suburban and rural areas, generally kept singly or in pairs. the sample areas included all the australian states and territories, with sera collected from to . kennel populations -in this group, serum samples were collected from three commercial breeders, a university study, three rescue kennels and four remote settlement colonies. all serum samples were stored at - °c before use. all samples were tested for igg antibodies and randomly selected igg positive sera were tested for igm antibodies to remote settlement colony (nt) . twenty-nine of the ( . %) randomly selected, igg antibody-positive sera were also found to be positive for igm antibodies to ccv. clinical histories were available for of these dogs and ( %) had clinical signs of diarrhoea. among the igg negative sera, fourteen samples were also tested for igm antibodies to ccv. these samples were selected on the basis of a high prevalence of gastroenteric disease in the kennelled population at the time of sampling (breeding colony , nsw, samples) or evidence from clinical records of a recent episode of gastroenteritis in the open population dogs sampled ( samples). seven ( %) of these dogs were positive for anti-ccv igm antibodies. the prevalence of ccv antibodies in different dog populations throughout the world has been found to range from to %, with as high as % reported in kennelled populations. [ ] [ ] [ ] we report the first serological study of canine coronavirus antibodies in the australian dog population. the prevalence of ccv antibody was . % ( to . %) in dogs housed singly or in small groups (open population) but a significantly higher (p < . ) prevalence of . % ( to %) was found among kennelled dogs. ccv has been reported previously in australia based on electron microscopic examination of faeces; these two studies showed . % of dogs positive, and . % of dogs positive. , in the open population of dogs tested we found . % positive for anti-ccv igg antibody, which reflects past exposure and infection with ccv whereas the electron microscopic studies detected only those dogs currently infected and shedding virus in their faeces. dogs positive for ccv antibody were found in every australian state and territory, including remote areas of the northern territory. schnagl and holmes had also previously reported cvlp in remote areas of western australia and northern territory. one dog with antibodies to ccv was found in tasmania, although no other sera were tested from that state. we conclude that ccv exists in dogs throughout australia. in the open populations, most elisa results fell within an od range of . to . (unpublished data). within the kennel populations, approximately % of od readings were greater than . (p > . ). the difference in the occurrence of exposure among dogs from kennel populations and the open population has been observed previously. [ ] [ ] [ ] the higher frequency of exposure found in kennel populations presumably represents the increased opportunity for exposure due to the different housing and social interactions. in the three commercial breeding colonies from nsw examined in this study, severe gastroenteric disease resulting in variable fatality among young pups had been previously observed (m lindsey personal communication). in these cases cpv was reported as the causative agent. the current study identified high titres of ccv antibody in these kennels during the same period. igm antibodies to ccv were also detected in these populations, indicating current ccv infection at the time of the gastroenteritis epidemics. it is probable that a mixed infection of ccv and cpv was occurring and the presence of ccv may have contributed to the severity of the enteritis. appel and brunner and swango demonstrated increased severity of clinical signs of both ccv and cpv in mixed infections, particularly in young and stressed animals. it is therefore difficult to assess the clinical implications of ccv infection alone, because mixed infections or other factors may influence the course of disease. the dogs that were found to be positive for ccv antibody and had signs of gastroenteritis, may represent examples of ccv induced disease as these dogs were also negative for many of the other common gastroenteric pathogens such as cpv (unpublished data). a highly significant correlation (p < . ) was found between the presence of anti-ccv igg antibodies and diarrhoea, with of dogs ( . %) that presented with diarrhoea being igg positive compared with of ( %) dogs that presented without diarrhoea. approximately % of dogs positive for igg antibodies to ccv were also positive for igm antibodies to ccv indicating infection within days of blood samples being collected. seven dogs were identified as igg antibody negative and igm antibody positive to ccv. these dogs represent examples of animals exposed to ccv within to days before blood samples were collected and that had not had time to produce a detectable igg antibody response. all seven of these dogs had signs of gastroenteritis. eighty-five percent of dogs that had clinical signs of gastroenteritis attributed to ccv infection were positive for igg and igm antibody to ccv. in comparison, for those dogs presented without any history of gastroenteritis only % were anti-ccv igm positive (p < . ). our study indicates that ccv is widespread in the australian dog population and suggests that the presence of ccv antibodies is associated with gastroenteritis. in , marjorie was invited back to south australia to organise the brucellosis and t u b e r c u l o s i s eradication scheme in cattle. again true to form, she was the first woman to join the department of agriculture in south australia. due to her efforts and management skills she orchestrated a successful eradication scheme. the effort to coordinate all aspects of the brucellosis campaign, with limited resources in a big state, was a difficult task. marjorie was appointed principal veterinary officer in and deputy chief veterinary officer for south australia in . she retired in to live in the adelaide hills, devoting some of her time on a voluntary basis to the guide dogs for the blind. marjorie was the first woman in many of her appointments and she was able to achieve this due to her strong character, abundant wit and sense of humour. she enjoyed a good argument or discussion and, with her formidable memory for dates and wide reading, she was not an easy person to get the better of. those who tried, felt the sharpness of her wit. an agitated marjorie was a sight to behold. there are still anecdotes circulating around png about marjorie, when someone accidentally locked her in the cold room. in she moved to sydney to be closer to her family and died on june , after a long illness during which she showed her characteristic strength of character. the veterinary profession is very proud of marjorie reid's contribution to the welfare of animals in australia and internationally. m arjorie reid was a remarkable woman who epitomised the wealth and breadth of veterinary training, by successfully accepting challenges that ranged from practice, laboratory, fieldwork and senior veterinary administration. marjorie, who was born in , graduated from the royal veterinary college in and was one of a small, but elite group of women in the veterinary profession in england. she had always had a strong sense of adventure and pioneering spirit and, in , she commenced practice in south africa. later, she moved to kenya and joined the well known veterinary research institute at kabete near nairobi. marjorie enjoyed working in kenya, where there was a constant challenge to use basic veterinary principles to provide a diagnostic service for commercial and wild animals. her two daughters were born during this period in kenya. during she immigrated to australia and became the first female veterinarian at the institute of medical and veterinary science in adelaide. it was during this period she became interested in bacteriology and began to develop her skills in this subject. always seeking adventure, in she joined the department of agriculture, stock and fisheries in papua new guinea and was based at the kila kila veterinary laboratory in port moresby. when she arrived the laboratory was a converted airport control tower from the second world war. this tested her strengths in more ways than one, not only in bacteriology, but also filling in as port moresby's veterinarian on occasions. with her colleagues, ifor owen and noel talbot, she showed a remarkable ability to use good practical commonsense to simplify what seemed to be highly complex issues. when the new laboratory was built, she was able to demonstrate her considerable laboratory skills to all and was a good but firm teacher to many. this was a happy and productive period of her life. marjorie was very good with animals of all species ranging from the usual to exotic species such as crocodiles. because of her combination of ability and character, she was the 'preferred vet' in a strongly male-dominated society, which reflected her high standing in the community. many at first underestimated her strength of character and discipline; everyone soon learnt not to loaf about when marjorie was around. recovery and characterization of a coronavirus from military dogs with diarrhea intestinal infection of neonatal dogs with canine coronavirus - :studies by virologic, histologic, histochemical and immunofluorescent techniques studies of respiratory disease in random source laboratory dogs: viral infections in unconditioned dogs viruses and diarrhoea in dogs infectious disease in the dog and cat coronavirus-like particles in stools from dogs, from some country areas of australia viruses and virus-like particles in the faeces of dogs with and without diarrhoea faecal viruses of dogs -an electron microscope study jw copland dogs in the netherlands studies on the epizootiology of canine coronavirus does canine coronavirus augment the effects of subsequent parvovirus infection canine parvoviral infection: effects on the immune system and factors that predispose to severe disease canine coronavirus infection in the dog following oronasal inoculation micro-neutralization test with canine coronavirus for detection of coronavirus antibodies in dogs and cats enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs the use of an enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in the netherlands van den ingh tsgam. canine viral enteritis: prevalence of parvo the authors would like to thank the various government and university departments, veterinary, private and veterinary pathology organisations for the serum samples and animal histories. we are also grateful to mark lindsey for comments on the manuscript and assistance in obtaining animal sera and case histories. mj naylor was supported by a cooperative education for enterprise development (ceed) grant funded by fort dodge australia pty limited and the university of western sydney, nepean. key: cord- -vkzya uq authors: ijaz, m. k.; sabara, m. i.; frenchick, p. j.; babiuk, l. a. title: effect of different routes of immunization with bovine rotavirus on lactogenic antibody response in mice date: - - journal: antiviral research doi: . /s - ( ) - sha: doc_id: cord_uid: vkzya uq summary the effect of different routes of immunization with either live or killed bovine rotavirus (brv) on the production of lactogenic antibody response in mice was evaluated. the routes of immunization were intramuscular (im), oral (o) or intradermal in the mammary region (imam). following immunization, serum antibody responses were monitored by an enzyme linked immunosorbent assay (elisa). following whelping, the mice were allowed to stay with their mother until sacrificed on alternate days post-parturition from day – . milk from their stomach was collected for antibody titration by elisa and virus neutralization test. regardless of the routes of immunization, a rapid increase in serum anti-rotavirus antibody titers was observed for the first wk after immunization followed by a gradual decline. after parturition, the mean antibody titer of lacteal secretions, as determined by elisa, increased gradually for days with the greatest increase on day , followed by a decrease in anti-rotavirus antibody. these titers also correlated with antibody titers in milk as measured by virus neutralization test. the best lactogenic antibody response was observed when imam × im × route of immunization was used with live brv as the antigen. interestingly, immunization via the oral route with killed brv also resulted in good antibody responses. in contrast, in the group where killed brv was used, animals receiving × orally had the highest antibody titer. the distribution of different antibody subtypes in milk samples revealed igg to be the predominant antibody followed by igm and iga. irrespective of the route of administration, there was an increase in iga on day as compared to day in most of the groups. the significant role played by mucosal immunity in passive protection and the possible ways to modulate subtype specific lactogenic immune response are discussed. animals models; lactogenic immunity; rotaviruses rotavirus has been identified as the single most important causative agent of acute neonatal enteritis in a variety of domestic and laboratory animals and humans. consequently, a world-wide effort is being made to develop an appropriate vaccine against this pathogen (babiuk et al., ; flewett and babiuk, ; hanson et al., ) . although previous studies have indicated no correlation between protection from rotavirus infection and the titers of passively acquired antibodies in the serum of newborns (saif and smith, ; m.k. ijaz et al., unpublished data) , it has been shown that the continuous presence of anti-rotavirus antibodies in the intestine of neonates is important in preventing clinical disease associated with rotavirus infections (offit and clark, . ; saif and smith, ; snodgrass et al., ; m.k. ijaz et al., unpublished data) . in several animal species, including humans, immunization during pregnancy has been shown to result in the presence of antibodies in colostrum and milk (goldblum et al., ; saif and smith, ) . in addition to the humoral immune response, the role played by the cellular component of immunity in the production of lactogenic immune response has also been documented (parmerly and beer, ; ripenhoff-talty et al., ) . cell transfer experiments in mice have revealed the migration of lymphoblasts from mesenteric lymph nodes into the intestine and mammary gland and other secretory organs and peripheral lymph nodes (mcdermott et al., ; mcdowell and lascells, ) . most cells migrating into secretory organs and mesenteric lymph nodes synthesized iga, but most of those migrating to peripheral lymph nodes produced igg or igm (bohl et al., ; mcdermott et al., ; roux et al.. ) . experiments carried out in porcine and bovine models have revealed the predominance of iga and igg subtypes in porcine and bovine milk, respectively, with the decrease of these subtypes occurring during the post-parturition period (saif and bohl, ; saif and smith, ) . thus it becomes imperative to direct efforts in order to stimulate the lactogenic anti-rotavirus antibody response to higher titers for longer periods after parturition to ensure protection against rotavirus infection in neonates receiving these antibodies in mother's milk. earlier experiments conducted to determine the kinetics of brv replication in a mouse model revealed that the neonates are most susceptible to infection at the age of seven days (ijaz et al., in preparation) . in our protection studies the neonates are challenged on day seven. taking this into consideration, we have utilized the murine model to study the influence of different routes of immunization with either live or killed brv on the rate of decline of antibody titers and different subtypes in the lacteal secretions of the da-m following parturition up to eleven days. this report forms the basis of our protection studies which are currently under way. rotavirus-free mice were obtained from charles river breeding laboratories (wilmington, ma). in order to ensure that they were free of anti-rotavirus antibodies, they were bled on arrival from the coccigial vein. the sera were tested for anti-rotavirus antibodies using an elisa assay (bidwell et al., ) and all were found to be seronegative. to maintain them rotavirus free, the animals were housed in isolation units throughout the experiment. bovine rotavirus (brv) isolate c- was grown in ma- cells in the presence of pg of trypsin/ml (difco laboratories, detroit, mi) (babiuk et al., ) . after h of incubation at °c. supernatant containing the virus was harvested and cell debris removed by centrifugation at rpm for min. the virus was concentrated from the clarified supernatant fluids by pelleting through a % sucrose cushion at rpm in an sw rotor (beckman model l - ) for l/ h at °c. the virus pellet was resuspended in double distilled water and the amount of virus protein measured spectophotometrically by using the formula: (alj,,) - . (a)zho) x = kg/ml where a ,, and a,,,, represents adsorbance at nm and nm, respectively. when live brv was used as the antigen. each dam was given l.r.g ( x lo' plaque forming units) of purified virus contained in adjuvant (freund's incomplete adjuvant [fia] ) or double distilled water, according to the immunization protocol (table ) . killed brv was prepared as described above. in order to inactivate the virus, it was subjected to ultraviolet light ( ergs) for h and the extent of inactivation was tested in vitro. in no instance was active bovine rotavirus found after this inactivation procedure. each dam was given kg of purified virus contained in adjuvant or double distilled water, according to the immunization protocol (table ) . for oral immunization, mice were inoculated with kg of brv followed and wk later with the same viral dose inoculated orally. one week before the final booster inoculation, mice were bred in a female-to-male ratio of :l. all the dams gave birth naturally and the pups remained with their dams throughout the experiment. sampling and sample processing all mice were bled prior to immunization and their sera were tested for the presence of rotavirus antibody by both an elisa and virus neutralization test. all of the mice were seronegative for rotavirus on initiation of the study. thereafter, blood samples were taken weekly during the course of immunization to monitor antibody responses as indicated in the immunization protocol (table ). the immunization intervals were chosen arbitrarily at three weeks, since in most instances. maximal immunity occurs three weeks post-vaccination. the newborn mice were allowed to stay with their mother throughout the period of the experiment. in order to collect samples of colostrum and milk, three neonates from each group were sacrificed every second day post parturition starting on days through i. milk from their stomach was collected and diluted twofold in mem. samples were spun in an eppendorf centrifuge and supernatants were aliquotted into eppendorf tubes and stored at - °c. enzyme-litlked ittmut~osorhet~r rrssup (elisa) for detectiotl o.f an(i-rotavirus antibodies antibody levels in serum and milk samples against brv were determined by the method described previously (bidwell et al., ) . briefly. -well microtiter plates (immunolon : dynatech laboratories inc., alexandria. va) were incubated at °c overnight with fg of brv (isolate c- ) antigen diluted in x carbonate-bicarbonate (ph . ). the volume of brv per well was ~ . after incubation of plates with antigen. unadsorbed protein was removed by extensive washing in distilled water. the antigen was then overlaid with ~ of mouse antiserum per well in an undiluted form or diluted in . m phosphatebuffered saline containing . % fetal bovine serum (fbs). incubation of antigen was carried out for h at room temperature, after which time unbound antibody was removed by washing in distilled water. a : dilution of rabbit antimouse horseradish peroxidase conjugate (boehringer mannheim biochemical, calgary, alberta) was then added per well, and incubation of the conjugate proceeded at room temperature for an additional hour. after extensive washing to remove excess conjugate. the bound conjugate was reacted with ~ of chromagen and enzyme substrate (recrystallized -amino salicyclic acid. mg/ml in . phosphate buffer, ph . . , aldrich chemical co., milwaukee, wn) per well. to which . c/c hydrogen peroxide was added immediately before use. the reaction was allowed to proceed for min at room temperature before the absorbance ( nm) of each well was determined with a micro-elisa reader (dynatech instruments, inc., ca) and titers were expressed as the reciprocal of the highest dilution with an od of > sd over mean background levels. for the quantitation of different classes of antibody in mouse milk, mouse monoclonal subtyping kit -k was used following the procedure recommended by the manufacturer (hyclone laboratories. logan, ut). micro-tieiwalizutiotl test neutralization of brv c- by milk samples taken from hyperimmunized dams was determined by a micro-neutralization test using -well plates containing confluent monolayers of bsc- cells grown in eagle's minimal essential media (mem) with % fbs (gibco laboratories. grand island, ny). milk samples were se-rially diluted and incubated for min at °c. virus dilutions representing tcid ,, were added :l with various dilutions of milk and the virus-milk mixture was transferred to the plates containing confluent monolayers of bsc- cells in each well in duplicate. the plates containing virus-milk mixture were incubated for h at °c. the cell monolayers were washed with mem and replaced with ~ of mem. the plates were incubated at °c for two days. the dilution sample giving % cytopathic effect (cpe), as observed under the microscope. was taken as end point. since passive protection is important for preventing rotavirus infection in neonates, it is important that neonates receive sufficient quantities of neutralizing antibodies in milk from their mothers. in this study we were interested in determining the best regimen of immunizations against brv which would result in the highest neutralizing antibody levels in milk. to achieve this goal. mice were immunized with either live or killed brv via different routes and the levels of serum antibody against rotavirus were monitored throughout the immunization schedule up to parturition. after parturition, anti-rotavirus antibodies were determined in lacteal secretions for days. using an elisa and virus neutralization test. antibody titers in serum, colostrum and milk are shown in figs. - . milk samples were also examined for determination of different immunoglobulin (ig) classes. data showing ratios of various subtypes in milk collected from dams immunized with either live or killed brv are summarized in tables and . the distribution of different subtypes in milk samples revealed igg to be the most predominant antibody followed by igm and iga. it is interesting to note that regardless of the route of immunization, a gradual increase in the antibody titer in the lacteal secretions was observed up to days post-partum, with the greatest increase on day . followed by a decrease in titer on day ; a unique observation compared with other species. ltntnunizatiotz with killed bovirle rotavirm to determine whether killed brv can produce lactogenic anti-rotavirus antibody response, experiments were performed using this antigen orally and in combinations of other routes. animals were immunized by the following route using killed brv: x , x im x , im x and imam x . freund's complete adjuvant (fca) and freund's incomplete adjuvant (fia) were used only with the im route of immunization (table ) . immunization with killed brv significantly increased anti-rotavirus antibodies in serum in all groups regardless of the route of immunization (fig. ) . the rapid immune response observed a week after the first immunization as compared to an almost negligible titer on day , does not reflect that animals were primed before immunization because mice used in this study were pre-tested for anti-rotavirus antibodies and all were found to be seronegative and control mice remained seronegative throughout the study. however, it does show that rotavirus is highly immunogenic. among all the groups immunized with killed brv, the group immunized times via the imam route showed the best anti-rotavirus serum antibody response. an interesting but unexplainable decrease in antibody occured days post-immunization in animals immunized via the imam route (fig. ). however. a second immunization at days increased the antibody titer above that of any other group. although the second immunization boosted the antibody titers in all groups, the third immunization at days did not show any effect on the level of antibody production in any of the groups under study (fig. ) . antibody titers against rotavirus in lacteal secretions as determined by elisa. varied from - log,,, between groups one day post-parturition ( fig. ) . animals immunized x had the highest titers whereas the im x had the lowest antibody titers. a gradual increase in antibody was observed in all groups with the maximum antibody titer in lacteal secretions being achieved days after parturition. unlike serum antibodies, the highest lacteal antibody titer occurred in the group immunized x and x im x . the virus neutralization antibody titers in milk showed a similar pattern throughout lactation as did the elisa titers. however, anti-rotavirus antibody titers, as determined by elisa, were consistently higher than virus neutralization titers, with the exception of the group immunized x which showed slightly higher neutralization titers on day and after parturition (fig. ) . immunization with live bovine rotavirus since killed antigen produced an excellent response following oral immunization, attempts were made to do similar studies with live virus. animals were immunized by the following routes using live brv: x , x im x , im x , imam x and imam x im x with or without adjuvant as indicated in the immunization schedule (table ) . immunization with live brv induced the production of high levels of anti-rotavirus antibodies in serum of dams in all groups. the pattern of immune response was rapid, similar to the groups immunized with killed brv. one exception was the group immunized imam x im x , which revealed significantly higher antia rotavirus antibodies days after primary immunization ( wk after the second immunization). there was a rapid decrease in antibody titer in this particular group days after the first immunization, but the second immunization at days induced a higher level of antibody than in the rest of the groups. the third immunization at days did not increase antibody production, and antibody levels in serum started declining towards the time of parturition (fig. ) . titers of anti-rotavirus antibodies in lacteal secretions, as determined by elisa, also revealed a similar pattern as seen in the groups immunized with killed brv. the colostrum and milk samples collected up to a week after parturition showed a gradual increase in antibody titers as determined by elisa. the highest antirotavirus antibody titer in all groups was observed on day post-parturition. the maximum titer of antibody at this point was observed in the group immunized imam x im x . the titers started declining thereafter (fig. ) . virus neutralizing antibody titers in the lacteal secretions exhibited a similar pattern throughout lactation. the anti-rotavirus antibody titers, as determined by elisa, were consistently higher than virus neutralizing antibody titers in all the groups (fig. ) . the distribution of antibodies associated with various immunoglobulin (ig) subtypes in mammary secretions are presented in tables and . throughout the duration of the experiment, igg antibodies predominated, followed by igm and iga in each group. irrespective of the route of immunization and type of antigen used, there was a gradual reduction in the distribution of igg and igm antibodies until the end of the experiment. in contrast, iga antibodies, after a gradual decrease up to day post-parturition, showed an increase on day before decreasing again. since neonates are often exposed to enteric viruses before they have sufficient time to develop active immunity, they are highly susceptible to infection unless passively immunized before exposure to these pathogens. lactogenic immunity has been shown to play a major role in providing protection to neonates from a variety of mammalian species against enteric infections (hanson et al., ; knight and peaker, ; offit and clark, ; opdebeeck, ) . this further underlines the significance of passively required antibodies from colostrum and milk in the neonatal gut for preventing these infections (hanson et al., ) . therefore, considerable efforts are underway in order to develop vaccines which will stimulate lactogenic immunity against enteric infections in order to improve the immune status of neonates and thereby, prevent early infection (babiuk et al., ) . the role of maternal antibodies in the protection of neonates against diarrhea, caused by a number of viral agents, has been shown to depend on the continual presence of antibody in the intestinal lumen (saif and smith, ) . in the case of rotavirus induced neonatal diarrhea in calves and lambs, it has been experimen-tally shown that by feeding colostrum, milk, or serum containing suffcient anti-rotavirus antibodies, infection of the intestine can be prevented (snodgrass et al., ) . the degree of protection not only correlated with the quantity of anti-rotavirus antibodies present but also with the subtype of the ig involved (snodgrass et al., ) . local immunity against rotavirus can perform two functions. first. the presence of antibody in the intestine should protect the neonate from disease. secondly, it should help in the reduction of rotavirus in feces and hence reduce environmental contamination (saif and smith, ) . since the mouse model being used in our protective-challenge experiments against rotavirus infection, utilizes neonates at days of age, the time when mouse enterocytes have the highest number of rotavirus specific receptors, thereby making them more susceptible to infection (riepenhoff-talty et al., ) . we studied lacteal antibody responses spanning this time frame, plus a few days beyond. to achieve this, different routes of immunization were evaluated. although administration of live or killed brv at mucosal sites (intestine and mammary regions) not only induced a significant elevation of iga, igm and igg antibodies in lacteal secretions there was also a marked increase in serum anti-rotavirus antibodies as determined by elisa. this might be due to rapid transmission of the viral antigen from mucosal sites to other body sites. therefore, the sites of inoculation of antigen might only serve as a "portal of entry". it could also be due to spillover of mucosally produced igs into the circulation (chang et al., ) . the most prominent feature of this study and other studies, employing the murine model (halsey et al., ) , is the increase of milk antibody production approximately days following parturition. the increase in milk igs on day could occur as a result of the production of antibodies by sensitized cells residing within the mammary glands, which are turned on by hormones along with the influx of antibodies from the serum (halsey et al., ) . it has been shown by halsey et al. ( ) that such a transfer does take place in the mouse with as much as % of the milk iga on the fourth day of lactation coming from serum as compared to % on the eighth day. the rest of the iga is likely produced locally by sensitized cells, in the mouse model. in contrast, there is no evidence for local production of iga in the mammary gland in early or mid-lactation in sheep (a.j. husband et al., ; th international congress of immunology, toronto, canada, . . ). hanson et al. ( ) could not observe any transfer of dimeric iga from the circulatory system to milk in rats. bohl and saif ( ) vaccinated pregnant cows via the imam route with live attenuated transmissible gastroenteritis (tge) virus and found that antibodies in milk from these animals were primarily of the igg class. in contrast, after natural infection with tge virus via the oral route, they observed that the antibodies in lacteal secretions are mainly of the iga class (bohl et al., ) . thus, it suggests that the route of infection or vaccinations with virus may influence the ig class in secretions. although in the present study the majority of antibody in milk was of the igg class, in natural infection with rotavirus it is the secretory iga which is closely related to protection in mice and humans because of its resistance to degradation by trypsin, chymotrypsins, and pepsin (offit and clark, ) . the reason for relatively higher igm titers than iga in lacteal secretions is not clear. it could, however, reflect the primary nature of the immune response, as rotavirus-free mice were used in this study. also in the case of imam route of immunization, antigen was administered intradermally in the mammary regions. even though the igm and igg antibodies were predominant in lacteal secretions, their effectiveness in preventing enteric infection in mice has been questioned (newby, ) . since iga is not absorbed from the intestine, it will remain locally and therefore should be more effective in preventing infection as compared to igg and igm which are absorbed (hanson et al., ) . in rats and mice, milk antibody is probably locally produced since a marked number of ig-containing cells are present in the mammary gland from late pregnancy through parturition and lactation to involution (lee et al., ) . thus, inoculation of antigen(s) into the mammary gland of the rat during pregnancy results in an increase of specific ig-containing cells of iga and igg subtype (lee et al., ) . in contrast, the majority of milk igs in ruminants, are essentially serumderived, and the lactating gland contains few ig-containing cells (saif and smith, ) . thus the physiology of the mammary gland of the specific species being immunized must be taken into consideration when designing immunization protocols for controlling enteric infections in neonates. the results of our study do not correlate with the work of oflit and clark ( ) particularly regarding the antibody titer in serum and milk. following oral immunization they reported that anti-rotavirus antibody response in serum and milk, even with homotypic virus. was approximately fold and go-fold lower, than that found after parenteral inoculation. whereas in our study, different routes of immunization did not seem to make any difference as far as antibody titer in serum and milk were concerned. the plaque reduction neutralization (prn) titer of milk in particular was much lower ( log,,,) after oral immunization compared to parenteral immunization (offit and clark, ) . even though one would expect high antibody titer in milk when immunizations are carried out using the mucosal versus the parenteral route. no explanation for this obvious difference was given by the authors. whether the differences in our study and their work is related to different quantities and quality of antigen remains to be determined. further studies are required to explore possible factors influencing the regulation of lactogenic antibody response in mice and other animals against rotavirus if effective control measures are to be implemented. these studies must address the effect of hormones and neuropeptides on the development of immunity and secretion of igs (diamond, ; halsey et al., ; stanisz et al., ; weisz-carrington, ) . recent studies clearly indicate that neuropeptides can modulate in vitro immune response either in a positive or a negative manner. furthermore, different antibody subtypes may be altered more than others (stanisz et al., ; th international congress of immunology, toronto, ontario, canada, . . ). these studies clearly indicate the potential of regulating specific lactogenic immune responses by neuroimmunoregulation. the role of neuropeptides and hormones in the regulation of anti-rotavirus lactogenic immunity is presently under investigation. finally, selective production of iga by an antisuppressor (as) mechanism may selectively enhance iga production and thereby contribute to intestinal and lactogenic immune regulation (ernst et al. ; th international congress of immunology, toronto, canada, . . ). an understanding of these mechanisms and appropriate exploitation should greatly aid in the enhancement of rotavirus immunity in the neonate and newborn and thus have a tremendous impact on the morbidity and mortality due to rotavirus infections in animals and humans. rotavirus isolation and cultivation i n the presence of trypsin rotavirus and coronavirus infections i n animals enzyme immunoassays for viral diseases we would like to acknowledge the excellent technical assistance of donna dent and barry carroll, glen gifford for preparation of the figures and irene kosokowsky for typing the manuscripts.financial support was provided by the medical research council, agriculturalcanada and the natural sciences and engineering research council of canada. key: cord- - n a authors: leu, sy-jye; lee, yu-ching; shih, neng-yao; huang, i-jen; liu, ko-jiunn; lu, hsu-feng; huang, shih-yi; yang, yi-yuan title: generation and characterization of anti-α-enolase single-chain antibodies in chicken date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: n a it was previously reported that up-regulation of α-enolase protein was detected in % of patients with non-small cell lung cancers (nsclc). moreover, a high titer of anti-α-enolase antibodies was developed in a smaller proportion ( . %) of these patients than in non-tumor-associated patients and healthy subjects. in the present study, we characterized polyclonal and single-chain variable fragment (scfv) anti-α-enolase antibodies from immunized chickens. the e. coli-derived recombinant α-enolase protein was purified to its high homogenicity as verified by sds-page. after the th immunization, a high titer of specific polyclonal anti-α-enolase antibodies was elicited in immunized chickens and specifically recognized the purified human α-enolase antigen as determined by western blot and elisa. the expressed heavy and light chain variable genes (vh and vl) were isolated from spleen b cells and amplified to construct phage antibody libraries containing scfv molecules. after four rounds of panning selection, the scfv antibodies of randomly chosen clones were expressed and their binding specificity to α-enolase protein was verified using competitive elisa, flow cytometry and immunofluorescence staining. nucleotide sequence analysis from α-enolase binding clones showed that ( %) clones used identical heavy and light genes for scfv antibody expression, as represented by enl . notably, amino acid changes in complementarity-determining regions (cdrs) were more frequently observed than those in framework regions (frs) in all clones, indicating a strong affinity selection through mutations. all together, it is believed that these polyclonal and scfv igy antibodies may be helpful in the development of molecular diagnostic and therapeutic agents for lung cancers in the future. enolase was originally characterized as an enzyme involved in glycolytic metabolism catalyzing the conversion of -phosphoglycerate into phosphoenolpyruvate. in mammals, there are three isoforms of enolase, called ␣-eno , ␤-eno and ␥-eno (redlitz et al., ) . the ␣-enolase is a major form of enolase present in the early stages of embryonic development; it is expressed ubiquitously in various types of tissue, whereas ␥-eno and ␤-eno are exclusively found in neuron and muscle cells (antikainen et al., ; chang et al., ) . by using an alternative translation start codon, eno can be translated into c-myc promoter binding protein (mbp- ), which is preferentially localized in cell nuclei (feo et al., ) . both eno and mbp- proteins are known to be negative regulators for c-myc expression. recently, it was reported that ␣-enolase is a multifunctional protein which exhibits enzymatic, structural, and receptor functions (chang et al., ; lee et al., ) . in addition to its glycolytic function, ␣-enolase has been found to play an important role in several biological and pathophysiological processes (sirover, (sirover, , . in particular, several studies have found ␣-enolase to play important roles in tumorigenesis. for example, redlitz et al. ( ) found ␣-enolase on the cell surface, functioning as one of the plasminogen receptors which may play a role in tumor invasion. up-regulation of ␣-enolase has also been reported in several highly tumorigenic or metastatic cell lines (chang et al., ; ito et al., ; peebles et al., ; wu et al., ; zhang et al., ) . ␣-enolase over-expression has been correlated with tumorigenicity in several types of cancer, which suggests its pathophysiologic role in cancer formation (altenberg and greulich, ) . furthermore, an autoantigen of ␣enolase was identified in non-small cell lung cancer and its over-expression was highly correlated with poor survival outcomes (chang et al., ) . in addition to its roles in cancer, ␣-enolase has been implicated in numerous diseases, including autoimmune disorders, ischaemia and bacterial infection (antikainen et al., ; bogdanos et al., ; gitlits et al., ; jiang et al., ; kinloch et al., ; saulot et al., ) . generation of monoclonal antibodies with high specificity to various target antigens through traditional hybridoma technology is an expensive and tedious process (groves and morris, ) . in recent years, a phage display system which is more cost and time effective involving the in vitro cloning of antibody heavy and light genes, has been extensively used to identify and isolate monoclonal antibodies from recombinant antibody repertories (barbas et al., ; winter et al., ) . smaller recombinant engineered antibody fragments, e.g. antigenbinding fragment (fab) or single-chain variable fragment (scfv), are now emerging as new applications in diagnosis and therapy for the biotechnology market. scfv molecules in which the vh and vl domains are joined with a flexible polypeptide linker usually retain the specific, monovalent antigen-binding affinity of the parent igg, while showing improved pharmacokinetics for tissue penetration (holliger and hudson, ) . among all the animals suitable for antigen immunization, domestic chicken is the most feasible and available host for scfv antibody library construction to screen efficient binders against various pathogen infections for diagnostic applications (fehrsen et al., ; finlay et al., ; park et al., ) . from the aforementioned studies, ␣-enolase might serve as a potential target for diagnostic or therapeutic application clinically. accordingly, in the present study, we generated and characterized the anti-␣-enolase polyclonal igy antibodies in chicken and monoclonal scfv antibodies by a phage display system using elisa, western blotting, flow cytometry and immunofluorescence staining. the gene encoding ␣-enolase protein was cloned from pe cells by reverse transcription-pcr using genespecific primers -ggtggaattctatctattctcaagatcc-atgcc- (forward) and -actccatggttacttggccaa-ggggtttct- (reverse). the pe cell line was originally obtained from effusion tumor cells of a -year-old patient with stage iv lung adenocarcinoma, which was kindly provided by dr. neng-yao shih from national institute of cancer research, national health research institutes, tainan, taiwan. the resultant pcr fragment was cloned into the pgex-kg vector at ecori and ncoi sites and transformed into the e. coli bl- (de ) strain for expression. the gene was also subcloned into the pet a vector using -ccgcgtgaattcggggatccatgtctattctcaagatcc- (forward) and -catggagtcgacctcgagcttggccaagg-ggtttctg- (reverse) primers and expressed as hisfused ␣-enolase. individual clones were grown in ml lb medium containing ampicillin ( g/ml) at • c overnight. the bacterial culture was diluted -fold in the same lb medium and further grown until the od reached between . and . . to induce gst-fused or his-fused ␣-enolase protein expression, isopropyl-␤d-thiogalactopyranoside (iptg) was added to a final concentration of . mm in the culture. the cell pellet was re-suspended in ml of × pbs containing % triton x- and lysed by three cycles of freezing (− • c) and thawing ( • c). after centrifugation, the resulting cellular lysate was incubated with glutathione sepharose b or a ni + -charged resin column to purify the ␣-enolase protein according to the manufacturer's instruction (general electronics, piscataway, nj, usa). female white leghorn (gallus domesticus) chickens were immunized with g purified ␣-enolase in an equal volume of freund's complete adjuvant by an intramuscular injection. three additional immunizations with incomplete adjuvant were performed at intervals of days. after each immunization, igy antibodies in sera and egg yolk were collected and titrated by an enzyme-linked immunosorbent assay (elisa) to determine the presence of humoral anti-␣-enolase immune response. egg yolk was separated from the egg white for igy purification using % dextran sul-phate as described previously (akita and nakai, a,b) . the purified total igy antibodies from each egg were dissolved in ml of tbs containing . % sodium azide and stored at − • c. the antibody library was established based on the previous report (andris-widhopf et al., ) . briefly, chicken spleens were harvested days after the final immunization. two hundred milligrams of spleen were homogenized in liquid nitrogen and placed in ml trizol solution for rna extraction using the manufacturer's protocol (invitrogen, usa). ten micrograms of total rna was reversely transcribed into the first-strand cdna using a super-script rt kit (invitrogen, usa). after amplification using chicken-specific primers (andris-widhopf et al., ) , pcr products of heavy and light chain variable (vh and vl) regions were subjected to a second round of pcr with a short-or long-linker (andris-widhopf et al., ) to form full-length scfv fragments, which were further digested with sfii and cloned into the pcomb x vector. recombinant dnas were transformed into e. coli xl- blue strain by electroporation. recombinant phage production was initiated by the addition of vcs-m helper phage, precipitated with % polyethylglycol and % nacl (w/v), and finally resuspended in phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) and stored at • c. then, plaque-forming units (pfu) of recombinant phages from the scfv antibody library were added to wells precoated with ␣-enolase protein ( . g/well), and incubated at • c for h. after removing unbound phages, bound phages were eluted with . m hcl/glycine (ph . )/ . % bsa, neutralized with m tris base buffer and used to infect the xl- blue strain. the amplified phages were precipitated and recovered as described above for the next round of selection. the panning procedure was repeated three or four times. a panel of clones were randomly selected and grown from the final panning process. after . mm iptg induction for h, bacterial cells were collected and lysed by three cycles of freezing and thawing and/or sonication. the supernatants were analyzed for their scfv antibody expression and binding reactivity to ␣-enolase using western blotting and elisa. scfv antibodies expressed in top f e. coli (invitrogen, a nonsuppressor strain) and purified using ni + -charged sepharose as described by the manufacturer (amersham biosciences, uk) were also prepared in flow cytometric and immunofluoresence analyses. to detect the scfv antibody expression, the cellular lysates were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page). all the proteins were transferred onto nitrocellulose membranes (amersham biosciences, uk), which were then blocked with % skim milk in tbst for h. polyclonal goat anti-chicken igy light chain antibodies (bethyl laboratories, montgomery, tx, usa) were added at : dilution and incubated for an additional hour. the membranes were washed with tbst three times for min each. the bound antibodies were detected by adding horseradish peroxidase (hrp)-conjugated donkey anti-goat ig antibodies (sigma, st. louis, mo, usa) at : dilution. after three washings, the membranes were developed with diaminobenzidine (dab) substrate until the desired intensity was reached. to examine their binding reactivity, the igy purified from chicken week after th immunization or the expressed scfv antibodies were incubated with the purified ␣enolase immobilized on nitrocellulose membranes or elisa plate wells. it was subsequently detected by adding goat anti-chicken igy light chain and hrp-conjugated donkey anti-goat ig antibodies as described (lee et al., a,b) . the elisa tests were done in the duplicated wells for each sample. the nucleotide sequence determination of heavy and light variable regions from chosen clones was carried out by an auto-sequencer machine (abi prism ; perkinelmer, national health research institute) using ompseq ( -aagacagctatcgcgattgcagtg- ) and hrml-f ( -ggtggttcctctagatcttcc- ) primers. the results were analyzed using alignment program blast and vector nti (http://www.ncbi.nlm.nih.gov/blast). the overall mutation rate was defined as: (number of amino acids that differ between cloned and germline genes/total number of amino acids in germline gene) × %. the nucleotide sequences of heavy and light chain variable regions of enl , enl , enl , enl and enl scfv clones were deposited in gen-bank (accession numbers are , , , and , respectively). the pe cells have been cultured in rpmi supplemented with % fetal bovine serum, mmol/l glutamine, and antibiotics for at least passages in vitro. a total of × cells were harvested and fixed with % paraformaldehyde as described (abcam, ma, usa). the ␣-enolase expressed in the pe cells was detected with purified scfv enl and enl antibodies, visualized with mouse anti-ha ( : ) and cy- -conjugated goat anti-mouse antibodies ( : ) (jackson immunoresearch laboratories, west grove, pa, usa). the results were analyzed using the facscan flow cytometer (becton dickinson, franklin lakes, nj, usa). negative controls were performed as described above, omitting the primary scfv enl and enl antibodies, while positive controls were performed using rabbit polyclonal anti-human enolase antibodies ( : ) (kelowna inc., taipei, taiwan) instead of scfv enl and enl antibodies. pe cells ( × cells/ml) were seeded on cover glass and fixed by incubating with % paraformaldehyde (freshly prepared by mixing equal volume of % paraformaldehyde in × pbs solution) (biotrin international ltd., dublin, ireland) on ice for min. after fixing, the cells were dehy- drated in a sequential treatment of %, % and % methanol and rehydrated with % and % methanol. the slides were then overlaid with blocking buffer ( %bsa in × pbs) at rt for hr. following washing with × pbs, scfv antibodies were incubated with cells at rt for one additional h. finally, their binding to ␣-enolase protein was detected by mouse anti-ha antibodies, followed by goat anti-mouse antibodies conjugated with cy- . nuclei were also counterstained with pi solution as suggested (invitrogen, usa). we examined the slides with a confocal spectral microscope imaging system (tcs sp , leica). to produce antigen for chicken immunization, the ␣enolase gene was cloned, expressed as both his-fused and gst-fused recombinant proteins and purified as described in section . after electrophoresis and coomassie blue staining, purified his-fused and gst-fused ␣-enolase were visualized as a single band of and kd (lanes and in fig. a, respectively) . the identity of gst-fused ␣-enolase was verified using anti-gst antibodies as shown in lane , fig. b . similarly, polyclonal igy antibodies produced in chickens immunized with purified his-fused ␣-enolase were able to clearly recognize both his-fused and gstfused ␣-enolase immobilized on western blots (lanes and in fig. c ). sera and eggs were collected from chickens before and after each immunization. we purified total igy antibod-ies in egg yolk and detected the presence of their heavy and light chain fragments using horse radish peroxidaseconjugated anti-chicken igy antibodies (data not shown). the purified igy antibodies were used to test for their binding activity to ␣-enolase immobilized on nitrocellulose membrane (fig. c ) or elisa plate wells. as shown in fig. , the igy antibodies prepared from the egg yolk after the th immunization specifically bind to ␣-enolase but not bovine serum albumin when titered at : , dilution, suggesting a strong humoral antibody response was elicited in the chicken host. in contrast, the igy antibod- fig. . humoral igy responses in chicken after the th immunization analyzed by elisa. purified ␣-enolase protein and bovine serum antigen (bsa) were coated on plate wells. a series of diluted igy antibodies were examined for their specific binding activity to either ␣-enolase or bsa. solid and open bars represent the binding of igy from th-or pre-immunized chickens to ␣-enolase, respectively. in addition, their binding to bsa presented by deep and light gray bars were shown in parallel as negative controls. ies from pre-immunized chicken eggs showed very little binding signal to either antigen. the chickens were sacrificed weeks after the last immunization and total rna was extracted from the enlarged spleens for antibody library construction. the amplification of full-length scfv gene fragments was carried out using consecutive pcr steps. in the primary pcr, vh gene products were amplified as bp in size using primers containing short (ggssrss) and long (ggssrssssggggsgggg) linkers as presented as envh.s (fig. a, lane ) and envh.l (fig. a, lane ) , respectively. accordingly, the vl gene was amplified as a band of bp and loaded in lane in fig. a (envl) . subsequently, the amplified vh and vl were joined to form full-length scfv gene fragments of approximately bp presented as enscfv.s (lane ) and enscfv.l (lane ) in fig. b . several phage displaying antibody libraries were constructed and two of which were used to screen the specific anti-␣enolase scfv antibodies (table ) . four rounds of panning cycles were carried out as described above. after each panning, fifteen clones were randomly selected and analyzed for bp fragment inserts in the pcom x cloning vector and for scfv antibody protein expression. our data (not shown) indicated that % ( / ), % ( / ), % ( / ) and % ( / ) of clones from each round of panning had the full-length inserts. moreover, ten clones with bp inserts from the last round of panning were induced to express their scfv antibodies. these results indicated that the expression level of immunoglobulin genes with highly conserved sequences could be dramatically different even under identical experimental conditions. the nucleotide sequences of the variable regions of heavy and light chain genes of clones were determined and aligned to the germline gene sequences of chicken immunoglobulin. the results revealed that % of the sequenced clones (enl , enl and enl ) shared identical heavy and light genes leading to the similar profiles of their scfv antibody expression as seen in lanes - in fig. . the overall mutation rates as compared to the germline gene sequences range from . % to . % and from . % to . % in heavy and light chain variable regions, respectively (fig. ) . the binding activities of the expressed enl table the size of anti-␣-enolase scfv gene libraries and eluted phage titer of each panning. to enl scfv antibodies against ␣-enolase were analyzed using elisa. we found that those scfv antibody fragments exhibit significant binding activity to ␣-enolase as compared to other scfv antibodies which were previously characterized and known to specifically recognize sars-cov spike protein. in particular, enl , enl , enl and enl scfv antibodies showed stronger positive reactivity than polyclonal igy purified from chicken immunized with the human ␣-enolase molecule (fig. ) . to test the binding reactivity of these cloned scfv antibodies, human ␣-enolase intrinsically expressed in pe tumor cells was analyzed for its expression on the membrane surface which was subsequently analyzed by flow cytometry. enl and enl scfv antibodies purified as a single band on sds-page (data not shown) were able to detect ␣-enolase protein in pe cells, and the binding signal is comparable to that of commercially available rabbit polyclonal antibodies specific for ␣-enolase as demonstrated in fig. . we also applied immunocytochemical staining to assess the binding ability of purified enl and enl scfv antibodies against ␣-enolase molecules expressed in pe cells. it has been demonstrated that the ␣-enolase molecule is mainly expressed and translocated on the nuclear membrane of pe cells (personal communication with dr. n-y shih). accordingly, our recombinant enl and enl scfv antibodies exhibited significant binding signal around the cell nuclear membrane as shown in fig. . in contrast, two negative controls using cy- -conjugated goat anti-mouse antibodies only or l clone expressing a scfv antibody specific for sars-cov spike protein instead showed no reactivity at all. the cell morphology and distribution under light microscopy are included in the most left panel for fig. . sequence analysis of vl and vh genes of scfv antibodies. the nucleotide sequences of vh and vl of clones were determined and translated into amino acid sequences to be aligned with those of the chicken germline gene. fr: framework region; cdr: complementarity-determining region. sequence gaps were introduced to maximize the alignment; these are indicated by blank spaces. dots indicate the consensus sequences. framework region (fr) and complementarity-determining region (cdr) boundaries are indicated above germline sequences. binding activity of scfv antibodies to purified ␣-enolase analyzed by elisa. cellular lysates containing scfv antibodies from randomly selected clones from the th panning cycle were examined for their binding to purified ␣-enolase coated onto the plate wells. binding activity was detected using the goat anti-chicken light chain antibodies at : dilution, followed by hrp-conjugated donkey anti-goat igg and measured at nm. two anti-sars-cov scfv antibodies (scos-s and scos-l ) were used as negative controls. one additional control experiment was carried out as described without adding primary recombinant scfv antibodies. polyclonal igy antibodies from chickens immunized with purified ␣-enolase were used as a positive control. the elisa data were represented as means of the duplicated experiments. comparison. taken together, the results provided further evidence to show that phage display technology might be a better alternative over hybridoma protocol for the cloning and generation of scfv antibodies against specific antigens. the objective of this study was to produce and characterize a panel of recombinant antibodies against human ␣-enolase for their potential diagnostic application in the future. the recombinant ␣-enolase proteins were successfully expressed in e. coli and purified to a great extent of homogeneity as shown in fig. . a specific humoral response in immunized chickens was steadily observed after a nd booster injection. the polyclonal igy antibodies from the th immunization showed a strong reactivity to ␣-enolase antigen using a : , dilution. moreover, the high titer of anti-␣-enolase igy antibodies was continu- fig. . binding activity of scfv antibodies to purified ␣-enolase analyzed by flow cytometry. surface associated ␣-enolase on pe cells was detected using purified enl and enl scfv antibodies, mouse anti-ha ( : ) and cy- -conjugated goat anti-mouse antibodies ( : ) . the grey thin lines indicate negative control, cells treated with dmso alone and stained with fluorescence-labeled abs against surface markers; the gray solid lines indicate cells stained with fluorescence-labeled ig isotype controls; and the black solid lines indicate cells stained with scfv antibodies enl and enl fluorescence-labeled abs against surface ␣-enolase. the results of one representative experiment of three separate experiments are shown. fig. . immunofluorescent staining of ␣-enolase protein in pe cells. cells were fixed and their ␣-enolase expression was detected using purified enl and enl scfv antibodies, followed by mouse anti-ha and cy- -conjugated goat anti-mouse antibodies. the nucleus (red) was visualized by pi staining. both enl and enl scfv antibodies clearly stained nuclear membrane (green) in pe cells. an anti-sars-cov scfv antibody, l , did not show any reactivity with nuclear membrane. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) ously detected months after final immunization (data not shown). taken together, our data indicated that ␣-enolase protein is highly immunogenic in experimental chicken, and this system has been applied to elicit polyclonal igy antibodies with high specificity against numerous genetically distinctive antigens (chalghoumi et al., ; mine and kovacs-nolan, ; shin et al., ) . the expression of scfv antibody in e. coli was examined on randomly chosen clones after the th panning step. as is often encountered in protein expression studies, the expressed levels and stability in our characterized scfv clones containing evolutionarily close immunoglobulin genes were also unpredictable and various (graves et al., ; jana and deb, ) . as shown in fig. , enl , enl and enl scfv antibodies were barely detected as determined on western blots as compared to enl , enl and enl clones. indeed, the amount of expressed scfv antibodies of enl , enl and enl was clearly visualized on coomassie blue-stained sds-page (data not shown). these clones exhibited identical protein expression patterns, suggesting they might be derived from one parental clone. this speculation was further supported by subsequent sequence analysis on the heavy and light chain variable genes of these clones. we did not detect any scfv antibodies expressed in clone enl or enl under identical conditions. combined with the fact that all the immunoglobulin genes are highly conserved as mentioned earlier, our data indicate that the levels of their expression as scfv molecules in e. coli cells vary tremendously from clone to clone. numerous antibodies composed of single-chain variable fragment (scfv) have been cloned and generated against broad tumor-associated antigens using a phage display system (robinson et al., ; sun et al., ) . we have constructed several immune antibody libraries from the spleen of chicken immunized with ␣-enolase by this novel technology. of these antibody libraries, two, one constructed with a short-linker ( × clones) and the other with a long-linker ( . × clones), were panned against recombinant human ␣-enolase. several specific scfv antibodies with high binding activity, as represented by clones enl and enl , were obtained from the long-linker containing library. on the contrast, the scfv antibodies characterized in the short-linker containing library are of low affinity and specificity. our pilot studies demonstrated that the specific anti-␣-enolase scfv antibody clones could not be readily detected in the original library constructed with a short-linker in a plaque lift assay (data not shown), suggesting the heavy and light variable genes specific for ␣-enolase might not be amplified, cloned, expressed or displayed on the surface of m viral particles. however, the reasons for such results are not exactly known. the power of the phage display technology is its effectiveness to enrich specific antibodies with high affinity in an in vitro panning procedure (barbas et al., ) . sequence analysis data indicated all the clones contained heavy and light chain variable genes derived from the chicken germline in which clones (enl , enl and enl ) use the same heavy and light genes for their scfv antibody expression together with the finding that the ratio of recombinant clones with full-length of scfv gene inserts increased from % to % during the panning cycles, the data supported very strongly that mono-specific anti-␣enolase with high affinity could be steadily generated using phage display antibody technology. notably, high mutation rates ranging from . % to % were found in cdr regions of both vl and vh genes; in particular the entire cdr region of vh genes of chicken germline was substituted by amino acids with various length and sequences in all the characterized clones (fig. ) . it was previously reported that somatic hypermutations to increase antibody affinity occurred more frequently in the cdr than fr regions of the rearranged v genes (gearhart and bogenhagen, ) . thus, all enl clones in the study accumulated more mutations in their cdr regions as a result of affinity selection of b cells. as compared to the traditional hybridoma method, our present study indicated that it is feasible to generate poly-specific igy antibodies in chicken and subsequently mono-specific scfv antibodies using phage display technology. these scfv antibodies could be applied to delineate the potential biological functions of ␣-enolase protein played in the lung carcinogenesis and to develop valuable reagents for clinical applications. comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic e. coli strain production and purification of fab' fragments from chicken egg yolk immunoglobulin y (igy) genes of glycolysis are ubiquitously overexpressed in cancer classes methods for the generation of chicken monoclonal antibody fragments by phage display enolases from gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits assembly of combinatorial antibody libraries on phage surfaces: the gene iii site antibodies to soluble liver antigen and alpha-enolase in patients with autoimmune hepatitis production of hen egg yolk immunoglobulins simultaneously directed against salmonella enteritidis and salmonella typhimurium in the same egg yolk identification of alpha-enolase as an autoantigen in lung cancer: its overexpression is associated with clinical outcomes serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scfvs in inhibition elisas eno gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with myc promoter-binding protein generation of high-affinity chicken single-chain fv antibody fragments for measurement of the pseudonitzschia pungens toxin domoic acid clusters of point mutations are found exclusively around rearranged antibody variable genes disease association, origin, and clinical relevance of autoantibodies to the glycolytic enzyme enolase enhancing stability and expression of recombinant human hemoglobin in e. coli: progress in the development of a recombinant hboc source veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas engineered antibody fragments and the rise of single domains differential expression of the human alpha-enolase gene in oral epithelium and squamous cell carcinoma strategies for efficient production of heterologous proteins in escherichia coli v-src induces expression of hypoxia-inducible factor (hif- ) and transcription of genes encoding vascular endothelial growth factor and enolase : involvement of hif- in tumor progression identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in behcet's disease chicken single-chain variable fragments against the sars-cov spike protein a dominant antigenic epitope on sars-cov spike protein identified by an avian singlechain variable fragment (scfv)-expressing phage chicken egg yolk antibodies as therapeutics in enteric infectious disease: a review development and characterization of a recombinant chicken single-chain fv antibody detecting eimeria acervulina sporozoite antigen proteomic analysis of a neoplastic mouse lung epithelial cell line whose tumorigenicity has been abrogated by transfection with the gap junction structural gene for connexin the role of an enolaserelated molecule in plasminogen binding to cells targeting erbb and erbb with a bispecific singlechain fv enhances targeting selectivity and induces a therapeutic effect in vitro presence of autoantibodies to the glycolytic enzyme alpha-enolase in sera from patients with early rheumatoid arthritis use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of helicobacter pylori infection emerging new functions of the glycolytic protein, glyceraldehyde- -phosphate dehydrogenase, in mammalian cells new insights into an old protein: the functional diversity of mammalian glyceraldehyde- -phosphate dehydrogenase phagedisplay selection on tumor histological specimens with laser capture microdissection making antibodies by phage display technology identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines this study was supported by grants nsc - -b- - , nsc- - -b- - , nsc - -b- - and nsc - -b- - -my (yi-yuan yang) from the national science council (nsc) and doh -td-g- - (neng-yao shih) from national health research institutes (nhri) in taiwan. key: cord- -t t p y authors: mathur, gagan; mathur, sweta title: antibody testing for covid- : can it be used as a screening tool in areas with low prevalence? date: - - journal: am j clin pathol doi: . /ajcp/aqaa sha: doc_id: cord_uid: t t p y nan severe acute respiratory syndrome coronavirus (sars-cov- ) is the virus that causes the respiratory illness coronavirus disease (covid- ) that has infected millions of people worldwide. soon after detection of spread of sars-cov- in the united states (us), focus was on developing molecular nucleic acid detection tests (real-time reverse transcriptase polymerase chain reaction [rt-pcr]) for early diagnosis of infection in symptomatic patients, patients with known exposure, and patients who are at risk. molecular testing is ramping up all over the us, and more than . million people have been tested by the end of april . different parts of the us have been hit with varying intensity. new york state has been hit hardest, with more than , confirmed cases and many more presumed to be infected and with a prevalence of covid- expected to be as high as % to %, similar to that seen in italy, spain, and some parts of china. serologic studies in other parts of the us that have a lower covid- attack rate have shown a lower prevalence (< %- %). with early signs of "flattening of the curve," as a result of more people adhering to social distancing and staying at home, the focus has now shifted to widespread antibody (serologic) testing of the population. antibody tests are blood tests that detect antibodies or immunoglobins (ig) that are produced as human immune response to sars-cov- infection. a positive result suggests that the individual has potentially been exposed to sars-cov- . when igm antibodies are present, they can indicate an active or recent infection. igg antibodies show up later in infection and can often indicate a past infection but does not exclude recently infected patients who can still be contagious, especially when igm antibodies are also concurrently detected. for viral infections igg antibodies usually persist longer than igm antibodies and provide immunity from reinfection, but this is not known for covid- yet. antibody tests are being developed to detect igg only, both igg and igm, or total antibodies. the trump administration and the media have been promoting antibody tests as a screening tool to allow individuals with positive results to get back to work and open our economy. the assumption is that the individuals with positive antibody tests have recovered from covid- (symptomatic or asymptomatic) infection and have developed immunity to the virus. furthermore, it is assumed that such individuals are thus no longer susceptible to infection and can return to work safely without fear of getting infected or spreading the infection. however, to be used as a robust and successful screening tool, an antibody test should have a high positive predictive value (ppv), ie, positive results can be trusted as true positive with confidence. ppv is dependent on the accuracy of the test (sensitivity and specificity) and the prevalence of disease in the population and can be calculated by using the following formula: as of april , , antibody tests have been approved by the us food and drug administration (fda) under emergency use authorizations. average sensitivity and specificity of fda-approved antibody tests is . % and . %, respectively. the details of fda-approved tests are shown ❚table ❚. approximately antibody tests offered by various manufactures are still under fda review but are available in the market, without established sensitivity and specificity, being used in hospitals and clinics as a screening ❚table ❚ tool. given variable prevalence of covid- ( %- %) in different parts of the us and differences in performance characteristics of antibody tests (fda approved and unapproved), statistically the ppv will vary widely and can be as low as % to % in areas with low prevalence ❚table ❚. the antibody tests can be a reliable screening tool in areas with high prevalence of covid- , such as the new york city tristate area (new york, new jersey, and connecticut) and massachusetts. however, in areas with lower prevalence, the antibody tests will need to have high accuracy ( % specificity) and consistent performance. this can be hard to achieve when there is limited availability of fda-approved antibody tests and many hospitals and clinics are using fda unapproved tests. prematurely promoting antibody tests as a screening tool all over the us will give individuals, who test positive and are not actually immune to covid- , a false sense of protection. these individuals can still get infected and further spread the infection, potentially leading to a second wave of sars-cov- infections. antibody tests are still essential and accurate tests should be developed, as they are critical to diagnose certain cases (negative molecular tests in patients presenting late in illness), identify asymptomatic infections, determine the seroprevalence in a given population, and track progression towards herd immunity over a longer period of time. antibody tests can also be used along with molecular tests for contact tracing. all these initiatives will help expedite reopening of the economy and returning the us population back to a "new" normal. summary of laboratory testing results reported to cdc covid- in italy: impact of containment measures and prevalence estimates of infection in the general population covid- antibody seroprevalence in us food and drug administration key: cord- -lgyc sro authors: stiehm, e. richard; orange, jordan s.; ballow, mark; lehman, heather title: therapeutic use of immunoglobulins date: - - journal: adv pediatr doi: . /j.yapd. . . sha: doc_id: cord_uid: lgyc sro nan medical science and thereby placed in the hands of the physician a victorious weapon against illness and death.' ' since then antibodies in multiple forms (animal and human serums, immune globulins and monoclonal antibodies) have been developed, primarily for prevention of infectious diseases, and less commonly for their treatment. these antibodies are presented in table . this section reviews their uses, with an emphasis on their value in the treatment of human infections, as summarized in table . antibody works by several mechanisms. it can neutralize viruses and bacterial toxins, lyse bacteria with the aid of complement, prevent the spread of microbes to adjacent cells or along nerve roots, coat bacteria for opsonization by phagocytes, block microbial attachment by saturating microbial receptors, and facilitate lysis of infected cells by binding them to cytotoxic cells with an fc receptor. antibody is particularly valuable in bacterial diseases associated with toxin production because much of the tissue damage results from action of the toxin; these can be neutralized rapidly by antibody before antibiotics kill the bacterium. anthrax (bacillus anthracis) anthrax is a rare but serious infection, predominantly of ruminant animals, caused by an aerobic gram-positive rod [ ] . humans are infected through the skin (cutaneous anthrax), by ingestion (gastrointestinal anthrax), or by inhalation of anthrax spores (inhalational anthrax) [ ] . the last often results from prolonged exposure to animal hides or carcasses or infected soil, and rarely by deliberate spore exposure in the bioterrorism setting. after inhalation the spores are ingested by alveolar macrophages and transported to regional nodes, where the spores germinate and release potent exotoxins. these toxins damage cell membranes, increase capillary permeability, cause pulmonary damage, and lead to shock and cardiovascular collapse. a vaccine is available for individuals at high risk for exposure and for the military. before the antibiotic era and as early as , anthrax antitoxin (usually equine) was used in therapy [ ] . an antitoxin is of value in a bioterrorism attack, both before and after exposure. the us government is collecting plasma from immunized donors to develop a human high-titer igiv [ ] . a human monoclonal antibody is being tested in animals and humans [ ] . diphtheria (corynebacterium diphtheriae). many of the adverse effects of diphtheria result from the action of its potent toxin on the heart, central nervous system, and other organs [ ] . thus the prompt use of antitoxin is indicated, in addition to antibiotics [ ] . the dose used depends on the localization and severity of infection, ranging from , units for mild infection of short duration to , units for severe illness with neck edema. the equine antitoxin is given intravenously, so must be preceded by skin testing for hypersensitivity and possible desensitization. the antitoxin is available through the us centers for disease control (cdc). a smaller dose of antitoxin can be used in asymptomatic, exposed, susceptible individuals. before the availability of diphtheria vaccine, antitoxins were given to health care workers caring for patients with diphtheria [ ] . tetanus (clostridium tetani). equine antitoxin for the treatment of tetanus was initiated by von behring in the s for toxin neutralization. extensive studies have been carried out to determine the optimal dose of antitoxin and the possible benefit of intrathecal antitoxin, particularly in tetanus neonatorum, a common problem in developing countries [ ] . since the s a human tetanus immune globulin (tig) has been available, but in some areas of the world equine antitoxin is still used. tig is given to unimmunized or incompletely immunized patients who sustain contaminated or deep puncture wounds [ ] . the recommended dose of tig is iu, along with initiation of active immunization. if tig is unavailable, human ivig can also be used; it contains variable titers of tetanus antitoxin but a minimal dose of - mg/kg is suggested for tetanus prophylaxis [ , ] . clostridium difficile gastroenteritis. clostridium difficile infection of the gastrointestinal tract is usually associated with antibiotic-associated diarrhea, often with pseudomembranous colitis and sometimes toxic megacolon [ ] toxic strains of clostridium difficile release distinct toxins, both of which have potent cytotoxic and inflammatory properties [ ] . infection generally leads to an antibody response to the toxin, and most individuals older than years have such antibodies. high levels of these antibodies acquired after colonization may result in the asymptomatic carrier state [ ] . some patients with symptomatic infection, many of whom are immunodeficient or immunosuppressed, develop antibiotic-resistant diarrhea; many have low or absent igg antibodies to toxin a. such patients may respond to ivig given to mg/kg every to weeks [ ] . such therapy increases antitoxin levels, controls the diarrhea, and prevents relapses [ , ] . controlled trials have not been performed. botulism (clostridium botulinum). botulism is a severe paralytic poisoning resulting for the ingestion or absorption of neurotoxin or spores of clostridium botulinum. several variants are recognized: food poisoning from ingestion of contaminated canned food, wound botulism from a contaminated soft-tissue infection, inhalational botulism among individuals working with the toxin or in a bioterrorist event, infantile botulism (see next section), and adult-type infant botulism in adults with preexisting gastrointestinal disease [ ] [ ] [ ] . in the last types, ingested spores multiply in the gastrointestinal tract to elaborate toxin; the absorbed toxin results in a paralytic disorder. a few cases of botulism have been associated with use of botulism toxin for cosmetic use [ , ] . an heptavalent fab fragment equine antitoxin (hbat) to types a, b, c, d, e, f and g is available in the united states through the cdc [ , ] . sensitivity testing must be conducted before their use. antitoxin to all types is given unless the toxin type is known. additional doses may be needed in severe wound botulism. antitoxin can also by used prophylactically in individuals known to have ingested contaminated food. it is not used for infantile botulism. infantile botulism (clostridium botulinum). this severe paralytic disorder of infants results from the ingestion of clostridium botulinum spores in baby formulae or food, resulting in slow onset of constipation, abdominal bloating, poor feeding, and respiratory paralysis [ ] . such infants must be hospitalized for prolonged periods for tube feeding and respiratory support, often for as long as to months. human iv botulism immune globulin is available for treatment of infantile botulism [ ] . despite its high cost ($ , per vial) it is cost-effective because of the shortened hospital stay needed. gas gangrene (clostridium perfringens). there is no antitoxin for gas gangrene. respiratory infections with streptoccocci, streptococcus pneumonia, haemophilus influenzae, and neisseria meningitides are reduced in immunodeficient patients receiving immunoglobulin therapy. these patients include young infants with poor antibody responses to polysaccharide antigens, patients infected with the human immunodeficiency virus (hiv), and patients with primary antibody immunodeficiencies. before antibiotics, immune serum or animal serum was used as therapy for severe bacterial infection [ , ] . other studies suggest that a large dose of ivig decreases the frequency of otitis in patients with recurrent otitis and normal immunity [ ] . thus regular use of ivig in antibody-deficient patients in doses of to mg/kg every to weeks or an equivalent amount given subcutaneously decreases the frequency and severity of otitis and other respiratory tract infections [ , ] . circulating antibody may play a role in the prevention and treatment of invasive group a streptococcal infection [ ] . newborns with transplacental antibody and patients on ivig rarely develop streptococcal illnesses. equine antitoxin was used with some success in the treatment of erysipelas and scarlet fever in the s and s [ ] . a preventive vaccine against the streptococcal m protein has been contemplated but is not yet unavailable. treatment with ivig, in addition to antibiotics, is probably beneficial [ , ] . streptococcal pyrogenic exotoxins types a, b, and c and mitogenic factor elaborated by certain strains of streptococci may be responsible for these complications. these exotoxins are potent superantigens that activate certain t lymphocytes directly, leading to synthesis and/or release of multiple cytokines with resultant shock, fever, and organ failure. ivig contains neutralizing antibodies to these antigens of varying titers from batch to batch [ ] . despite this variability ivig is recommended, in addition to antibiotics, in the management of these infections, not only to neutralize pyrogenic toxins but to dampen cytokine storm and release [ ] . controlled trials are unavailable but case reports and large series compared with historical controls are encouraging [ ] . large doses of ivig are recommended (eg, - g/kg over several days). staphylococcal infections are ubiquitous and of varying severity, ranging from superficial skin infections to deep-seated cellulitis, osteomyelitis, and overwhelming shock [ , ] . these severe infections occur when the organism is resistant to antibiotics or is a strain associated with toxin production. one well-recognized syndrome is toxic shock associated with tampon use in menstruating women [ ] . this syndrome results from release of the toxic shock syndrome toxin- , a potent superantigen that initiates the release of multiple cytokines and a clinical picture of rapidly progressive fever, shock, and organ failure. most authorities recommend a high dose of ivig to neutralize the toxin and dampen cytokine storm [ , ] . a second situation in which ivig may be of value is in neonatal staphylococcal infection, usually coagulase-negative staphylococcus epidermidis. this is the most common cause of sepsis in premature infants and is aggravated in part by the use of catheters and central lines [ , ] . one controlled study indicated that ivig was of value in decreasing the incidence of this infection [ ] . other studies were not confirmatory, possibly because of differences in titer for the protective antibodies [ ] . immunoglobulin is also used in the treatment of antibiotic-resistant staphylococcal infection. older studies from waisbren [ ] and current studies from russia suggest clinical benefit [ ] . animal studies support such a combined approach [ ] . newborns, particularly premature newborns with birth weight less than g are potential candidates for immunoglobulin therapy in view of the frequency and severity of infections. all newborns have low levels of igm and iga, and, if premature, a deficiency of transplacental maternal igg, the deficiency of which is proportional to the degree of immaturity [ ] . premature infants also have defects in antibody synthesis, complement levels, opsonic activity, neutrophil mobilization and killing, and cellular immune responses [ ] . accordingly several studies sought to determine the value of igiv in the prevention or early treatment of infection in premature infants. these studies differ in terms of entry criteria, immunoglobulin dose and duration, and end points (eg, type and severity of infection, survival). meta-analyses of prospective, randomized, placebo-controlled prevention studies suggest a slight reduction ( %) in the frequency of sepsis but no difference in mortality, length of nursery stay, or other complications of prematurity [ ] [ ] [ ] . by contrast meta-analysis of controlled studies for the treatment of proven sepsis, involving premature infants, showed that igiv therapy reduced mortality from % to %, a significant difference [ ] . there was a suggestive benefit for infants with suspected sepsis also. infants with neutropenia may particularly benefit. because a common cause of neonatal sepsis is staphylococcus epidermidis, a hyperimmune staphylococcal ivig may be of particular benefit in the prevention of neonatal sepsis. two recent studies of igiv from either immunized donors (altastaph) [ ] or selected donors with high titers to a fibrinogen-binding protein (veronate) [ ] did not show a significantly decreased incidence of infection. studies of monoclonal antibodies to staphylococcal antigens are in progress. thus the national institutes of health consensus statement that igiv should not be given routinely to infants of low birth weight but that it may be of value in selected premature newborns with proven or suspected infection remains valid [ ] . patients undergoing severe stress associated with trauma, extensive surgery, or intensive care have profound exposure to and susceptibility to infection, usually as a result of enteric gram-negative infections [ , ] . monoclonal antibodies, igm-enriched igiv, and regular igiv have been studied in these situations with inconclusive results [ ] . laupland and colleagues [ ] reviewed randomized trials of igiv and found suggestive benefit in terms of length of stay in the intensive care unit (icu) and mortality. similar studies in pediatric patients in the icu have not been performed. despite the lack of controlled trials, igiv is often used in critically ill patients, particularly neutropenic patients, because of possible benefit and rare side effects. although many viral diseases are prevented by immunoglobulin, just a few are amenable to antibody therapy, as presented in table . this section focuses on some viral diseases in which antibodies can be used in therapy. although smallpox (variola) has been eradicated from the world since , immunization with live vaccinia virus (cowpox virus) is still used by the military and by certain laboratory personnel working with vaccinia [ ] . further, smallpox is a potential bioterrorism weapon so a supply of vaccinia immune globulin (vig) is being stockpiled by the us government for complications of smallpox vaccine and for a response to biological warfare. kempe [ ] used immune globulin from vaccinated individuals (vig) to prevent the spread in a outbreak of smallpox in madras, india. he also showed that vig could be used to treat the not infrequent complications of smallpox vaccine including vaccinia eczematum, generalized vaccinia, autoinoculation, and prevention of spread to high-risk individuals exposed to a recently vaccinated individual. vig, both for iv and intramuscular (im) use, is prepared from vaccinated donors and is commercially available. the usual dose is mg/kg [ ] . parvovirus is a dna virus that causes fifth disease (slapped cheek syndrome, a common exanthem of childhood that usually provides lifelong immunity to subsequent exposure [ ] ). parvovirus infects erythroid progenitors (its receptor is the common red cell p antigen) to cause red cell aplasia in patients with congenital or acquired immunodeficiencies including hiv, immunosuppressed organ transplant recipients, and patients with sickle cell disease [ ] [ ] [ ] . igiv contains neutralizing antibody to parvovirus such that prolonged highdose therapy can eradicate the infection. parvovirus infection during pregnancy can also cause fetal hydrops [ ] . arthritis and chronic fatigue syndrome are uncommon manifestations of chronic parvovirus infections [ , ] . the ivig dose needed to eradicate parvovirus in not established but is large ( - g/kg) and should be repeated until the virus is eradicated as indicated by serum polymerase chain reaction analysis [ , ] . antibodies to cytomegalovirus (cmv) either in the form of hyperimmune iv cmv immune globulin (cmvig-cytogam) or regular igiv have been used for more than a decade to prevent cmv infection in recipients of bone marrow and solid organ transplant [ ] . cmvig is prepared from donors with high anti-cmv titers but regular igiv also contains cmv antibodies at lower titers. testing of donor and recipient for cmv infection, the use of cmv antibodynegative blood donors, and the use of antiviral drugs have greatly reduced the indications for cmv antibody [ ] . cmvig is still used in heart and heartlung transplants (along with antivirals) if either the donor or the recipient is cmv-seropositive [ ] . cmvig is also of suggestive benefit in severe cmv pneumonitis along with antiviral treatment [ ] . cmvig may also be of value for in utero cmv infection; infusions of cmvig were given intraperitoneally at and weeks to a cmv-infected fetus, with possible benefit [ ] . nigro and colleagues [ ] gave pregnant women with primary cmv infection cmvig during pregnancy; some women received additional cmvig into the amniotic sac or umbilical cord. only one woman gave birth to an infant with cmv infection compared with cmv infection in of infants of control women who did not receive antibody therapy. these data are encouraging but are not from well-controlled studies. thus the use of cmvig in recipients of organ transplant, severe cmv infections, or in utero cmv infections is unproved but of suggestive therapeutic benefit. transplacental maternal antibody has a proven preventive effect in herpes simplex virus (hsv) infection in the newborn period: mothers with a reactivated herpex infection (ie, preexisting infection) during delivery are -fold less likely to transmit hsv to their newborn infants during vaginal delivery than are mothers with primary hsv infection acquired during late pregnancy [ ] . masci and colleagues [ ] used ivig to prevent recurrent genital hsv infection with suggestive benefit. the value of hsv monoclonal antibody or ivig is being evaluated for treatment of disseminated neonatal disease. epstein-barr virus (ebv) antibodies are present in variable titers in ivig, particularly in cmvig, because donors with high titers of cmv often have high titers of ebv. a few patients with posttransplant ebv-induced lymphoproliferative syndrome or hepatitis have been treated successfully with a combination of igiv or cmvig, antiviral therapy and interferon-a [ ] [ ] [ ] . similar results have been achieved in ebv infection in x-linked lymphoproliferative syndrome: such patients have a hereditary predisposition to overwhelming ebv infection [ ] . varicella-zoster immune globulin (vzig), available since , is prepared from plasma with high titers to vz virus [ ] . the commercial product vari-zig is used for the prevention or modification of susceptible high-risk immunodeficient or immunosuppressed children exposed to chickenpox or shingles. it is also used in susceptible women during late pregnancy, newborn infants whose mother develops chickenpox perinatally, and exposed premature infants of less than weeks' gestation. it is not of benefit in established chickenpox or zoster infection [ ] . encephalomyelitis. before poliovirus vaccine was introduced, immunoglobulin was used in the prevention of poliomyelitis [ ] . immunodeficient individuals are susceptible to chronic enteroviral encephalitis, usually echovirus or coxsackievirus or less commonly, attenuated poliovirus vaccine strains [ ] [ ] [ ] [ ] . regular doses of igiv given to antibody-deficient patients have markedly reduced the frequency of enterovirus encephalitis in these patients. attenuated poliovirus has been replaced in many countries by inactivated (salk) vaccine. high-dose ivig (sufficient to increase the serum igg levels to mg/ml) has been used successfully in immunodeficient patients with enteroviral encephalomyelitis [ ] [ ] [ ] [ ] . some patients have been given intrathecal infusions [ , ] . not all ivig-treated patients are cured: some may have viral strains for which the ivig has no neutralizing antibody. for these instances typing of the cerebrospinal fluid and treatment with selective ivig units with antibodies to the infecting serotype may be necessary. antiviral therapy with pleconoril has also been used [ ] . neonatal enteroviral infection. severe and sometimes fatal disseminated enterovirus infection can develop in neonates [ ] [ ] [ ] . high-dose ivig has been used in such infants with suggested benefit in decreasing the severity of the illness [ ] . maternal plasma may also be used in the likelihood that the mother has antibody to the organism involved [ ] . ivig has also been used to prevent spread to unaffected infants in a nursery [ ] . unless the titer in the ivig is known, large doses are recommended. an increasingly important use of hyperimmune hepatitis b immune globulin (hbig) is to prevent hepatitis b recurrence in hepatitis b-seropositive recipients of liver transplant, many of whom are transplanted because of complications of hepatitis b [ , ] . hepatitis b reoccurs in half of the patients in years [ ] . such recurrences can be reduced significantly by giving large doses of hbig for a prolonged period beginning at the time of transplantation and continuing indefinitely after transplantation [ ] . antiviral agents such as lamivudine are also given simultaneously. the dose of hbig after transplantation is varied so as to maintain a continuous serum anti-hbs titer. hepatitis b vaccine can also be given to induce active immunity. the types of hbig available include the % igim used for prophylaxis in newborns of hepatic b-positive mothers and for unimmunized exposed susceptibles and a % hbig for iv use in liver transplantation. the use of the latter adds a considerable cost to liver transplantation. the university of california at los angeles medical center spends $ , per year on hbig, nearly all for the liver transplant program. a hyperimmune hepatitis c immune globulin for hepatitis c liver transplantation is also under study. monoclonal antibodies to hepatitis b and c are under development. west nile fever. west nile fever, caused by the west nile virus, is common in many tropical regions where culex mosquitoes are endemic. it has spread to europe and the united states, and can also be transmitted by infected blood and organ transplantation. several case reports and animal studies suggest that ivig prepared from seropositive donors modifies the severity and mortality [ , ] . ebola. ebola virus, a filivirus, causes severe and often fatal hemorrhagic fever in tropical africa. there is no effective antiviral agent. goat hyperimmune serum protected guinea pigs from experimental infection if given within hours of exposure. this product was used for emergency prophylaxis in patients exposed by a laboratory accident. only one developed mild infection [ ] . equine serum has protected monkeys against low-dose virus challenge but not high-dose virus challenge [ ] . blood from convalescing patients has also been used with promising results [ ] . other animal antisera have been developed, as have monoclonal antibodies. tick-borne encephalitis. tick-borne encephalitis caused by a flavivirus is endemic in central europe. a vaccine is available as is a hyperimmune immune globulin. a combination has been also used [ , ] . argentine hemorrhagic fever. argentine hemorrhagic fever caused by the junin virus has a high mortality from vascular or neurologic complications. maiztegui and colleagues [ ] found that immune plasma given before the ninth day of illness reduced mortality to % among patients given immune plasma compared with . % mortality among patient given normal plasma. severe acute respiratory distress syndrome. convalescent plasma and ivig have been used in the treatment of severe acute respiratory distress syndrome caused by a corona virus. studies were inconclusive [ ] . antibody is a time-honored way to prevent viral infection after exposure, and has a crucial role in the treatment of bacterial diseases associated with toxin production. it is also of value in prevention of certain viral infections as well as in the treatment of parvovius, enterovirus infection, and certain regional viral infections. polyclonal immunoglobulin, now used in scores of diverse disorders [ ] [ ] [ ] , was first used in the prevention of infectious diseases. in , ogden bruton [ ] reported a child with agammaglobulinemia and initiated the first use of repeat injections of immunoglobulin as replacement therapy. in his report c-globulin fractionated from human plasma was administered subcutaneously to an -year-old boy who had no known c-globulin in a serum protein electorophoresis. this child had multiple infections, including episodes of septicemia, which were ameliorated by chronic treatment with the immunoglobulin. this experience represented the dawn of immunoglobulin therapy for primary immunodeficiency and defined its use in a disease for which no therapeutic alterative was available. since then, the study of primary immunodeficiency has expanded markedly. there are now more than distinct diagnoses, most of which have defects of humoral immunity [ ] . approximately in people are living with a primary immunodeficiency in the united states, of whom greater than % have an antibody deficiency potentially requiring immunoglobulin replacement therapy [ ] . other primary immunodeficiency registries confirm that greater than % have an antibody deficiency [ ] [ ] [ ] [ ] . treatment with immunoglobulin remains the best therapeutic option for most of these patients. characteristics of antibody immunodeficiencies appropriate for replacement therapy are presented in table . the clearest indications for immunoglobulin therapy are those associated with an absence of b cells (category i). these patients are unable to make antibodies or immunoglobulin i. examples include agammaglobulinemia and certain types of severe combined immunodeficiency. several gene defects may be responsible for these illnesses [ ] , but all need immunoglobulin replacement therapy. the next category (ii) of patients needing immunoglobulin are those who have b cells but cannot make igg and generate specific igg antibodies. because igg represents the major defense of humoral immunity against infection, these patients also require immunoglobulin replacement therapy. this diagnostic category includes the hyper igm syndrome (higm) and common variable immunodeficiency (cvid). higm is caused by several specific gene mutations [ ] , but most cvid cases have no identifiable genetic lesions [ ] . diagnosis can be made by either identifying a specific gene mutation, or by defining the quantitative and qualitative deficit of igg [ ] . as in patients in category i, continuous and uninterrupted replacement therapy with immunoglobulin is warranted. if the diagnosis is confirmed molecularly, immunoglobulin therapy must be continued. in a few cases, it may be clinically appropriate to stop immunoglobulin therapy once during a lifetime to determine if the defect is fixed [ ] . this strategy should not be repeated if the single trial indicates a persistent deficit. if a trial off immunoglobulin therapy is considered, this should be performed in late spring or summer, when respiratory infections are less prevalent. a third diagnostic category (iii) of antibody deficiencies is those associated with qualitative defects in humoral immunity [ ] . these patients have b cells and produce normal quantities of igg but the quality of igg is diminished. these individuals are unable to respond appropriately to specific antigenic challenges such as vaccinations or infections. this category includes those with specific antibody deficiency with normal immunoglobulins [ ] and certain patients with nemo (nf-kappa;-b essential modulator) deficiency [ , ] . diagnosis is made after documentation of an ineffective vaccination response, a failed humoral response to an infection, or a specific molecular/ genetic diagnosis linked to this category [ ] . a fourth category (iv) includes patients with lower than expected levels of igg but who are able to mount effective antibody responses. this category forms a subset of individuals referred to as having ''isolated hypogammaglobulinemia'' when only the igg level is low. although hypogammaglobulinemia can be a component of many immunologic defects, in isolated hypogammaglobulinemia antibody quality is adequate, with normal responses to vaccination or infection. because the normal age-specific ranges of igg define the lower limit at the . th percentile, one of individuals has low levels of igg. the question becomes, when there is no deficit of antibody quality, is isolated hypogammaglobulinemia clinically a problem? it is also important to discern when hypogammaglobulinemia represents a primary versus a secondary problem with increased loss of igg. examples of the latter include draining chylothorax [ ] or intestinal lymphangiectasia [ ] . in these individuals, the hypogammaglobulinemia is less likely to cause a problem because antibody synthesis is intact and often accelerated. in patients with primary hypogammaglobulinemia, the level of igg that is associated with a definitive risk for infection is not defined, especially when antibody quality is intact [ ] . some insurance companies recommend replacement therapy for patients who have an igg level less than mg/dl and a history of recurrent infection. although that situation may be reasonable, questions still exist about how to manage the patient recognized as having primary hypogammaglobulinemia with low igg levels (ie, < ) but no history of infection. diagnostic examples include transient hypogammaglobulinemia of infancy (thi) [ ] [ ] [ ] or otherwise unexplained primary hypogammaglobulinemia [ ] . the former diagnosis is established in retrospect, as the igg level normalizes with age. thus, in select cases of thi immunoglobulin replacement may be considered as a temporizing measure. however, primary hypogammaglobulinemia remains a difficult diagnostic and therapeutic dilemma. other patients (a fifth diagnostic category [v]) have a deficiency of one of the major igg subclasses, igg , igg , or igg . igg deficiency is common and should not be considered an abnormality [ ] . although a deficiency of one of the major igg subclasses indicates some immunologic deviation, most of these patients have a normal total igg level, intact responses to specific antigens, and are not candidates for immunoglobulin replacement therapy. those with impaired antibody specificity do not fall in to this category, but into the third category. however, even without impairment in antibody quality, immunoglobulin replacement in some patients in a deficiency subclass does reduce the incidence of infections [ , ] . nevertheless, most insurers in the united states have additional criteria for justifying therapy in patients with igg in deficiency subclasses. a final diagnostic category is patients with recurrent infection who do not have hypogammaglobulinemia subclass deficiency or deficits of antibody quality. thus, they have infectious susceptibility without evidence of identifiable immune abnormality. the infectious burden in these individuals can be high and most certainly has an explanation, so nonhumoral diagnoses should be aggressively sought. there are also patients an explanation of whose infectious susceptibility presently evades clinical science. immunoglobulin replacement therapy has been considered in these individuals under certain circumstances. immunoglobulin preparations for antibody immunodeficiencies although bruton [ ] gave immunoglobulin to his patient by the subcutaneous (sc) route, subsequent patients until received immunoglobulin by weekly im injections [ ] . this strategy was necessary because the immunoglobulin preparations were not purified to the degree required for iv administration. in the early s, immunoglobulin preparations with low quantities of immunoglobulin aggregates were developed for iv administration. ivig and igiv have numerous advantages, including achieving high peak and trough igg levels and convenient monthly dosing regimens. although limited studies have compared ivig with imig, the iv route has become the preferred route of immunoglobulin administration worldwide [ ] . seven ivig preparations are currently approved by the us food and drug administration (fda) for replacement therapy in primary immunodeficiency (table ). each has been studied in a licensing trial in patients with primary immunodeficiency and found to be safe and effective. the primary end point in most of these clinical trials has been the prevention of serious bacterial infection compared with the expected frequency of such infections before diagnosis [ ] . the rate of infection can be surprisingly high, as shown by bruton's [ ] first patient mentioned earlier. the early diagnosis and treatment of primary immunodeficiency with immunoglobulin products has reduced morbidity and mortality and considerable savings of health care expenditures [ , ] . all ivig products are purified from human plasma pools under strict manufacturing guidelines. although each manufacturer has its own process there are more similarities than differences in the various methods. all processes remove non-igg impurities and igg aggregates and add stabilizers to prevent in vitro aggregate formation. despite these efforts, adverse reactions during ivig administration are not uncommon [ ] . all immunoglobulin manufacturers have robust measures to screen donors and to inactivate blood-borne pathogens; the safety of immunoglobulin preparations in the last decade has been superb [ ] . there are subtle differences among different ivig products from different companies; several companies have more than one product on the market [ ] . this situation can lead to confusion about which ivig to administer to which patient. in general, most ivig products are tolerated by most patients. the characteristics of the individual ivig preparations, as outlined in table , may help in selecting the best product for each patient. they differ as to concentration, stabilizers, sugar content, iga content, sodium content, and osmality. the volume of individual vials, storage requirements, need to reconstitute a lyophilized product before use, local availability, and price are also variable. many patients may tolerate one product more effectively than another. thus, when a patient tolerates a particular immunoglobulin product it is advisable to continue with that product whenever possible [ ] . three other preparations of immunoglobulin are approved by the us fda (table ) . one is approved for im administration and two for sc administration. few patients receive their immunoglobulin by the im route. the sc administration of immunoglobulin resurfaced in in the united states [ ] . subcutaneous immunoglobulin (scig) is usually given in the abdominal wall or thigh with a thin bored needle and an infusion pump, delivered over several hours. although its initial use in the united states was limited, the sc route gained popularity in europe; extensive clinical experience indicated that it was equivalent to ivig therapy [ , ] . a crossover trial with ivig and a us fda licensing trial showed that scig was equivalent to ivig in preventing infection in primary immunodeficiency [ , ] . scig has advantages and disadvantages compared with ivig therapy (many related to patient preferences) and these have been reviewed extensively [ ] [ ] [ ] . one advantage of scig over ivig is the markedly decreased incidence of systemic reactions [ , ] . another is eliminating the need for iv access or indwelling iv access devices. the most serious disadvantage is the need for more frequent administration (at least weekly) to administer sufficient immunoglobulin [ ] . another disadvantage is less frequent physician encounters because most scig infusions are given at home by caretakers or home infusion companies. the dose and frequency of immunoglobulin therapy is a complex topic and draws on both evidence-and experience-based sources. these recommendations are presented in a several reviews and consensus statements [ , , , , ] . the recommendations include starting doses of to mg/kg/mo. after several months this dose can be altered depending on the trough level and the clinical response. patients vary as to their requirement to maintain reasonable resistance to infection [ , ] . scig is typically used after the patient has been on ivig for several months. the weekly scig dose is usually one-fourth of the previous monthly ivig dose. some immunoglobulin-naive patients are started on immunoglobulin therapy with scig so the number of initial doses may need to be increased. the amount of scig given at a single site for an adult is usually ml of the % solution (ie, . g). more than one site can be used simultaneously to deliver the target dose. this procedure has been facilitated by the availability of special tubing, needle sets, catheters, and pumps. infusion site reactions are not uncommon but are rarely severe [ ] . ivig is usually administered monthly and scig is usually administered weekly, but other schedules are often used. these schedules include shorter or longer intervals between infusions of ivig to achieve a satisfactory clinical response. scig can be given biweekly, or divided into more frequent injections, even small daily doses. the latter is generally self-administered at home, well tolerated, and preferred by some patients because of the small daily dose needed [ , ] . trough levels of immunoglobulin achieved must be considered. several studies have correlated resistance to infection with specific igg trough levels. targeting a specific trough level may be feasible for patients with agammaglobulinemia who have a profound deficiency of igg [ ] but more difficult for other antibody deficiencies [ ] [ ] [ ] . in agammaglobulinemia, a trough level of mg/dl is a minimally acceptable level and mg/dl a more desirable trough level [ , ] . these recommendations may not be appropriate in other disorders in which baseline igg levels and antibody titers are variable; in these cases the clinical response must be considered. polyclonal immunoglobulin is essential therapy for the primary antibody immunodeficiency diseases. the different disorders in which immunoglobulin therapy are used are reviewed. several immunoglobulin products are available for their treatment; they have similar therapeutic properties but there are individual differences among the available products. immunoglobulin can be given either intravenously (ivig) or subcutaneously (scig). dosage, frequency of infusions, achieved trough levels, and advantages and disadvantages of ivig and scig are discussed. the early s witnessed an increase in the use of ivig as an immunomodulator for inflammatory and autoimmune disorders. more than % of the ivig prescribed is for patients with autoimmune and inflammatory diseases, despite the fact that ivig is approved for just a handful of indications (box ). in the late s, this situation led to an ivig shortage, compromising those patients who depend on igg replacement therapy to correct their underlying antibody deficiency. in , the american academy of allergy, asthma and immunology's committee on primary immunodeficiency evaluated the use of ivig for multiple disorders. the strength of the evidence for a beneficial effect and the basis for this recommendation were classified (box ). this section reviews the use of ivig for the autoimmune and inflammatory conditions in this report (box ), in the context of a review of the mechanisms of action of ivig in these conditions. the multiple effects of ivig on the innate and adaptive immune system are illustrated in fig. . historical note: ivig in immune thrombocytopenic purpura the first use of ivig for an autoimmune process was in children with immune thrombocytopenic purpura (itp). imbach and colleagues [ ] observed that antibody-deficient patients receiving ivig who also had itp had a marked increase in platelet count after ivig infusions. subsequently, these investigators examined the therapeutic effects of ivig in children with a primary diagnosis of itp; they used high-dose ivig ( mg/kg) for consecutive days. the investigators reported a dramatic increase in platelet count within hours of the administration of ivig. in some patients, the increase in platelet count was sustained; in others, repeat ivig treatments were necessary. box presents the indications for ivig in autoimmune cytopenias as well its likely benefit. several hypotheses have been proposed to explain the rapid increase in platelet count (or other antibody-coated cells) after ivig administration. the most accepted hypothesis is that high-dose ivig induces an fc receptor blockade of reticuloendothelial cells in the liver and spleen, preventing them from removing antibody-sensitized cells. debre and colleagues [ ] provided evidence for this hypothesis when they infused fcc fragments in children with itp, and showed an increase in platelet count after the infusion. the fc receptor blockade theory may account for the rapid increase in platelet count after the ivig infusion, but not for the longterm benefits of ivig. thus additional mechanisms have been sought. one such mechanism, supported by animal studies, is that ivig stimulates inhibitory fccriib receptors found on a variety of cell types including b cells that in turn inhibit antibody and immune function [ ] . samuelsson and colleagues [ ] showed in a mouse model of itp that ivig suppresses or inhibits antiplatelet antibody production through this fccriib receptor. subsequently ravetch and colleagues [ , ] identified distinct motifs in the ivig that have a propensity to engage and activate the fccriib inhibitor receptor that inhibits antibody synthesis. these distinct properties were attributed to the carbohydrate moiety in the ivig molecule, representing about % of the total igg molecule. more than different covalently attached carbohydrate glycans in the igg molecule have been identified. glycosylation of the igg is essential for binding to all fcc receptors. the important glycan moiety in the igg molecule is attached to the asparagine (asn ) in the second domain of the constant region of the igg molecule. using a k/bxn serum-induced arthritis model in mice, kaneko [ ] showed that igg at g/kg inhibited the inflammatory arthritic process. deglycosylated or neuraminidase-treated ivigs were unable to inhibit this inflammation. kaneko then showed that ivig enriched for the sialylated glycan moiety had comparable inhibitory effects on the inflammatory process at one-tenth of the dosage used with intact ivig. this investigator showed that this inhibitory activity resided in the igg fc fragment, and was dependent on fccriib expression on effector macrophages. anthony and colleagues [ ] have engineered a recombinant/sialylated human igg fc protein that had the same immune modulating activity as native ivig. these investigators showed that the action of sialylated fc in the rheumatoid arthritis mouse model is mediated through the interaction of sialylated fc with the sign-r receptor on macrophages [ ] . the investigators propose that the interaction between sialylated fc and sign-r produces an antiinflammatory state that upregulates inhibitory fccriib receptors on effector cells, making these cells more resistant to triggering by immune complexes. they suggest that dc-sign, the human homolog of sign-r , has a comparable role for the antiinflammatory effects of igg fc fragments. another mechanism proposed by yu and lennon [ ] suggested that the administration of high-dose ivig augments the catabolism of endogenous serum igg. igg catabolism occurs through a process by which the igg molecule binds to a specialized fc receptor found on endothelial cells (eg, fcrn), which protects the igg molecule from normal catabolism and its removal from the plasma. this process accounts for the long serum igg half-life ( days). high-dose ivig saturates the fcrn receptor, resulting in the accelerated catabolism of autoantibodies [ ] [ ] . hansen and balthasar [ ] have supporting data in a rat model of immune thrombocytopenia using monoclonal antibodies. the uses of ivig in several autoimmune inflammatory neuropathies are presented in box . the fda has recently approved the use of ivig in chronic inflammatory demyelinating polyneuropathy. this table also shows the evidence-based efficacy of ivig in rheumatic disorders. aside from the mechanisms involving the fccriib inhibitory receptor and the accelerated catabolism of autoimmune antibodies through the fcrn receptor, it has also been proposed that the administration of ivig can regulate autoreactive b cells by restoring the idiotypic-antiidiotypic network. other autoimmune diseases may be associated with a deficiency of these antiidiotypic antibodies, which are believed to regulate the production and activity of these autoantibodies (box ). kazatchkine and colleagues [ ] showed that f(ab ) fragments prepared from ivig could bind to several autoantibodies (eg, antifactor viii, antithyroglobulin, anti-dna, antiintrinsic factor, neutrophil cytoplasmic antigens), and thus lead to increased catabolism of these autoimmune antibodies and prevent them from inducing tissue injury [ ] . these investigators postulated that ivig may work, at least in part, in certain autoimmune diseases by neutralizing the functional activity of various autoantibodies or inhibiting their binding to their respective autoantigens [ ] . another mechanism by which ivig may benefit autoimmune disease is by preventing the uptake of complement on target tissues. berger and colleagues [ ] showed that high concentrations of igg inhibit the uptake of c on antibodysensitized erythrocytes. thus, any inflammatory or autoimmune process that involves a c b-or c b-dependent process could be modulated by ivig therapy. this situation is best exemplified in patients with dermatomyositis in whom the disease is mediated by activation of c and deposition of the membrane attack complex on the endomysial capillaries [ ] . treatment with ivig inhibits complement-induced inflammation by decreasing complement deposition on the endomysial capillaries of muscle tissues [ , ] . this mechanism of ivig is relevant not only in dermatomyositis but also in guillain-barré syndrome and myasthenia gravis [ , ] . as shown in box , ivig is used in many other inflammatory diseases. however, the evidence-based data for several of these diseases are not so strong as some of the autoimmune disorders discussed earlier. nevertheless, one inflammatory disease in which ivig may be beneficial is toxic epidermal necrolysis or stevens-johnson syndrome. patients with toxic epidermal necrolysis have high levels of serum-soluble fas ligand that bind to fas receptors on keratinocytes to induce apoptosis (cell death). viard and colleagues [ ] showed that the anti-fas antibodies in ivig block the interaction of fas ligand with fas receptors on the keratinocytes, preventing destruction of the epithelium. ivig contains antibodies to several cell-surface molecules [ ] including antibodies to a -peptide sequence containing the (arg-gly-asp) motif that is expressed on cell surfaces and matrix proteins that are part of the integrin adhesion system. ivig inhibits the adhesion of b cells to fibronectin and inhibits platelet aggregation [ ] . turhan and colleagues [ ] and chang and colleagues [ ] investigated the effect of ivig on a mouse model of sickle cell acute vasoocclusive crisis, in which the adhesion of sickled red blood cells to leukocytes causes the vasoocclusive disease. in this model, high-dose ivig given after the onset of a crisis resulted in improved blood flow and prolonged survival. these investigators showed that ivig reverses acute vasoocclusive crisis in sickle cell mice by inhibiting neutrophil adhesion to the capillary endothelial cells. the various mechanisms of the antiinflammatory and immunomodulatory properties of ivig are reviewed. the first use of ivig was in the treatment of immune thrombocytopenia, presumably because of fc receptor blockade. other mechanisms are reviewed as well as the evidence for the value of ivig in multiple disorders. ivig may have yet undiscovered immunomodulating properties on both the innate and adaptive immune systems. future advances will include a better understanding of its mechanisms of action and modification of the igg molecule to enhance its immunomodulating properties. idiopathic thrombocytopenic purpura (ia-a) might provide benefit autoimmune neutropenia autoimmune hemolytic anemia (iii-d) posttransfusion purpura (iii-d) inflammatory neuropathies definitely beneficial: guillain-barré syndrome (ia-a) chronic inflammatory demyelinating polyneuropathy multifocal motor neuropathy (ia-a) probably beneficial: myasthenia gravis (ib-iia-b) eaton myasthenic syndrome (ib-a) igm antimyelin-associated glycoprotein paraprotein-associated peripheral neuropathy (ib-a) stiff man syndrome (ib-a) might provide benefit: relapsing-remitting multiple sclerosis (ia-a) intractable childhood seizures rasmussen syndrome (iib-b) acute disseminated encephalomyelitis (iii-c) lumbosacral or brachial plexitis human t-lymphotropic virus- -associated myelopathy (iii-c) postinfectious cerebellar ataxia acute idiopathic dysautonomia (iii-d) unlikely to be beneficial: demyelinating neuropathy associated with monoclonal igm (ib-a) amyotrophic lateral sclerosis (iii-c) poems syndrome paraneoplastic neuropathies (iii-c) rheumatologic and organ-specific autoimmune diseases definitely beneficial: graves ophthalmopathy (ib-a) probably beneficial: autoimmune uveitis (iia-b) might provide benefit: severe rheumatoid arthritis autoimmune diabetes mellitus (iib-b) vasculitides and antineutrophil antibody syndromes systemic lupus erythematosus (iii-d) unlikely to be beneficial: antiphospholipid antibody syndrome toxic epidermal necrolysis/ stevens-johnson syndrome (iia-b) might provide benefit: steroid-dependent asthma (ib-a) prevention of acute humoral rejection in renal transplants (ib-a) treatment of acute humoral rejection in renal transplants pediatric autoimmune neuropsychiatric disorder associated with streptococcus (pandas) (iib-b) subset of women (repeat second-trimester loss) with spontaneous recurrent abortions unlikely to be beneficial: nonsteroid-dependent asthma (ib-a) prevention of chronic gvhd after bone marrow transplantation (ib-a) chronic fatigue syndrome atopic dermatitis (iia-b) use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology principles and practice of infectious diseases studies on anthrax: clinical report of ten human cases hhs to buy , courses of anthrax antitoxin. available at: cidrap.umn raxibacumab 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infectiosum and idiopathic thrombocytopenic purpura parvovirus infection and its treatment report of the committee on infectious diseases persistent parvovirus-associated chronic fatigue treated with high dose intravenous immunoglobulin persistent b parvovirus infection in patients infected with human immunodeficiency virus type (hiv- ): a treatable cause of anemia with aids chronic pure red cell aplasia caused by parvovirus b in aids: use of intravenous immunoglobulin: a report of eight patients cytomegalovirus infection in kidney transplantation: prophylaxis and management clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation comparison of combined prophylaxis of cytomegalovirus hyperimmune globulin plus ganciclovir versus cytomegalovirus hyperimmune globulin alone in high-risk heart transplant recipients immunotherapy of cmv infections intraperitoneal administration of cytomegalovirus hyperimmunoglobulin to the cytomegalovirus-infected fetus 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and active immunization chronic enteroviral meningoencephalitis in agammaglobulinemic patients intraventricular gamma-globulin for the management of enterovirus encephalitis successful treatment of echovirus meningoencephalitis in sex-linked agammaglobulinaemia by intrathecal and intravenous injection of high titer gammaglobulin chronic enteroviral meningoencephalitis, in agammaglobulinemia: case report and literature review enteroviral meningitis: natural history and outcome of pleconaril therapy neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin investigation of treatment failure in neonatal echovirus infection use of normal immunoglobulin in an echovirus outbreak in a special-care baby unit prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis b immunoglobulin liver transplantation in hbsag-positive hbv-dnanegative cirrhotics: immunoprophylaxis and long term outcome hbv-infection in liver transplantation in hbsag positive patients: experience with long-term immunoprophylaxis hyperimmune gammaglobulin for the treatment of west nile virus encephalitis using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection preparation and use of hyperimmune serum for prophylaxis and therapy of ebola virus infections evaluation of immune globulin and recombinant interferon-a b for treatment of experimental ebola virus infections treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients tick-borne encephalitis vaccination against tick-borne encephalitis (tbe): influence of simultaneous application of tbe immunoglobulin on seroconversion and rate of adverse events efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome sars: systematic review of treatment effects use of intravenous immunoglobulin in human disease: a review of 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infections and low immunoglobulins: characteristics and analysis of normalization therapy for patients with recurrent infections and low serum igg levels efficacy of intravenous gammaglobulin for immunoglobulin g subclass and/or antibody deficiency in adults the gamma globulins. iv. therapeutic uses of gamma globulin efficacy of intravenous immunoglobulin in primary humoral immunodeficiency disease early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: a retrospective survey of patients impact of a physician education and patient awareness campaign on the diagnosis and management of primary immunodeficiencies pharmacoeconomics of immunoglobulins in primary immunodeficiency adverse reactions and pathogen safety of intravenous immunoglobulin european surveillance of immunoglobulin safety-results of initial survey of patients with primary immunodeficiencies in countries differences between igiv products: impact on clinical outcome 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protects against recurrent infection replacement igg therapy and self-therapy at home improve the health-related quality of life in patients with primary antibody deficiencies quality of life and health-care resource utilization among children with primary immunodeficiency receiving home treatment with subcutaneous human immunoglobulin the effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia. a randomized, double-blind, multicenter crossover trial comparison of the efficacy of igiv-c, % (caprylate/chromatography) and igiv-sd, % as replacement therapy in primary immune deficiency. a randomized double-blind trial efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency high dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood infusion of fc gamma fragments for treatment of children with acute immune 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autoantibodies to factor viii (antihaemophilic factor) by high-dose intravenous gammaglobulin intravenous and standard immune serum globulin preparations interfere with uptake of i-c onto sensitized erythrocytes and inhibit hemolytic complement activity modulation of complement-mediated immune damage by intravenous immune globulin intravenous immune globulin for dermatomyositis controlled studies with high-dose intravenous immunoglobulin in the treatment of dermatomyositis, inclusion body myositis, and polymyositis increased in vitro uptake of the complement c b in the serum of patients with guillain-barré syndrome, myasthenia gravis and dermatomyositis intravenous immune globulin therapy for neurologic diseases inhibition of toxic epidermal necrolysis by blockade of cd with human intravenous immunoglobulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action inhibition of cell adhesion by antibodies to arg-gly-asp (rgd) in normal immunoglobulin for therapeutic use intravenous immune globulin prevents venular vasoocclusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion key: cord- - q oopaz authors: dobaño, carlota; vidal, marta; santano, rebeca; jiménez, alfons; chi, jordi; barrios, diana; ruiz-olalla, gemma; melero, natalia rodrigo; carolis, carlo; parras, daniel; serra, pau; de aguirre, paula martínez; carmona-torre, francisco; reina, gabriel; santamaria, pere; mayor, alfredo; garcía-basteiro, alberto; izquierdo, luis; aguilar, ruth; moncunill, gemma title: highly sensitive and specific multiplex antibody assays to quantify immunoglobulins m, a and g against sars-cov- antigens date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: q oopaz reliable serological tests are required to determine the prevalence of antibodies against sars-cov- antigens and to characterise immunity to the disease in order to address key knowledge gaps in the context of the covid- pandemic. quantitative suspension array technology (qsat) assays based on the xmap luminex platform overcome the limitations of rapid diagnostic tests and elisa with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy and multiplexing capacity. we developed three qsat assays to detect igm, iga and igg to a panel of eight sars-cov- antigens including spike (s), nucleoprotein (n) and membrane (m) protein constructs. the assays were optimized to minimize processing time and maximize signal to noise ratio. we evaluated the performance of the assays using plasmas obtained before the covid- pandemic (negative controls) and plasmas from individuals with sars-cov- diagnosis (positive controls), of whom were asymptomatic, had mild symptoms and were hospitalized. pre-existing igg antibodies recognizing n, m and s proteins were detected in negative controls suggestive of cross-reactive to common cold coronaviruses. the best performing antibody isotype/antigen signatures had specificities of % and sensitivities of . % at ≥ days since the onset of symptoms and . % at ≥ days since the onset of symptoms, with auc of . and . , respectively. combining multiple antibody markers as assessed by qsat assays has the highest efficiency, breadth and versatility to accurately detect low-level antibody responses for obtaining reliable data on prevalence of exposure to novel pathogens in a population. our assays will allow gaining insights into antibody correlates of immunity required for vaccine development to combat pandemics like the covid- . in a globalized world where emerging infectious diseases of broad distribution can put at stake the health and economy of millions of people, there is a need for versatile and reliable serological tools that can be readily applicable (i) to determine the seroprevalence of antibodies against any new pathogen and, more importantly, (ii) to characterise immunity to the disease at the individual and community levels. in the case of the covid- pandemic caused by sars-cov- , one of the main priorities since the beginning of the epidemics in china by the end of ( ) was to ascertain the percentage of the population that had been exposed to the virus, considering that a considerable number of people could have been asymptomatic ( ) ( ) . the lack of sensitive and specific serological tests early in the covid- pandemic delayed the precise estimation of the burden of infection for the rational implementation of public health measures to control viral spread ( ) . furthermore, immunological assays that can measure a high breadth of antibody types and specificities are needed to dissect which are the naturally acquired protective responses and identify correlates of immunity ( ) . additionally, when a vaccine becomes available, such assays would be valuable to evaluate immunogenicity of candidate vaccines and monitor duration of immunity at the population level ( ) . common tools for antibody studies are (i) rapid diagnostic tests (rdt) as point of care (poc) devices that usually measure either total immunoglobulins or igg and igm, qualitatively ( ), (ii) traditional enzyme-linked immunosorbent assays (elisa) ( ) that can quantify different isotypes and subclasses of antibodies against single antigens at a time, and that require certain previous expertise, personnel and equipment, and (iii) chemiluminescent assays (clia), widely used in clinical practice, faster and with higher throughput than elisa ( ) . the performance characteristics of the commercial kits available in the early months of the covid- pandemic were questionable ( ) , while external evaluations validating their reliability and accuracy were not published. a number of in-house elisa assays have also been developed in hospital and research laboratories ( ) , but they have the limitations that (i) a relatively large amount of sample is required, (ii) the large surface area of the individual microplate wells and the hydrophobic binding of capture antibody can lead to non-specific binding and increased background, and (iii) most elisas rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity ( ). an alternative technique that offers the benefits of elisa but also a larger dynamic range of antibody quantification and higher sensitivity ( ) ( ) is based on the xmap luminex® platform (www.luminexcorp.com/bibliography). secondary antibodies are labelled with fluorescent phycoerythrin (pe) directly or with biotin that mediates binding to streptavidin-rphycoerythrin (sape), which does not depend on an additional reaction. the technique has the added value of higher throughput (up to -well plate format), increased flexibility, and lower cost with the same workflow as elisa, particularly if using magnetic magplex® microspheres. paramagnetic beads allow for automation of workflow and better reproducibility compared to the previous generation of microplex® microspheres. since the beads have the capture antigen immobilized on their much smaller surface area compared to a -well microplate well, reduced sample volumes are required and non-specific binding is diminished ( ) . furthermore, a chief advantage over elisa is the multiplex nature of the assay that allows measuring antibodies to different antigens simultaneously. this increases the probabilities to detect a positive antibody response due to the heterogeneity of the human response and therefore it has a higher sensitivity relevant for identifying seropositive individuals. the luminex technology, capable of measuring simultaneously antibodies against (magpix®), (luminex / ®) and up to different antigens (flexmap d®), makes it an invaluable tool for antigen and epitope screening. finally, its versatility to set up adapted antigen panels makes luminex an excellent platform to ensure better preparedness for faster response to future emerging diseases and pandemics. here we report on the establishment and validation of three quantitative suspension array technology (qsat) assays to measure igm, iga and igg antibodies against eight sars-cov- antigens, based on the adaptation of previous in-house protocols that measured antibodies to other infectious diseases, including malaria ( )( )( ) ( ) . due to the need to process a large amount of samples with the minimal time and cost during a pandemic like the covid- , we optimized several conditions to reduce the duration of the assays and report them here for the three main isotypes that have proved useful for seroprevalence studies ( ) . positive samples were plasmas from individuals with a confirmed past/current diagnosis of covid- . one hundred and eleven had sars-cov- infection confirmed by real time reverse-transcriptase polymerase chain reaction (rrt-pcr). fifty-five were recruited in a study of health care workers in hospital clínic in barcelona, most of them with mild symptoms, of them hospitalized and without symptoms, all rrt-pcr positive ( ) . fiftyseven were covid- patients recruited at the clínica universidad de navarra in pamplona (spain), of which had severe symptoms and were hospitalized and had mild symptoms (one clinically diagnosed with positive radiology and serology, and negative rrt-pcr); were asymptomatic health workers with positive diagnosis confirmed by four serological tests but no rrt-pcr data. time since onset of symptoms ranged from to days. positive samples were used individually or as pools of up to samples depending on the tests. for optimization tests, only a subset of samples were used. negative controls were plasmas from healthy european donors collected before the covid- pandemic, and were used individually. numbers of positive and negative samples were in line with protocol recommendations from the foundation for innovative new diagnostics (find). ethics. samples analyzed in this study received ethical clearance for immunological evaluation and/or inclusion as controls in immunoassays, and the protocols and informed consent forms were approved by the institutional review board (irb) at hcb (refs. ceic- and hcb/ / ) or universidad de navarra (ref. un/ / ) prior to study implementation. the receptor-binding domain (rbd) of the spike (s) glycoprotein of sars-cov- , the leading vaccine candidate target, was selected as the primary antigen to develop the initial qsat assay because (i) s is one of the most immunogenic surface proteins together with the nucleocapsid protein (n) ( ) (ii) rbd is the fragment of the virus that mediates binding to the host receptor ace in the lung cells ( ) (iii) antibodies to rbd correlate with neutralizing antibodies ( )( ) that could be associated with protection based on studies of other coronaviruses and animal models ( ) ( ) ( ) ( ) , and (iv) an elisa based on this same protein has received fda approval for covid- serology ( ) . the rbd was from the krammer lab different test concentrations of protein antigens were coupled to magnetic magplex . µm cooh-microspheres from luminex corporation (austin, tx) in reactions of a maximum of , beads, at , beads/µl ( ) . first, beads were washed twice with . µl of distilled water using a magnetic separator (life technologies, d), and resuspended in µl of activation buffer, mm monobasic sodium phosphate (sigma, s ), ph . we compared the performance of the assays when a subset of positive and negative plasma samples were incubated at different dilutions with the antigen-beads for h or h at rt in relation to our previous protocol on at ºc. antigen-coupled beads, initially including rbd singleplex, were added to a -well µclear® flat bottom plate (greiner bio-one, ) at beads/well in a volume of µl/well pbs-bn. next, individual positive plasma samples (range of dilutions tested from / to / ) and individual negative controls (at the same dilutions as the positive samples), were added per plate in a final volume of µl per well. two blank control wells with beads in pbs-bn were set up in each plate to control for background signal. plates were incubated on a microplate shaker at rpm and protected from light, and then washed three times with µl/well of pbs-tween . %, using a magnetic manual washer (millipore, . for more accurate igm measurements, we tested whether diluting samples : with gullsorb™ igg inactivation reagent (meridian bioscience™) prior to testing for igm levels could reduce high responses observed in some negative samples ( ) . additionally, we tested the levels of rbd and s antibodies obtained at different plasma dilutions when incubated in a multiplex panel with additional antigenbeads including s , s , m and n constructs, compared to those obtained in singleplex, to check for potential interferences. finally, since viral proteins have diverse immunogenicity, definitive plasma dilutions were established with titration experiments in individual positive and negative samples once the final multiplex antigen panel and all assay conditions had been selected. we compared the performance of the assays when using biotinylated secondary antibodies days (top markers), ≥ days (top markers) and ≥ days (top markers). for each model, we calculated the auc and selected three seropositivity cutoffs aiming at specificities of i) %, ii) ≥ % and < %, and iii) ≥ % and < %, and obtained the corresponding sensitivity. models with % specificity and the highest sensitivities were selected for roc curve representations. the analysis was carried out using the statistical software r studio version r- . . ( ) (packages used: randomforest ( ) and proc ( )). the characteristics of sars-cov- infected participants whose plasma samples have been used in the study, with regards to age, sex, days since rrt-pcr diagnosis and days since onset of symptoms, are included in table s . the optimal amount of protein to be coupled to beads depended on the antigen and needs to be tested with each new lot. among the concentrations tested ( , or µg/ml protein), titration curves did not usually change substantially, in which case the lower concentration was chosen for the subsequent experiments. an illustrative example is shown for which the medium concentration was slightly superior when tested for igg and igm and thus selected ( figure s ). plasmodium falciparum antigens were based on on incubations at ºc ( ) . for the covid-incubations at ºc versus shorter times at rt. we tested that the range of dilutions was still adequate when reducing the incubation time (figure a ) and compared antibody levels and number of seropositive samples incubating on at ºc versus h rt at / ( figure b) . although the mfi readings in positive samples generally diminished with shorter times, the mfi readings in the negative samples also reduced, i.e. the signal to noise ratio was the same or sometimes better, maintaining or increasing the overall proportion of seropositive among the positive samples and thus the sensitivity. based on these data, we adopted the h incubation time for an initial covid- seroprevalence study ( ) . we subsequently tested shorter incubations more extensively and found that h was non-inferior to h incubation ( figure c ) and thus h was selected for the optimized sop. reduction of background in igm assay. treatment with gullsorb™ reduced or did not change the mfi signal, depending on the sample, antigen and dilution ( figure s a ). this additional incubation generally increased the signal to noise ratio and thus sensitivity and number of seropositive igm responses among the positive controls, particularly at the lower dilutions, therefore the gullsorb™ incubation was adopted for this assay ( figure s b ). igm reactivity in negative controls was lower against s-based antigens than against m-or nbased antigens and thus gullsorb™ treatment benefited the signal to noise ratio the most in these later proteins. singleplex versus multiplex antigen testing. multiplexing the antigens ( -plex panel) did not significantly decrease the mfi antibody levels to rbd or s compared to singleplex testing ( figure a ) neither for any of the other antigens ( figure s a) . interestingly, there was no evidence of any interference between rbd, s, s or s antigens despite sharing epitopes within the same multiplex panel. a number of negative pre-pandemic samples had preexisting antibodies recognizing sars-cov- proteins for certain isotypes and dilutions ( figure s a ): igg to s , s , m and n constructs, and iga to s and n-term & c-term of n. furthermore, testing plasmas against multiple antigens increased the sensitivity of the assay since some individuals who were seronegative or low responders to rbd, responded with higher antibodies to s (figure b) . once the multiplex antigen panel was established, a set of positive and negative samples were tested at different dilution(s) covering the diverse immunogenicity of the proteins, and / and / were selected for the assay performance evaluation (figures c and s b) . secondary antibodies conjugated to pe performed as well as a two-step secondary antibody conjugated to biotin followed by sape incubation (figure ) . the pe-antibody reagent that resulted in a shorter assay was selected as the preferred option. finally, min incubation was non-inferior to min incubation ( figure s ). we sought for the combination of ig and antigen responses that yielded the highest specificity (primarily), sensitivity and auc to detect seropositive responses. for rbd and s, igg and iga at / dilution, and igm responses at / , gave higher percentages of seropositive responses among the positive controls and thus were selected for the calculations; for n constructs, igg and iga performed better at / except for n c-term in which igg was better at / . antibodies to m, s (igg & iga) and n n-term (igm) did not discern well positive from negative responses and were not included in the rf models. the contribution of each antibody/antigen variable was ranked according to an rf algorithm at different periods since onset of symptoms ( figure s ) and the top - variables were selected. we performed rf for all the combination of variables and assessed the sensitivity of each combination at three different seropositivity thresholds aiming at specificities of %, % and %. the specificity of the qsat assays in samples from participants with sars-cov- positive diagnosis with ≥ days since the onset of symptoms (n= ) was up to % with sensitivity up to . %, and auc up to . , for the best combinations of ig isotypes/antigens. the top performing antibody signatures for three different seropositivity thresholds targeting specificities of %, % and % are shown in table , and their roc are shown in figure . in samples from participants with ≥ days since the onset of symptoms (n= ), the specificity was up to % and the sensitivity up to . %, with auc up to . for the best combinations of ig isotypes/antigens (table , figure ). in samples from all participants regardless of time since symptoms onset (n= ), the specificity was up to % and the sensitivity up to . %, depending on the combinations of ig isotypes/antigens, with auc up to . for the best combinations (table s , figure ). the performance of the qsat assays to predict positivity was clearly superior using combinations of multiple ig isotypes/antigens to using single isotype/antigen markers ( figure ). higher sensitivities were obtained when specificities were set to % or % ( tables , & s ), reaching % for samples ≥ or ≥ days since the onset of symptoms. we developed three novel multiplex immunoassays for quantifying igm, iga and igg to eight sars-cov- protein constructs and evaluated by machine learning classification algorithms the performance of several isotype/antigen combinations to detect any positive antibody response to infection, obtaining specificities of % and sensitivities of . % (≥ days since symptoms onset) or . % (≥ days since symptoms onset), and very high predictability (auc ≥ . ). our qsat assays, based on the xmap technology, provide the best precision, accuracy and widest range of detection compared to classical qualitative (rdt) or quantitative (elisa) assays. for any given test, there is usually a trade-off between sensitivity and specificity. to evaluate the performance of the assays here, we prioritized specificity over sensitivity for the implications that false positives may pose at a personal level and the impact that specificity has in seroprevalence studies. particularly when prevalence of infection is low, the positive predictive value of a test strongly relies on a high specificity. for example, in a scenario of % prevalence and % sensitivity, the positive predictive value of the test decreases from % to %, with a reduction in specificity from % to %. however, other seropositivity thresholds can be used to have a balanced specificity/sensitivity or to maximise sensitivity. a time period after the onset of symptoms is usually established for these analyses, because antibodies take an average - days since infection to be produced and detected depending on the isotype and test (igm - days, iga - days, igg - days) ( )( )( )( )( ) ( ) . thus, it is not necessarily expected to detect antibodies in individuals who are acutely infected and diagnosed around the time of plasma collection. accordingly, when considering all samples, which included and individuals with less than and days since onset of symptoms, respectively, sensitivity was lower (up to . %) at specificities of %. however, we detected igm or iga as early as days, and igg as early as day, from onset of symptoms. in fact, since samples were collected in the early days of the covid- pandemic, it is expected that igm and iga, which are induced upon primary infection earlier than igg, could contribute to a higher sensitivity of detection. most of the best signatures identified included igm and iga besides igg, regardless of the time period since onset of symptoms, also beyond days. however, over time, the only antibodies that would be expected to remain in blood are igg due to the decay of igm and iga, e.g. igm levels may become undetectable by the fifth week after symptoms onset ( ) . therefore, with longer days since infection, the serological assays to detect maintenance of antibodies could focus on igg detection. the superior performance of the qsat assays is partly based on direct fluorescence detection as opposed to colorimetric detection mediated by an enzyme. also, antigens are covalently coupled to beads as opposed to passive coating of the elisa plates, leading to a higher density of antigen per surface area and less antigen wash off during the assay. the higher background of elisa microplates is related to the fact that they have a much larger surface area than the combined area of microspheres, which is more prone to the binding of non-specific antibodies if blocking is not performed correctly ( ) . the sensitivities and specificities of other sars-cov- serological assays externally validated with > positive and > negative samples (as recommended by find protocols), some of them approved by the usa fda, are summarized in table s ( ) ( ) . while luminex assays generally have high correlation to elisas in singleplex (r ~ . ) ( ), it is important that the assays perform equally well in multiplex format, with no interference noted between antigens, even if they had overlapping epitopes. a key value of multiplexing is that it allows to capture a wider breadth of responses and this is needed because some individuals may not respond to one antigen (e.g. rbd) but may do so to other antigens (e.g. s or n proteins) ( )( ) ( ) . here, we substantially increased the sensitivity of the assay when combining isotypes/antigens compared to using only one isotype/antigen. the addition of n was more beneficial to detect seropositive responses when the onset of symptoms was recent, as this antigen is the most abundant and immunogenic and specific antibodies appear to be elicited earlier ( ) . in contrast, combinations of s antigens seemed to be sufficient to detect seropositive responses with longer periods since the onset of symptoms. an added advantage of multiplexing is the reduced usage of sample volume, resources and time, if antibodies to several antigens are to be evaluated. the possibility to perform miniaturized assays in small amounts of blood is very attractive in paediatric studies, in large field surveys where fingerpick may be more logistically feasible, and to test special tissues of interest including mucosal fluids. those combined advantages have a direct impact on the cost-efficacy of the qsat assay, that is overall cheaper than rdt or elisa assays. the cost of the xmap assay can be less than one-fifth of the least expensive commercial elisa and less than one-sixteenth of the most expensive commercial kit. cost is reduced because there is less protein used due to the smaller surface area and less amounts of other materials and reagents. we reduced the dilutions of plasma and titrated the secondary antibody to use the minimal amounts of samples and reagents, without compromising sensitivity. the economy of scale will improve further when the assays are adapted to high throughput flexmap d -well plate format but they are also easily adaptable to the bench top magpix -well format that is more affordable and easy to maintain even in remote laboratory settings. interestingly, positive antibody responses to m, s , s and n antigen constructs were detected in samples collected before the covid- pandemic. the presence of such antibodies has been interpreted as cross-reactivity with antigens of coronaviruses causing the common cold ( )( ) ( ) . indeed, higher sequence homology at the protein level between sars-cov- and coronaviruses has been reported for n (particularly n-terminal and central regions), m and s ( )( ) ( ) . pre-existing sars-cov- -specific t cells have been recently reported and also attributed to cross-reactivity to human coronaviruses previously encountered ( ) ( ) . the multiplex nature of the assay will allow to test this hypothesis in the future with the addition of antigens to related coronaviruses e, hku , nl and oc in the same assay panel, by comparing the patterns of antibody reactivity, in order to address the significance of this in immunity to here, antibody responses to m were very marginal and did not contribute to higher assay sensitivity and this could partly be because the purity of the protein was not high. however, this antigen may be valuable in studies establishing the antibody correlates of protection since at present the targets of immunity have not been elucidated. it is possible that, in addition to neutralizing antibodies directed to the rbd region of s, antibodies of other specificities with non-neutralizing functions, for example fc-mediated opsonisation and phagocytosis, could be relevant in protection. in fact, t cell responses to epitopes located on m have been detected at high frequencies ( ) , and it is possible that antibodies to this or other less immunogenic antigens may also have a role in protection in some individuals. in our study, the addition of s from a commercial supplier did not have any added value but for future versions of the assay we will test s from different sources, as this subunit is expected to not cross-react with other beta-coronaviruses and be specific for sars-cov- diagnostics ( ) ( ) . the assays performances were excellent but further testing needs to be performed with longer periods of time since onset of symptoms, although we do expect to maintain high specificity and sensitivity albeit antibody signatures would be different and based on igg only. future studies will include additional positive samples of asymptomatic individuals, who probably have lower antibody levels than mild or severe cases and are rarely included in the validation of commercial kits. in addition, it will be interesting to include negative controls reacting with other coronaviruses or other infections (e.g. malaria) and pathologies known to induce polyclonal responses or rheumatoid factor, which may increase background responses. in conclusion, we developed % specific and fast assays with possibly one of the best diagnostic characteristics reported in the published literature to assess seroprevalence of covid- . considering their high sensitivity, these qsat assays would be suited to identify individuals with levels of antibodies below the lower limit of detection of rdt or the lower limit of quantification of elisa, such as asymptomatic children or immunosuppressed individuals, or long-term decaying antibodies ( ) . in addition this approach would be particularly suited to identify hyper immune donors with very high levels of antibodies and the largest antigenic breadth for immunotherapy. the assays are highly versatile, being easily adaptable to quantify other antibody igg and iga subclasses and avidity with the use of chaotropic agents, and even functional activity like binding inhibition to the virus receptor ace . the multiplex capabilities make them also ideal for sizeable peptide screenings to accelerate epitope mapping and selection for identifying fine-specificity of immune correlates of protection for vaccine development, and would also be applicable in vaccine evaluation when the first candidates reach larger-scale phase and clinical trials. the assays development and sample collection were performed with internal funds from the investigators groups and institutions, and the performance analysis received support from a novel coronavirus from patients with pneumonia in china covid- : four fifths of cases are asymptomatic, china figures indicate asymptomatic sars coronavirus infection among healthcare workers, singapore. emerg infect dis the important role of serology for covid- control what policy makers need to know about covid- protective immunity immune surveillance for vaccine-preventable diseases lateral flow assays enzyme-linked immunosorbent assay (elisa) chemiluminescent immunoassay technology: what does it change in autoantibody detection? auto-immun highlights. / / serological assays for emerging coronaviruses: challenges and pitfalls luminex corporation. overcoming the cost and performance limitations of elisa with xmap(r) technology. tech note characterization and development of a luminex(®)-based assay for the detection of human il- simultaneous quantitation of cytokines using a 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sensitive than antibody to spike protein in covid- patients. medrxiv analysis of serologic cross-reactivity between common human coronaviruses and sars-cov- using coronavirus antigen microarray. biorxiv antigenic crossreactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses e and oc sars-cov- specific antibody responses in covid- patients beyond the spike: identification of viral targets of the antibody response to sars-cov- in covid- patients. medrxiv targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals presence of sars-cov- reactive t cells in covid- patients and healthy donors. medrxiv serological signatures of sars-cov- infection: implications for antibody-based diagnostics. medrxiv we thank the volunteers who donated blood for covid- studies and the clinical and laboratory staff who participated in the sample collection and processing. special thanks to key: cord- -nnm n a authors: varadé, jezabel; magadán, susana; gonzález-fernández, África title: human immunology and immunotherapy: main achievements and challenges date: - - journal: cell mol immunol doi: . /s - - - sha: doc_id: cord_uid: nnm n a the immune system is a fascinating world of cells, soluble factors, interacting cells, and tissues, all of which are interconnected. the highly complex nature of the immune system makes it difficult to view it as a whole, but researchers are now trying to put all the pieces of the puzzle together to obtain a more complete picture. the development of new specialized equipment and immunological techniques, genetic approaches, animal models, and a long list of monoclonal antibodies, among many other factors, are improving our knowledge of this sophisticated system. the different types of cell subsets, soluble factors, membrane molecules, and cell functionalities are some aspects that we are starting to understand, together with their roles in health, aging, and illness. this knowledge is filling many of the gaps, and in some cases, it has led to changes in our previous assumptions; e.g., adaptive immune cells were previously thought to be unique memory cells until trained innate immunity was observed, and several innate immune cells with features similar to those of cytokine-secreting t cells have been discovered. moreover, we have improved our knowledge not only regarding immune-mediated illnesses and how the immune system works and interacts with other systems and components (such as the microbiome) but also in terms of ways to manipulate this system through immunotherapy. the development of different types of immunotherapies, including vaccines (prophylactic and therapeutic), and the use of pathogens, monoclonal antibodies, recombinant proteins, cytokines, and cellular immunotherapies, are changing the way in which we approach many diseases, especially cancer. the knowledge of human immunology has improved exponentially in recent years, and more advances in the near future are certainly imminent. the immune system is extremely complex, but we are now developing new tools and skills to study it. several factors have been involved in these advancements, and the most important ones include the development of thousands of different monoclonal antibodies that allow the identification of a large variety of cell subpopulations and the functional analysis of immune cells. these tools, together with new and sophisticated technologies, such as single-cell analysis, imaging techniques, omics (including massive dna-rna sequencing, proteomics, and metabolomics data and new tools for processing these data, such as artificial intelligence and machine learning approaches, mathematical modeling, etc.), newly designed animal models (using conventional transgenic/knockout/knock-in mice or new technologies such as crispr-cas (clustered regularly interspaced short palindromic repeats-crispr-associated protein ), are increasing our knowledge about how our immune system functions. the study of the interaction between the immune system and other systems, such as the nervous and endocrine systems or the microbiome, in several illnesses has produced interesting results with important clinical applications. all of these advances can be applied to several immunemediated pathologies, but overall, the success achieved with some types of immunotherapies in recent years is revealing new ways to explore and manipulate the immune system for our benefit. writing a review about human immunology is a significant challenge, but we have attempted to bring together recent knowledge about the immune system, immune-mediated illnesses and types of immunotherapies. the last two decades have witnessed a major revolution in the field of immunology. the traditional classification of the immune system into two different arms, namely, innate and adaptive components that collaborate to respond to foreign antigens or to perform self-/nonself-discrimination, has become much more complex. the development and application of new technologies have provided new findings and created a new landscape in which the immune system establishes cross talk, not only between immune components but also with commensal microorganisms , and other important systems, such as the endocrine and nervous systems [ ] [ ] [ ] . these developments have forced immunologists to reformulate the immunological architecture that confers protection, which has made the study of the immune system especially attractive. moreover, these advances have led to an increased interest in better understanding, managing, and manipulating the immune response in both health and disease. the characterization of new immune cell subsets has been a constant feature in the immunology field. this evolution is clearly reflected in the discovery of an innate counterpart of t lymphocytes, collectively named innate lymphoid cells (ilcs) , and in the identification of different types of effector cd and regulatory t cells . innate lymphoid cells (ilcs). ilcs are lymphocytes, but in contrast to adaptive immune cells, they can colonize lymphoid and barrier tissue sites during fetal development, do not undergo somatic recombination and do not express antigen-specific receptors , . in addition to lymphoid organs, ilcs are enriched in barrier tissues, such as the gastrointestinal tract, airways, and skin , . these innate cells have been considered to be tissue-resident cells, but recent studies suggest that ilcs can migrate through the lymphatic system during homeostasis or enter into the circulation upon infection and inflammation , . currently, five different ilcs are defined on the basis of their transcription factor expression, different cytokine production and/or developmental patterns : natural killer (nk) cells (discussed below), lymphoid tissue inducer cells (ltis) and three subsets of helper-like ilcs (ilc s, ilc s, and ilc s), which are considered to be the innate counterparts of t helper (th) , th , and th cells, respectively. the main focus of this review is ilcs. ilc s are dependent on the t-box transcription factor t-bet and produce interferon gamma (inf-γ), but they differ in the expression of eomesodermin transcription factor . ilc s express cd in humans and cd r in mice, but the natural cytotoxicity receptor nkp (also known as ncr ) is expressed in both species , . ilc s constitute the most homogeneous class of ilcs; they are dependent on gata and rorα, and they produce type cytokines, mainly interleukin (il- ) and il- . ilc s are involved in immune responses to parasite infection, and in humans, they express chemoattractant receptor-homologous molecule expressed in t h cells (crth ) and high levels of cd , whereas most mouse ilc s express st (a member of the il- receptor family) , . the development and function of ilc s depend on the transcription factor rorγt. both human and mouse ilc s can produce granulocyte macrophage colony-stimulating factor (gm-csf), il- , and/or il- , . in humans, two major ilc subsets can be distinguished on the basis of the expression of the natural cytotoxicity receptor nkp (also known as ncr ) , . both types can produce il- , but the production of il- is mainly confined to nkp + ilc s. extensive research has focused on deciphering the role of ilcs to ensure the maintenance of tissue homeostasis and immune protection , . ilcs express particular sets of receptors in a tissuespecific manner, and these allow the detection of host-derived signals (including those from alarmins, neuronal mediators, microbia, and the diet) . the integration of these endogenous signals is essential for the maintenance of tissue homeostasis, but dysregulation of ilc responses leads to inflammation and disorder , . ilc are mainly involved in early protection against viruses and bacteria , , but their response to dysregulated local proinflammatory cytokine production in adipose tissues leads to the development of metabolic disorders and obesity . il- and il- produced by ilc s induce goblet cell differentiation and the recruitment of eosinophils, basophils, and mast cells , which are involved in protection against infection by helminths and viruses, but when uncontrolled, these cells drive allergic responses and metabolic disorders. moreover, the depletion of ilc s in animal models suggests a role for these cells in atopic dermatitis and asthma . ilc s are abundant in mucosal tissues, and ncr + ilc s have been proven to be essential for regulating the balance between commensal and pathogenic bacteria through the production of il- . in contrast, ncr − ilc s can promote colitis in a model of inflammatory bowel disease . the lack of immunodeficiency in ilc-deficient patients led to the proposal that ilcs are dispensable in the presence of functional t cells and b cells . however, recent studies support the idea that ilcs cannot be considered to have functions that only duplicate those of the adaptive immune system. in addition to those showing the essential role of lti cells in the formation of secondary lymphoid organs during embryogenesis and the postnatal development of intestinal lymphoid clusters, recent studies also provide evidence that subsets of ilcs express multiple factors that modulate the adaptive immune response in health and disease , . in particular, ilc s and ilc s modulate the t-cell response. studies in mice suggest that in healthy intestine, ilc s express major histocompatibility complex (mhc) class ii molecules but lack the expression of costimulatory molecules; therefore, they inhibit microbiota-specific t-cell responses, thus preventing intestinal inflammation . it seems that the interaction between ilc s and tfh cells limits il- secretion and the production of iga by mucosal b cells . studies with murine models have significantly contributed to the classification and understanding of the role of ilcs in the immune system, especially since similarities have been observed between ilcs identified in mice and humans . however, the differences between these two species present real challenges , because human ilcs have unique attributes that are only now being elucidated, with further work required in this exciting field. the roles of ilcs in immunity and their cross talk with other components of the immune response await further analysis. detailed coverage of this topic is beyond the scope of this review, and we refer the reader to recent reviews that provide more information on the biology of human and mouse , ilcs. t cells and plasticity. t cells are categorized as tα/β and tγ/δ cells, depending on the type of t-cell receptor (tcr) that they express . human tγ/δ cells, similar to their murine counterparts, are a minor population ( - % of nucleated cells) in peripheral blood, but are especially abundant in barrier tissues such as the epidermis [ ] [ ] [ ] . the three main subsets of t cells carrying α/β receptor are the cd +t helper cells and cd +cytotoxic and cd + cd + regulatory t cells . new effector cd + helper t-cell subsets (initially classified as th and th ) , have been recently described, and at least six human th cell subsets have been identified to date: th , th , th , tfh, th , and th cells , . all of these cells recognize foreign peptides presented by class ii mhc molecules on antigenpresenting cells (dendritic cells, macrophages, and b lymphocytes). th cells are required to activate macrophages and cellmediated immunity to kill intracellular pathogens , whereas th cells are important in facilitating eosinophils to fight against parasitic helminths and b cells for antibody production and antibody class-switching to generate iga or ige . th cells are required to mobilize neutrophils for the clearance of fungi and extracellular bacteria, and they are also involved in mucosal protection . th and th cells are also involved in mucosal immunity; th cells protect against parasites , , and th cells prevent microbial translocation across epithelial surfaces and promote wound healing , . as mentioned in the introduction to ilcs, studies on human th cells isolated from lymphoid organs and blood samples, along with recent observations on the developmental mechanism of distinct th cell subsets, have revealed both similarities and differences of human and mouse th cells , , . tfh cells are very important for germinal center reactions, antibody class switching, affinity maturation, and the development of high affinity antibodies and memory b cells , . at the surface marker level, tfh cells are generally characterized by the expression of cxcr , the chemokine receptor for cxcl , which is highly expressed on b-cell follicles for expressing inducible t-cell costimulator (icos) and programmed death protein (pd- ) , , which enable their involvement in the interaction of tfh cells and b cells . the definition of a given t cell lineage is based on its ability to sense different inductive cytokines, to produce particular cytokines or to express a lineage-specifying transcription factor. th cells produce ifn-γ and express t-bet ; th cells are characterized by il- , il- , and il- production and gata- expression , ; ptregs, which are induced in the periphery from naïve precursors, produce tgf-β and express foxp (tr cells are il- -secreting tregs that do not express foxp ) . th cells produce il- a, il- f, and il- and express rorγt , , and tfh cells produce il- and il- and express the bcl transcription factor. in addition, th cells, which produce il- and express the aryl hydrocarbon receptor (ahr) , , and th cells, are characterized by the expression of il- and the transcription factor pu. . additional levels of regulation, such as the differential expression of micrornas, long noncoding rnas (lncrnas), and protein stability and function, have been found to control various aspects of th cell differentiation and effector function , . cd + cytotoxic t cells express the dimeric cd marker and have specific lytic capacity to target cells through several mechanisms, including the release of cytotoxic granules, secretion of cytokine tumor necrosis factor alpha (tnfa) and interferon gamma, and the induction of cell death through the interactions of fas and the fas ligand , . their tcrs are restricted to interactions with peptides presented by class i mhcs. regulatory t cells (tregs) include thymically derived and peripherally induced regulatory t cells (ttregs and ptregs, respectively), and they produce either il , tgf-beta, il- or combinations of these proteins . ttregs express the transcription factor foxp and secrete il and tgf-β; ptregs, which are induced in the periphery from naïve precursors, can also be subdivided into il- -induced tregs [tr cells] (which secrete large amounts of il- and moderate levels of tgfβ), th cells (which produce il- and tgf-β), and tgfβ-induced tregs [itregs], which may or may not express foxp . moreover, new subsets of regulatory t cells have been described. they include follicular regulatory t cells (which express foxp and bcl- and cxcr ), which modulate the function of tfh cells and fine-tune the germinal center response [ ] [ ] [ ] , and a il- dependent regulatory population of cells (referred to as itr cells), which show potent suppressive potential in several mouse disease models . other regulatory populations have also been described, including bregs and cd + tregs, which are the analogous counterparts of tregs [ ] [ ] [ ] . recent studies have revealed the capacity of differentiated t cells, particularly th cell and ptreg subsets, to change their phenotype in response to changing contexts [ ] [ ] [ ] [ ] [ ] . becattini et al. found that human memory cd t cells primed in vivo by pathogens (e.g., candida albicans and mycobacterium tuberculosis) or vaccines (tetanus toxoid) are highly heterogeneous, both at the population and clonal levels. with respect to studies on human arthritis, nistala et al. proposed that th cells are recruited to the joint and converted to th / or th cells in response to local il- levels. this plasticity has also been observed with in vitro assays under conditions that mimic a disease site, namely, low tgf-β and high il- levels . these results are inconsistent with the original idea of th lineage stability and provide new possibilities for disease treatment aimed at inducing particular th subsets to modulate the immune response against pathogens or to control detrimental immunity , , . trained and adaptive immune memory other classical concepts in fundamental immunology, such as immune memory, are also changing. the specificity and the capacity to generate long-lived memory cells are two properties that have been classically used to distinguish innate immunity from adaptive immunity. adaptive immunity is clearly based on the specific recognition of antigenic determinants by somatically diversified receptors (b cell and t cell receptors (bcr and tcrs, respectively)) and on its capacity to respond more effectively to restimulation with the same antigen. in contrast, innate immune responses have traditionally been considered nonspecific and without the capacity to adapt . however, the discovery of germline-encoded pattern recognition receptors (prrs) and the "trained innate" immunity (or innate immune memory) have provoked a shift in our understanding of the immune response. in , medzhitov et al. demonstrated that pattern recognition receptors (prrs) expressed on innate cells recognize invariant molecular structures expressed by invading pathogens . after the interaction, prrs trigger the expression of costimulatory molecules and activate important signaling pathways to induce the activation of innate and adaptive immune cells. prrs mainly belong to four families: toll-like receptors (tlrs), nod-like receptors (nlrs), c-type lectin receptors (clrs), and peptidoglycan recognition proteins (pgrps) , . the profiles of prrs expressed by innate cells can lead to partially specific recognition of a type of microorganism; e.g., innate cells can distinguish between gramnegative and gram-positive bacteria and modulate the immune response based on this recognition, although they cannot differentiate between bacterial species . the idea that only jawed vertebrates developed immunological memory has also been challenged by the observation of resistance to reinfection in organisms that lack an adaptive immune response, such as plants and invertebrates , . recent studies have shown that monocytes and macrophages exposed to candida albicans or β-glucans exhibited an enhanced secondary response . in addition, immunization of mice with bacillus calmette-guérin (bcg, the tuberculosis vaccine) induces t cellindependent protection against secondary infections by candida albicans, schistosoma mansoni or influenza virus [ ] [ ] [ ] [ ] . thus, organisms are protected not only against the original microorganism but also to unrelated pathogens. the mechanisms underlying the establishment of this innate immune memory differ from those involved in adaptive immune memory . after infection or vaccination, innate immune cells (such as monocytes and macrophages) display long-term functional changes through epigenetic and metabolic reprogramming, including histone acetylation, methylation and modulation of noncoding rnas [ ] [ ] [ ] . in turn, the faster and more pronounced reactivity of adaptive immune cells (t and b lymphocytes) upon reinfection is characterized by permanent changes in the genome of cells, such as mutations, gene rearrangement, clonal expansions, as well as epigenetic modifications, all of which ensure a more persistent effect than is endowed by trained immunity , , . other cells for which immunological memory has been described include tγ/δ cells and innate lymphoid cells . recently, some authors have proposed that nk cells are also capable of immunological memory [ ] [ ] [ ] [ ] . antigen-specific recall responses by human nk cells were observed by nikzad et al. in humanized mice and in varicella zoster virus (vzv)-exposed adult human volunteers, in which cytotoxic nk cells were recruited to sites of an vzv test antigen challenge on the skin. sensitization with haptens using mice lacking t cells and b cells led to the generation of hapten-specific memory nk cells . the recall response persisted for more than four months after priming, and was adoptively transferred to naïve mice . interestingly, nk cells exhibit memory that is not only specific to a given virus, such as cytomegalovirus , , but that is also induced in the absence of a defined antigen , . furthermore, new studies suggest that trained immunity is not a phenomenon that is restricted to immune cells, because epithelial stem cells also retain memory of previous inflammatory challenges by displaying an enhanced wound-healing capacity upon skin damage . given the data outlined above, immunological memory is now recognized to be highly diverse and not restricted to b cell-or t cell-mediated adaptive immunity. much remains to be learned in this field, but the different manifestations of immunological memory described above offer an important basis for clinical applications, such as the development of novel vaccination strategies or new therapies for pathological situations in which immunological memory can be detrimental, such as allergies or autoimmune diseases , , . interaction of the immune system and the microbiome the immune system has evolved in the presence of commensal microorganisms that colonize barrier surfaces of vertebrates and invertebrates , . the cross talk between the natural host microbiome and immune system is particularly interesting in the gastrointestinal tract, where the density and diversity of indigenous bacteria, viruses and fungi are greatest compared to those of other anatomical sites . in the literature, reports of observed changes in microbial community composition during diseases are diverse and include those in inflammatory bowel disease (ibd), obesity, metabolic syndrome, and multiple sclerosis [ ] [ ] [ ] [ ] [ ] . however, the microbiome can be influenced by different factors, such as the specific niche that it occupies, diet, stress, environmental factors, and host genetics, and a specific correlation does not necessarily infer causation. the presence of these commensals in mucosal tissues has been known since before metchnikoff, but the current knowledge on the role of the microbiome in shaping the immune system throughout life came mostly from the development of next-generation sequencing (in particular, the reduction in the cost of s ribosomal rna gene sequencing) and the use of germ-free animal models, which can be colonized even with human microbiota . germ-free mice are characterized by atrophy of peyer's patches with few germinal centers and isolated lymphoid follicles, a lower number of b, t, and dendritic cells and a decreased level of immunoglobulins, particularly iga and igg . these effects are observed at the mucosal and systemic levels, and they can be reversed within weeks after the colonization of germ-free mice with commensal bacteria . moreover, colonization with commensal bacteroides fragilis revealed the immunomodulatory effect of bacterial polysaccharides in restoring systemic cells and the differentiation of cd + t cells into regulatory t cells (foxp + tregs), which in turn favor mucosal immunomodulation . the induction of th cell maturation by segmented filamentous bacteria has also been reported . these important examples emphasize the major roles of the commensal microbiome in the maturation of mucus-associated lymphoid tissue and the systemic immune system. the development of new technologies to better track the locations and activities of distinct microbial populations is essential to elucidate host-microbe interactions, through which other systems, such as the nervous system, seem to play important roles , - . the better characterization of some immune cell subsets, trained immunity, and host-microbiome interactions provides a few very good examples that prove the maturation of immunology in the last few decades. in this sense, studies with mouse models have significantly contributed to the increase in our fundamental knowledge; however, the differences between murine and human immunology are notable, and conclusions drawn from mouse studies are sometimes not fully translated to humans . if we want to fully exploit the power of the immune system for human health, greater effort is required for understanding human immunology. immunologists, in cooperation with experts from other fields, have developed a variety of protocols and tools to achieve greater selectivity in the identification and analysis of human cell subsets, types of cytokines and receptors, chemokines, etc. these tools range from biological approaches that rely on next-generation sequencing, mass spectrometry, and bioinformatics to immune monitoring technologies based on multiparameter flow cytometry and single-cell gene expression analysis. although not without limitations, these techniques provide a much better picture of the whole immune system than individual and independent approaches. immune-mediated illnesses comprise a wide variety of diseases characterized by the dysregulation of a normal immune response. most of these illnesses are complex disorders believed to arise from a combination of genetic and environmental factors . infectious diseases infectious diseases are caused by pathogens (viruses, bacteria, fungi or parasites that infect the host body), and they remain a leading cause of mortality worldwide. prominent examples include illnesses produced by mycobacterium tuberculosis, human immunodeficiency virus (hiv), plasmodium falciparum or the current coronavirus disease (covid- ) outbreak caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), which has already infected millions of people and produced thousands of deaths in many countries. for a number of years, many people believed koch's postulates, which implied that virulence traits reside solely in the pathogen. however, recent advances in molecular biology have shown that host genes play major roles in infection, together with a wide range of environmental variables . to date, six gene products endowing infectious disease susceptibility have been validated in the literature: ( ) hemoglobin subunit beta; ( ) band -anion transport protein; ( ) duffy antigen/ receptor, which is associated with plasmodium spp. infections; ( ) the prion protein associated with creutzfeldt-jakob disease; ( ) fucosyltransferase and , which is associated with norwalk virus infections; and ( ) c-c motif chemokine receptor (ccr ) coreceptor, encoded by an immune-related gene and leads to the impairment of the entry of the human immunodeficiency virus (hiv) into helper t cells, thus avoiding/decreasing the progression to acquired immunodeficiency syndrome . another gene associated with infectious disease and the immune system is the natural-resistance-associated macrophage protein (nramp ), which encodes an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. it is a susceptibility locus for increased ratios of infection with leishmania spp. parasites and certain strains of salmonella spp., mycobacterium bovis and mycobacterium tuberculosis , . in addition, it has been suggested that functional variants of immunoglobulin fc gamma riia (cd ) are related to the development of invasive encapsulated bacterial infections . moreover, because of recently acquired genomic data, new human polymorphisms have been discovered, some of which play roles in changing immunoglobulin levels, seroconversion rates or the intensity of antigen-specific immune responses. in addition, they also contribute to human susceptibility to infection by viruses such as influenza, rhinovirus and respiratory syncytial virus . these polymorphisms are mapped within the mhc (hla-dqb * , hla-drβ , or hla-dpβ ), natural killer cell immunoglobulin-like receptors and (kir dl and kir ds ) and natural killer lectinlike receptor d (kldr- ) . several recent studies available as preprints have analyzed certain genes that may explain the differences in the variable expression of and susceptibility to covid- by patients, either by affecting the host receptor for the virus (angiotensin i converting enzyme (ace- )) , immune genes (tlr and others) or blood groups (group o seems to be the most protective) , and more extensive omics studies are now underway with larger numbers of patients. in , the physician paul ehrlich first used the term "horror autotoxicus" to describe the way autoimmunity contradicts the natural aversion to self-injury ("living with the enemy", reviewed in ). currently, according to the american autoimmune related disorders association, more than autoimmune diseases have been identified. historically, these diseases were considered to be rare, but current epidemiological data have shown that they affect approximately - % of the population worldwide. some of the most common autoimmune diseases include type diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (https://www.aarda.org/diseaselist/). although significant progress has been made in understanding the mechanisms of autoimmune diseases and the nature of selftolerance, these disease remain major burdens on health systems around the world. autoimmune diseases arise when the immune system attacks normal components of the body . the concept of immune tolerance is defined as the ability of the immune system to prevent the targeting of self-molecules, self-cells or self-tissues. on the other hand, the failure to distinguish self from nonself is often termed a break of tolerance, and it is the basis for an autoimmune disease . what are the mechanisms that lead to a break in tolerance? autoimmune diseases are complex disorders that are believed to arise from a combination of genetic (mutations and higher inheritance frequency of some types of major histocompatibility complex alleles), epidemiological (age and sex) and environmental (infections, microbiota, tobacco, chemicals and pharmaceutical drugs). factors these factors trigger a break in self-tolerance with the activation of self-reactive lymphocytes through several mechanisms, such as molecular mimicry, the overexpression and abnormal expression of mhc class ii molecules in peripheral tissues, thymic aging, and immunodeficiencies (discussed below) and many others. some lymphocytes escape control due to polymorphisms in several genes that affect the routes of lymphocyte activation. other causes may include defective antigen presentation by some mhc variants with specific polymorphisms. therefore, the self-reactive lymphocytes that have escaped control and react against self-constituents initiate the autoimmune process . although a large number of genome-wide association studies (gwas) have led to the identification of hundreds of polymorphisms associated with the development of different autoimmune diseases, it has proven difficult to define the role of most of these polymorphisms in the breakdown of tolerance to a selfantigen [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is worth highlighting, however, that the mhc remains the main genetic factor associated with human autoimmunity , . other gene variants identified are common to many autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type i diabetes, ulcerative colitis, autoimmune hepatitis and numerous other autoimmune diseases. for example, the protein tyrosine phosphatase nonreceptor type (ptpn ) gene encodes a protein that inhibits t-cell activation in the adaptive immune system, whereas it promotes myeloid cell activation; interferon regulatory factor -transportin (irf -tnpo ) is involved in the accumulation of lymphocytes within lymphoid organs and failed elimination of autoreactive naïve t cells; btb domain and cnc homolog (bach ) has a critical role in immunoglobulin class-switching recombination, somatic hypermutation of immunoglobulin encoding genes and the activation of tissue macrophages. a more complete list of genes associated with autoimmunity can be found in the review by wang et al. researchers are currently looking for the missing heritability in autoimmune diseases by focusing on the study of methylome profiles, genetic cargos in extracellular vesicles, genetic alterations, and ways in which the microbiome may affect these diseases. immune-mediated rejection of tissue allografts was first described in by the british immunologist peter medawar , . only three years later, george snell described the mhc, which carries the histocompatibility genes, and one decade later, jean dausset described the human leukocyte antigen (hla); each of these scientists was recognized with the nobel prize in physiology and medicine . since its discovery, mhc has emerged as the most polymorphic gene locus in eukaryotes with hla and related alleles, more than nucleotide variants reported in the individual-participant data-international immunogenetics/ human leukocyte antigen (ipd-imgt/hla) work group database (https://www.ebi.ac.uk/ipd/imgt/hla/), release . . , / / . although the main barrier for long-term organ and tissue grafting is driven by hla incompatibilities, other important players play roles in transplant rejection. in particular, minor histocompatibility antigens, which are peptides derived from allelic variants of normal cellular proteins, presented by class i or ii mhc antigens induce cellular immune responses in hla-matched individuals who lack the same allelic variant . natural killer (nk) cells also play important roles in transplantation through their killer cell immunoglobulin-like receptors (kirs), which are receptors for hla class i molecules. nk cells expressing an inhibitory kir-binding self-hla can be activated when exposed to allografts that lack a ligand for the inhibitory receptor . the locus that codifies these receptors displays a considerable degree of polymorphism, with alleles reported in the individual-participant data-international/killer cell immunoglobulin-like receptors (ipd/kir) work group database, release . . , / / . more recently, we have begun to appreciate the importance of non-hla genetic factors in the development of transplant rejection; examples include polymorphisms in the genes encoding cytokines, such as tumor necrosis factors (tnf), interleukins (il- , il- and il- ), interferon gamma (ifn-γ), and transforming growth factor-β (tgf-β ). other genes encode pathogen recognition receptors, with nucleotide-binding oligomerization domaincontaining (nod (card )) being the most widely studied, although conclusive data have not been obtained to date . primary immunodeficiencies (pids) comprise a heterogeneous group of more than genetic disorders that result in defects in the immune response . pids are considered mendelian disorders because they are mainly autosomal recessive disorders that often display incomplete penetrance, which affects the severity and onset of the disease. with the exception of immunoglobulin a (iga) deficiency, pids are considered to be rare disorders, as their prevalence worldwide ranges from to among , people . unsurprisingly, these types of diseases are not uncommon in highly consanguineous populations such as those in the middle east/northern africa (mena) region. the incidence of consanguinity marriage in these areas ranges between and %, which leads to a unique population in which autosomal recessive diseases arise, with the prevalence of pid in these countries as high as in , people . although more than genes have been described for pids, approximately % of the causal genes remain unknown, and next-generation sequencing studies performed in mena populations are contributing to the search for currently unknown genes that cause pids . a complete and updated list of pid-causing genes and diseases can be found at the european society for immunodeficiencies (esid) webpage (https://esid.org) . clinical manifestations of pids are highly variable; many disorders involve an increased susceptibility to several types of infections, but some patients develop autoimmune diseases. patients usually present recurrent sinus or ear infections or pneumonia within a one-year period; other indicators are failure to thrive, poor response to prolonged use of antibiotics, and persistent thrush or skin abscesses . depending on the affected pathway, pids are associated with varying levels of severity, times of onset, and risks of infection by certain groups of microorganisms. according to the international union of immunological societies (iuis) (https://iuis.org/ committees/iei/), inborn errors of immunity can be classified as follows: (a) immunodeficiencies that affect cellular and humoral immunity; (b) combined immunodeficiency (cid) with associated or syndromic features; (c) predominant antibody deficiencies; (d) diseases of immune dysregulation; (e) congenital defects of phagocyte number, function, or both; (f) defects in intrinsic and innate immunity; (g) autoinflammatory disorders; (h) complement deficiencies; and (i) phenocopies of a pid , . however, pids are broadly classified as follows according to the component of the immune system affected: • t-cell immunodeficiency, e.g., defects in the ifn-γ/il- pathway and mutations in the autoimmune regulator (aire) gene. • b-cell (antibody-mediated) immunodeficiency: gamma-globulinemia, x-linked common variable immunodeficiency (cvid), selective iga deficiency, specific antibody deficiency, and igg subclass deficiency. • combined immunodeficiency: wiskott-aldrich syndrome, ataxia telangiectasia, digeorge syndrome and severe combined immunodeficiency (scid). • phagocyte defects: chronic granulomatous disease, hyperimmunoglobulin e (ige) syndrome and leukocyte adhesion deficiency. • complement defects (deficiency in early, late or regulatory complement components) . autoinflammatory diseases systemic autoinflammatory diseases (aids) are characterized by recurrent acute inflammatory episodes secondary to a dysregulated inflammatory process that typically develops during childhood, with recurrent episodes of fever, rashes, and disease-specific patterns of organ inflammation. genetically speaking, these are hereditary disorders, andto date, more than genes (table ) have been identified as causes of aids, which can be grouped according to the pathway that is altered . ( ) inflammasome. the inflammasome is a multiprotein intracellular complex that detects pathogenic microorganisms and stressors and activates the highly proinflammatory cytokines il- β and il- . genes affected in this group are mefv (mediterranean fever pyrin innate immunity regulator), which is related to familial mediterranean fever (fmf); nlrc (nlr family card domaincontaining ); nlrp (nlr family pyrin domain-containing ) and wdr (wd repeat domain ) . ( ) type-i interferon (ifn)-mediated disorders. these disorders are characterized by the upregulated expression of genes induced by ifn. the gain of function by variants of tmem (transmembrane protein ) is the core manifestation of this disorder group, but other genes have been identified, including ddx (dexd/h-box helicase ), dnase (lysosomal deoxyribonuclease ), pola (dna polymerase alpha subunit) and usp (ubiquitin-specific peptidase ) , . ( ) ubiquitination disorders. ubiquitination is a process that marks proteins for degradation via the proteasome, which is required for the processing of intracellular antigens (such as virus proteins or mutated tumor proteins) and their presentation by class i hla molecules. ubiquitination involves three main steps: activation, conjugation and ligation, which are performed by ubiquitin-activating enzymes (e s), ubiquitin-conjugating enzymes (e s), and ubiquitin ligases (e s). ubiquitination disorders are caused by variants of the psmb , psmb , psma and psm genes (proteasome s subunit beta , subunit beta , subunit alpha and subunit alpha , respectively), affecting the proteasome subunits, proteasome maturation protein gene (pomp) and/or proteasome assembly chaperone (psmg ), by encoding proteasome assembly molecules . in addition, other genes in this group, such as otulin (otu deubiquitinase with linear linkage specificity), encode ubiquitin peptidases, i.e., proteins involved in ubiquitination assembly complexes, such as hoil- (hemeoxidized irp ubiquitin ligase ) and hoip (nhp -like protein homolog). finally, the loss of function due to variants of the tnfaip (tnf-alpha-induced protein , also known as a ) gene, which encodes a protein with ubiquitin ligase and ubiquitinase activity, has also been described . ( ) inflammatory or innate immune regulators. a large number of genes have been found to affect the pathways/ mechanisms involved in macrophage and b-cell differentiation and lymph node development, among many functions. genes in this group include ada (adenosine deaminase ), tnfrsf a (tnf receptor superfamily member a), adgre (adhesion g protein-coupled receptor e ), trnt (trna nucleotidyltransferase ), lacc (laccase domain-containing ) and ap s (adaptor related protein complex subunit sigma ) . allergy allergic diseases can be termed complex diseases that involve both genetic and environmental factors, and they influence not only the development of ige-mediated sensitivity in the case of hypersensitivity type i allergies but also the subsequent development of clinical symptoms in a range of tissues, including skin, nose, and lung tissue . since the first report of a link between chromosome q and atopy in , knowledge about the common risk variants for allergic diseases has increased exponentially, mainly because of gwas. most allergic diseases have allergy-related traits such as asthma, with the strongest association mapped to chromosome q . however, the disease-associated gene at this locus remains unclear; one of the candidate genes is ormdl (sphingolipid biosynthesis regulator ) due to its role in sphingolipid synthesis and the regulation of eosinophils. other genes associated with asthma are interleukin (il ) and its receptor, il rl (interleukin receptor-like ), hla region, smad (sma-and mad-related protein ) and il rb (interleukin receptor subunit beta) . as asthma and other allergic-associated traits could be present in patients without allergies, some researchers performed gwas analysis on cohorts of patients who had high levels of allergenspecific immunoglobulin e (ige) or a positive skin prick test. as a were identified, and the strongest association was on chromosome q . this locus has been associated with two genes: c orf (emsy transcriptional repressor, brca interacting), a potential regulator of interferon-stimulated gene, and lrrc (leucine rich repeat-containing ), which is involved in transforming growth factor beta (tgfβ)-signaling in t regulatory cells. the rest of the associated loci involved in the pathogenesis of allergy highlight the importance of the th responses (stat (signal transducer and activator of transcription ), tslp (thymic stromal lymphopoietin), bcl (b-cell lymphoma protein), il rl (interleukin receptor-like ), il (interleukin ), gata (transacting t-cell-specific transcription factor binding protein )); innate immunity (tlr / / (toll-like receptor / / )); tgfβ-signaling (lrrc (leucine rich repeat-containing ), smad (mothers against decapentaplegic homolog )); t-cell (il (interleukin ), ptger (prostaglandin e receptor )) and t regulatory box (lrrc (leucine rich repeat-containing ), il- , nfatc (nuclear factor of activated t cells ), foxa (forkhead box a )) . in the last two years, researchers have focused on epigenomewide association study (ewas) of allergy processes. the epigenetic landscape is specific for a given cell; thus, ewas requires careful selection of the relevant cell type for a given biomedical condition. for allergies, ewas has mainly been performed on nasal mucosal cells and whole blood (although the result was later normalized by the number of circulating eosinophils). nasal mucosal cells comprise cd + t cells, cd + t cells, myeloid cells, innate lymphoid cells, b cells, double-negative t cells, granulocytes, cd + cells, and plasma cell populations . in all of these studies, cpg-associated regions were identified, from which the smad gene, coding for an important regulator of t-cell differentiation, was replicated in three independent cohorts . of all of the genes in whole blood identified using ewas, only the acot (acyl-coa thioesterase ), epx (eosinophil peroxidase), gja (gap junction protein alpha ) and mettl (methyltransferase-like ) genes were confirmed in the nasal cell populations . in , ehrlich proposed the idea that mutant cells arise continuously and that the immune system scans for and eradicates these mutant cells before they manifest clinically . however, immune surveillance remained a controversial topic until its acceptance in the s . immune surveillance is the recognition and elimination of cancerous cells by lymphocytes, which act as sentinels that recognize transformed cells. ultimately, during tumor progression, cancer cells show low immunogenicity and resistance to immune effector cells, thus expanding and escaping immune control. the way in which cancer cells modify the immune system has been called immune editing . the key of immunosurveillance is cancerous cell expression of tumor antigens that can activate various immune cell phenotypes; for simplicity, any overexpressed, mutated, dysregulated, or rearranged gene product expressed by a cancerous cell may be considered a tumor antigen. it is critical to consider that most of these proteins, except those derived from virus-infected cancer cells, are primarily self-proteins, but they are expressed with mutation(s) or minor changes in their antigenic structure . one mechanism by which cancer cells escape from immune recognition is antigenic modulation. for example, the loss of mhc class i molecule expression leads to aberrant antigen masking, which is one of the mechanisms described for tumor cells that escape specific antitumor t-cell immune responses . in addition, the mhc-peptide-t cell receptor complex elicited by a tumor antigen shows weak stability, since high-affinity t-cells tend to be rendered tolerant to these antigens . another mechanism is the direct inhibition induced by cancer cells due to their interaction with surface regulatory molecules, table . also called checkpoint molecules. these molecules include programmed cell death- (pd ) and cytotoxic t-lymphocyteassociated protein (ctla- ), which induce the inhibition of host t cells. although these checkpoints usually help conventional immune responses control immune activation, they can also be used by tumor cells to inhibit antitumoral t-cell responses . pd ) is a transmembrane protein expressed on t, b, and nk cells, and it binds to pd ligands (pd-l and pd-l ) on target cells. when it binds to its ligand on tumor cells, pd inhibits tumor cell apoptosis, causes peripheral effector t-cell exhaustion, and promotes the conversion of effector t cells into regulatory t cells , . ctla is also a physiological negative regulator of t-cell activation. the interaction with cd /cd in the tumor leads to the inhibition of t-cell function and suppressed effector activity . knowledge of these two checkpoint inhibitors has opened the door to new antitumoral therapeutic approaches, such as the use of monoclonal antibodies that block the aforementioned interactions (anti-pd , anti-pd-l , or anti-ctla- ), which are called checkpoint inhibitors . in addition, tumor cells create an inhibitory microenvironment around them. malignant cells can recruit other cells, such as immune cells and fibroblasts, which can be corrupted by tumor cells. the interaction between tumor and nontumor cells creates the tumor microenvironment, which is mostly driven by the dynamics of the tumor promoting the proliferation/expansion of cancer cells. for example, tumor and stromal cells release multiple factors, such as the chemokine ccl (c-c motif chemokine ligand ), which inhibits effector t-cell functions and attracts tregs to the microenvironment . tumor cells use different mechanisms to promote cancer progression and further metastasis. the complete immunological eradication of cancer is the goal of antitumoral immunotherapy and is discussed later in this review. immunosenescence and inflammaging aging is accompanied by the decline and dysregulation of immune efficacy, which results in an increased vulnerability to infectious diseases, diminished responses to vaccination, and reduced tumor clearance. immune alterations mainly manifest as a reduction in the number of naïve peripheral blood cells and a relative increase in some types of memory cells . natural aging causes progressive atrophy of the thymus, which is called thymic involution. the endpoint is a significant decrease in naïve t cells, which reduces the diversity of the t-cell antigen receptor (tcr) repertoire and culminates in disrupted t-cell homeostasis . the cellular and molecular hallmarks of aging have been described as genomic instability, telomere attrition, epigenetic alterations, sarcopenia, changes in intracellular communications, cellular senescence, immunosenescence and mitochondrial dysfunction . the process of aging alters the innate and adaptive immune systems. in terms of innate immunity, aging results in a decreased number of circulating monocytes and dendritic cells, reduced phagocytic properties of macrophages and neutrophils, and impaired antigen presentation by dendritic cells . as mentioned above, aging also generates a reduction in the t-cell and b-cell receptor repertoire due to the accumulation of senescent or exhausted lymphocytes, together with a decrease in the number of circulating naïve t and b cells , . on the other hand, nk cell cytotoxicity is maintained in centenarians, and an increase in the number of these cells is observed in healthy aging people . moreover, cd + t cells exhibit cytotoxic features in centenarians; this is an acquired characteristic for cd + t cells that usually have helper, but not cytotoxic functions under physiological conditions . in addition to these features, chronic inflammation is considered the key that underlies the phenomenon called 'inflammaging', which is related to elevated self-reactivity and results in the typical chronic low-grade, systemic inflammatory phenotype observed in the elderly in the absence of acute infection. currently, it is believed that self-reactive t cells are the main contributors to this process. it has been proposed that this basal inflammatory state contributes to the development of some diseases, such as type ii diabetes, alzheimer's disease and atherosclerosis . understanding the mechanisms of age-related disorders in immune regulation is important for identifying more efficient strategies of immune rejuvenation and for the effective induction of vaccination-mediated immunity in older individuals . immunotherapy includes the use of certain components of the immune system (antibodies, cells, cytokines, etc.) for the treatment of various cancers and autoimmune diseases and the manipulation of the immune system through vaccines for the prevention and treatment of infectious and allergic diseases (fig. ) . immunotherapy using microorganisms or their components in vaccines was first practiced centuries ago; soluble substances such as poly-and monoclonal antibodies, as well as cytokines, have been used for many years, but recently, cellular immunotherapy has emerged in clinical practice. although immunotherapy can be used for many diseases (infections, autoimmune diseases, macular degeneration, allergic diseases, etc.), it is being used most expansively in the cancer field. the main goal is to destroy the tumor, either directly or indirectly (by enhancing the patient's immune system), while offering greater specificity and fewer side effects than conferred by conventional therapies. pathogens and vaccines for infectious diseases immunotherapy associated with pathogens was first linked to the prevention of infectious diseases, starting from variolization (in the x century), followed by edward jenner's vaccination against smallpox (in the xviii century) and subsequently many other preventive vaccines for infectious diseases. the great advances in the knowledge about infectious diseases took place in the nineteenth century, but the xx and xxi centuries are clearly the vaccination centuries, as many new successful vaccines (with attenuated or dead pathogens, subunits, recombinant proteins, carbohydrates or dna) introduced against a variety of pathogens. more recently, and with the increased knowledge of the human microbiome, the use of microorganisms in therapy has seen a resurgence. some intestinal infections, such as those produced by clostridium difficile, can be cured with the transfer of intestinal bacteria from healthy people (feces transplantation) . numerous other attempts to use microorganisms to cure inflammatory illnesses (crohn's disease, ulcerative colitis, etc.) have met with limited success , which indicates that this type of therapy is much more complex than initially anticipated. as a consequence, many more studies are required to ensure that this approach can be used for curative immunotherapy. researchers are also working on genetically modified or artificial bacteria (e.g., based on salmonella enterica, listeria monocytogenes or lactobacillus lactis), but only limited effects have been observed to date . oncolytic viruses (ovs). although the use of bacteria in antitumoral therapy has been largely restricted, the use of therapeutic viruses is increasing. virus-based therapy was introduced in the s with the use of adenovirus, but only in recent years has it been used in practice in the clinic. oncologic viruses have the capacity to attack tumor cells in a preferential manner and induce immunogenic cell death (icd) and host antitumor immunity (fig. ) . the first virus approved for use in therapy was a recombinant oncolytic adenovirus named h , which was licensed in by the china food and drug administration (cfda) for treating head and neck carcinoma in combination with chemotherapy . ten years later, the oncolytic attenuated-modified virus herpes simplex i-talimogene laherparepvec (t-vec, imlygic®) was approved by both european (emea) and american (fda) agencies for the treatment of melanoma . the virus is modified by the insertion of human gm-csf and deletion of the icp gene. since the approval of t-vec, a new era has dawned on the use of ovs in cancer therapy , . currently, oncolytic viruses from the adenoviridae, herpesviridae, picornaviridae, reoviridae and poxviridae families are in different phases of clinical studies for several types of tumors , . for example, reovirus against brain tumors (alone or combined with other therapies) or maraba virus against triple-negative breast tumors , offer some hope to patients with these types of cancer. viral sequences can be modified by genetic engineering techniques, thus making the virus more prone to infect some cells and enhancing viral infiltration and tumor tropism. combinations with other components (immunomodulators, drugs, and cytokines) are also being explored to suppress antiviral immunity and enhance antitumoral cytotoxicity . vaccines for cancer prevention. it is clear that certain viruses and bacteria play roles in cancer development. viruses such as genital herpes, hepatitis b, epstein barr or human papilloma and bacteria such as helicobacter pylori have been associated with cancers of the uterus and liver, in burkitt's lymphoma, and oral/genital and stomach cancers, respectively . therefore, immunization against these pathogens offer protection not only from infection but also from cancer. therapeutic vaccines. once an illness has developed, the intention of a therapeutic vaccine is to eliminate or decrease its pathology. thus, vaccines are used for cases of allergies, cancers and autoimmune diseases. allergy (type ): allergen-specific immunotherapy (ait) aims to modulate the immune system against an allergen, thus modifying the natural course of the allergic disease and conferring longlasting benefits . the basic ait involves the introduction of repeated doses of allergen (either injectable or sublingual allergen extract tablets) and often in escalating doses in a controlled manner, followed by a maintenance phase. in cases for which long-lasting tolerance is acquired, therapy may be discontinued. allergen extracts can be obtained from different sources, such as cat hair and pelt, mites, different types of pollen, venom protein, foods, etc. allergy vaccines are currently the only effective therapy that can stop the progression of the illness because treatment with anti-inflammatory drugs, such as anti-histaminic drugs or corticoids, mitigates the symptoms of the allergic processes but does not modify the natural course of the disease , . ait has been shown to induce the activation of antigen-specific tregs and il- -producing bregs (br ) subtype cells, which is combined with anergy caused by th cells and the production of allergen-specific igg antibodies that can compete with ige for binding to allergens . in the past, most vaccines were developed using natural allergen extracts. however, significant progress has been made in recent years to correctly characterize the allergen at the molecular level, and some of these allergens are now being produced by recombinant technologies, nucleic acid-based strategies, or synthetic peptide chemistry . cancer: another therapeutic approach for vaccines is in the field of cancer. therapeutic cancer vaccines that contain self-or nonself-patient tumor lysates, viral vectors, mutated tumor proteins or peptides, among other types administered in the presence of adjuvants can activate the immune system to induce antitumoral responses . the goal is to activate the th and tc cell compartments to expand specific cytotoxic t and nk cells directed against tumor cells. some vaccines are more immunogenic than others, and this effect can be related to several factors, such as the types/numbers of genetic mutations in the tumor, expression of neoantigens, production of viral proteins, an immunosuppressive environment, lack of expression of histocompatibility complex molecules, etc., which together may explain the large variability in tumor elimination . therapeutic cancer vaccines are generally very safe, and major secondary effects have not been observed, although large differences in patient responses are detected. moreover, this strategy may be used in conjunction with other complementary therapies , such as monoclonal antibodies, chemotherapy or cellular therapy , . several patients are currently taking part in clinical trials and are receiving therapeutic cancer vaccines against different types of tumors, such as lung (clinicaltrials.gov identifier: nct ), prostate (clinicaltrials. gov identifier: nct ) or pancreas (clinicaltrials.gov identifier: nct ), using individual or combined therapies. autoimmunity: in the case of therapeutic vaccines for autoimmune diseases, such as multiple sclerosis, diabetes, myasthenia gravis or guillain barré syndrome, the intention is to induce tolerance to self-antigens through the activation of regulatory cells (tregs and bregs) and tolerogenic dendritic cells, thus avoiding the immune response to self-components . due to the large variety of autoimmune diseases, the different etiologies and extensive variability, even in the same type of disease, designing a vaccine that can be useful for a wide range of patients is very difficult. however, several researchers are obtaining good results in animal models with nanostructures and peptides that induce specific tolerance, and it is predicted that, in the near future, these types of therapies will be applied to patients suffering from autoimmune diseases (reviewed by serra and santamaria ). polyclonal antibodies (pabs)-serotherapy the discovery of antibodies by dr. e. von behring and kitasato at the end of the xix century highlighted the potential of antibodies to neutralize tetanus and diphtheria toxins. this discovery opened the way to exploring the potential clinical applications of conventional antiserum-containing polyclonal antibodies from immunized animals/humans . this "serotherapy" was initiated by dr. roux and dr. yersin, who used antidiphtheria serum to treat several children . after this initial success, the use of serotherapy was increased for use against diphtheria and other diseases but also led to the identification of problems, such as immunogenicity with the formation of immune complexes (arthus reaction), the variability and limitation of the antibody batches, the content of a mixture of classes and subclasses of antibodies with different biological activities, and their temporal effects. for all of these reasons, therapy with polyclonal antibodies was very much restricted to special cases, such as the use of gamma-globulins for the prevention of rhesus (rh) maternal-fetal incompatibility and tetanus or snake venom toxicity . with the identification of gamma-globulin-deficient patients by dr. bruton in , the use of immunoglobulins as therapeutic molecules for the treatment of humoral immunodeficiencies was initiated. however, some problems were encountered in the initial phases, mostly related to the serum preparation and aggregation/ fragmentation of antibodies. since their initial use, several efforts have been made to avoid impurities and to improve the purification process, and several commercial products are now available (as intravenous or subcutaneous preparations). currently, many patients with humoral immunodeficiencies are successfully being treated to prevent them from catching infectious diseases. more recently, the therapeutic applications of immunoglobulins have expanded to other diseases, such as against covid- caused by sars-cov- infection (see below), autoimmune disorders and kawasaki syndrome in children . the beneficial effects seem to be mediated by several immunological mechanisms, including viral neutralization, inhibition of inflammatory cells and activation of immune regulators . monoclonal antibodies (mabs) the development of monoclonal antibodies (mabs) by c. milstein and g. köhler in (nobel prize winners for physiology/ medicine in ) changed medicine and immunology completely, along with many other disciplines. monoclonal antibodies are produced from the fusion of two cells to generate a hybrid cell or hybridoma with two characteristics, i.e., the production of one specific antibody and immortality. dr. milstein is considered to be the father of modern immunology for his crucial contribution . the development of many different mabs has enabled the identification of new molecules and the development of more accurate diagnostic approaches; specific, fast and inexpensive technologies; processes for the purification/concentration of compounds; and better and more specific therapy. mabs can now be used against specific targets according to the concept of the "magic bullet", a term coined by dr. paul ehrlich at the beginning of the xx century (reviewed in ref. ). numerous different mouse and rat mabs were produced against several molecules, but due to their murine origin, patients treated with these mabs suffered from hypersensitivity and immune responses , . thus, most mabs currently used in clinical applications are linked to radioactive elements and used for diagnostic purposes (table ) . in an effort to avoid immunogenicity, mabs were subsequently modified by genetic engineering approaches to carry mostly sequences of human origin. several research groups and companies developed chimeric and humanized mabs ( table ) , and these mabs included additional modifications, such as changes in the carbohydrates (glycosylation) and/or antibody regions, with the aim of improving their therapeutic action [ ] [ ] [ ] [ ] [ ] . moreover, fragments of recombinant antibodies (fabs, single-chain fvs, different v regions, fusion proteins, smaller antibodies, etc.) increased the variability of these potential therapeutic agents. the generation of fully human mabs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or p vectors. the generation of knockout mice (in which mice lack ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mabs , . other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies ( table ) . sir greg winter, nobel prize winner in chemistry in , , became the pioneer of mab humanization through the genetic engineering of an antibody (campath ), later developing a fully human antibody (antitumor necrosis factor alfa, tnf-a) using recombinant phage technology , , . several companies are currently developing human antibodies using these and new technologies (reviewed in , , , ) . since , the list of approved mabs for human therapy has continued to increase (table ) , and many more mabs are in clinical trials [ ] [ ] [ ] . the versatility of mabs is based on a different mechanism of action : human immunology and immunotherapy: main achievements and challenges j varadé et al. -neutralization/blocking of soluble elements. for example, the neutralization of cytokines (tnf-α) and growth factors (vascular endothelium growth factor) prevents the exhibition of their effects, i.e., inflammatory and angiogenic effects, respectively , . -complement activation. igg/igm antibodies activate the complement cascade by the classical route, which leads to the death of the target cell , . -cytotoxicity mediated by nk cells. nk cells can facilitate mab killing of target cells. the mab, after binding to a target cell, can attach to fc receptors on the surface of nk cells to trigger the release of granzymes and perforin, thus inducing cell target death , . -induction of cell death by apoptosis. certain mabs directed against some membrane molecules can directly activate apoptosis . -blocking activation signals. antibodies can block some membrane receptors and avoid cell activation/proliferation activation/proliferation , . -carriers of toxins, pro-drugs, enzymes, and radioactive elements. mabs are able to concentrate select compounds around target cells, providing a much more selective therapy than conventional chemo-or radiotherapy . -check point inhibitors. leading to a recent revolution in cancer therapy, the identification of several inhibitory molecules can be blocked by mabs, thus leading to the activation and proliferation of antitumoral t cells. molecules such as ctla- and pd and its ligand pdl- , maintain immune cells under controlled conditions. however, it is possible to reactivate the antitumoral immune responses by blocking some of these molecules with mabs, either directed to only one of them or by using various antibodies in combination (for example, against ctla- and pd ) . the results obtained with these therapeutic mabs against checkpoint inhibitors in some types of cancer have been amazing. for their contribution to the understanding of the roles of ctla- and pd- , the swedish academy gave the nobel prize in to dr. j.p. allison and dr. t. honjo, respectively . however, this therapy is not efficacious in all types of cancers for several reasons, such as the expression of these and other checkpoint inhibitors in immune cells, the number of antitumoral cells in each patient, an immunosuppressant microenvironment, the rate of cancer mutations, and the expression of histocompatibility molecules. there is a large list of recombinant proteins that are currently being used for human therapy, including interleukin (il- ), interferons (ifns) and gm-csf. il- was identified in as a growth factor for t lymphocytes, and soon after dr. rosenberg started to use it in antitumoral therapy , . years later, in , il- was approved by the fda for patients with metastatic renal cancer and in for the treatment of metastatic melanoma . interferon (ifn) was described in by isaacs and lindenmann . the interferon family is the largest family of cytokines and is classified into three different types (i, ii, and iii). type i ifns (including ifn-α and ifn-β) exhibit several molecular actions that may be very relevant for use in therapy for a range of pathologies (such as autoimmune diseases and cancers) . in , the fda approved human ifn-α a and ifn-α b for patients with hairy cell leukemia and later on for patients with multiple sclerosis. since their initial use, these interferon species have been approved for many other diseases, including chronic hepatitis b and c, lymphoma, advanced melanoma, and as adjuvants together with other therapies for several types of cancers , . another cytokine is gm-csf, which activates the production of granulocytes and monocytes from bone marrow myeloid progenitors and has shown adjuvant antitumoral effects , . other cytokines, such as il- , il- , il- , il- , il- , and il- , , are being tested in several clinical trials, and it is expected that some of them, either alone or in combination, can be used in future antitumoral therapy. other recombinant proteins are already on the market, some of which are derived from antibodies, with some advantages such as small size, low immunogenicity and general ease of production. examples are etanercept and abatacept (ctla- ig), which were approved by the european medicines agency in and , respectively. the former is a chimeric protein that carries the external portion of the tumor necrosis factor (tnf) receptor linked to the igg fc region, which captures soluble tnf to block its inflammatory effects . the latter example is a fusion protein that combines the extracellular portion of human ctla- and igg fc. abatacept is a competitive inhibitor that blocks t-cell activation and can be used in the treatment of inflammatory illnesses such as rheumatoid arthritis . natural killer (nk) and lymphokine-activated killer (lak) cells. natural killer (nk) cells were described in the s based on their capacity to eliminate tumor cells without prior sensitization, with differences observed compared with specific cytotoxic t cells (which are activated based on the recognition of the target cells) , . in , piontek et al. reported that nk cells have the ability to preferentially kill cells that had lost the expression of the major histocompatibility complex class i molecules , . lymphokine-activated killer (lak) cells are a heterogeneous population that includes not only nk but also nkt and t cells, which can be generated in an in vitro culture of peripheral blood mononuclear cells (pbmcs) in the presence of il- . dr. rosenberg and collaborators carried out studies using these cells in the presence of il- (reviewed by rosemberg ). these lak cells showed good antitumoral responses in % of the melanoma patients who received them as therapy . however, secondary effects such as liver toxicity and the expansion of the treg population limited their therapeutic effect. researchers started to design new recombinant il- with some mutations to avoid the activation of tregs , with linking it to polyethylene glycol (peg) to increase its half-life and efficacy . another cytokine described later, il- , showed similarities to il- in many respects , and it had some unique advantages, such as the capacity to activate nk and cytotoxic t cells (tc) but not tregs. il- is being used in different versions (alone, as a heterodimer with receptor il- /il ra or il rα igfc, or in an agonist complex with alt- ) and in combination with other therapies in several clinical trials (examples: nct , nct , and nct ). more recently, researchers have focused their attention on other cytokines and combinations (such as il- , il- , and il- ) , which are able to activate nk cells in vitro and induce a good responses in animal models. in some human clinical trials, remission has been observed for patients with acute myeloid leukemia , , which broadens the options for the use of nk cells in the treatment of this pathology. the properties of nk cells reveal their versatility as treatments against tumors. nk cells are able to kill tumors through several mechanisms, including receptor-mediated cytotoxicity, antibodydependent cell-mediated cytotoxicity (adcc) and death receptormediated apoptosis, but they also secrete cytokines such as interferon gamma that enhance the antitumoral adaptive immune response. nk cell adoptive transfer (either autologous or allogenic nks) is currently being tested in clinical trials for hematological diseases and solid tumors, and numerous research groups have recognized their potential in other situations, such as transplant rejection and pregnancy. nk cell lines, memory-like nk cells and stem cell-derived nk cells are additional types of cells that can be used for tumor immunotherapy . regarding other cellular therapies, nk cells as substitutes for t cells for use upon transformation with an chimeric antibody receptor (car) are being explored (see below). dendritic cells. paul langerhans identified dendritic cells in human skin in , but these cells were not named until by dr. ralph m. steinman (nobel prize in ) and dr. zanvil a. cohn, who chose the term because the cell morphology, with long extensions, resembles that of neuronal dendrites . in humans, dendritic cells are obtained from different sources that vary in origin, maturation state and tissue distribution (skin, lymphoid tissue, circulating cells). among the main types of dendritic cells, plasmocytoids are conventional myeloid dc and dc , pre-dc and monocyte-derived dendritic cells. in the epidermis, there are three types: langerhans cells (lc), monocyte-derived lc-like cells and inflammatory dendritic epidermal cells (idecs) . as indicated above, dcs are antigenpresenting cells and are the only cells that are able to activate naïve t lymphocytes. a subpopulation of dcs also carries out a process known as cross-presentation. in this way, they facilitate the activation of both helper and cytotoxic t lymphocytes . in addition to their participation in the immune response, they can be used in antitumoral therapeutic vaccines , . it is possible to generate a type of blood monocyte-derived dendritic cell in the presence of a mixture of cytokines in culture -a process that induces their subsequent maturation and activation in the presence of tumor antigens (cell lysates, recombinant or purified antigens, peptides, rna, dna, and viral vectors ). these cells can also be obtained from bone marrow hematopoietic cd + progenitor cells . other sources, such as circulating or skin dendritic cells, are relatively scarce and are therefore not usually used. after their differentiation and activation in vitro , , dcs are exposed to tumor antigens and infused back into the patient (either by blood infusion or injected into areas near the lymph nodes or even directly into them) to reach the secondary lymphoid organs as soon as possible, at which point they can present antigens to the t cells. this approach is a type of individualized therapy and is therefore expensive. the first approved vaccine in which autologous dendritic cells were used was sipuleucel-t (provenge) , which was a treatment for prostate cancer refractory to hormonal treatment. immunotherapy with dendritic cells is currently being tested in more than clinical trials for various tumors: brain, pancreas, mesothelioma, melanoma and many others (clinicaltrials.gov identifiers: nct , nct , nct , and nct , respectively). the data indicate that the therapy is well tolerated and has led to increased patient survival in some trials. furthermore, complete cure and partial remission outcomes have also been observed. the lack of efficacy on other tests was probably due to the presence of immunosuppressive factors in the tumor environment. another therapeutic use of dendritic cells involves their induction of immunosuppression both in transplants and in autoimmune diseases . in an autoimmune pathology such as multiple sclerosis, the intention is to achieve stable tolerogenic dendritic cells that can act against some autoantigens (such as myelin peptides) in the presence of vitamin d , dexamethasone, or other agents . phase i clinical trials have generally shown good tolerance to this therapy without serious adverse effects . however, greater control of this treatment is necessary in several respects to obtain the best therapeutic results ; e.g., the human immunology and immunotherapy: main achievements and challenges j varadé et al. type of dendritic cells and ex vivo differentiation, the antigens used, and the injection route are important considerations. gamma/delta t cells (tγ/δ). human t cells expressing γ/δ tcr cells have interesting properties, including the capacity to kill a broad range of tumor cells. the advantages of these cells in cancer therapy are based on their independence from mhc expression on tumor cells and that their relative insensitivity to some inhibitor molecules (such as pd- ). the initial clinical application, with the adoptive transfer of autologous vδ + cells after ex vivo expansion, showed only sporadic responses , and different exploratory studies are currently being carried out to increase their clinical therapeutic use. allogeneic vδ + cells are also being explored in cancer therapy; e.g., they are being used against refractory hematological malignancies and advanced cholangiocarcinoma . although the basis of immune regulation was suggested centuries ago, regulatory t cells were described by sakaguchi et al. as cd + cd + natural regulatory t cells that expressed the forkhead box p transcription factor (foxp ) . later, induced or adaptive regulatory t cells were also identified, including different subsets that carry several phenotypic markers and express various cytokine secretion profiles . all of these factors play crucial roles in the maintenance of immunological self-tolerance by suppressing autoreactive t cells. the manipulation of tregs to achieve therapeutic outcomes is a field of great interest, because of both their expansion and activation in diseases, such as allergic and autoimmune diseases, and as a potential targets for cancer immunotherapy . tumor-infiltrating lymphocytes (tils). lymphocytes that infiltrate solid tumors are called tumor-infiltrating lymphocytes (tils). in , thomas and burnet proposed that the immune system performs tumor immune vigilance, with lymphocytes as sentinel cells leading to the elimination of somatic cells transformed by spontaneous mutations , . since the end of the s, dr. rosenberg has been trying to prove and improve the effective use of tils. the process starts with surgery and the isolation of tils from a tumor, followed by til activation in culture in the presence of cytokines, cellular expansion and, finally reinfusion into the patient. since its inception, this therapy has been improved markedly, with an increase in optimal responses from less than % to the current - %, in some cases. these higher success rates are due, in particular, to the prior preparation of the patient, including the depletion of lymphoid tissues, to avoid an expansion of regulatory cells , myeloid suppressor cells and other cells that can compete with the transferred tils. currently, there are more than trials in which tils are being used alone or in combination with other immunotherapies on several tumors, such as melanoma, metastatic colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, ovarian cancer and breast cancer. this individualized therapy has limitations; it can only be used on solid tumors, and the number and specificity of the tils and the type of tumor and microenvironment make standardizing this therapy difficult. chimeric antigen receptor (car). since tils include a variety of t lymphocytes with different specificities, the next step was to obtain t cells of a single type (monoclonal cells) carrying a clonal receptor capable of recognizing tumor antigens. this effort was carried out for the first time in mice and subsequently, in , in humans with a transgenic tcr against the mart- melanoma antigen , . these types of receptors are known as ttcrs, but their ability to recognize antigens is restricted since they can only identify the peptides presented by antigen-presenting cells on self-histocompatibility molecules. this situation changed because of one of the latest revolutions in antitumor therapy, the development of t lymphocytes that carry a chimeric antigen receptor (car) based on a specific antibody directed to a target surface molecule , . these modified t cells can directly recognize tumor cells without required antigen processing or presentation by professional antigen-presenting cells. moreover, the car includes all of the necessary elements for intracellular signaling and activation of helper and cytotoxic t lymphocytes. car therapy was developed by one of its pioneers, dr. carl june at the university of pennsylvania in the united states , who used modified t lymphocytes that carried a chimeric antigen receptor to target cd + leukemic b cells. after interacting with cd + cells, these modified car t cells were activated and able to proliferate and exert cytotoxic functions against target cells. in this case, both tumor and healthy b cells were affected. although bone marrow continues to produce b lymphocytes, in cases of severe b lymphopenia, it is possible to provide exogenous immunoglobulins periodically. the whole process of the current car t-cell therapy begins with blood donation, from which lymphocytes are purified and genetically modified in vitro by a viral vector, which carries the genes coding for the chimeric antigen receptor. the cells are expanded in the presence of cytokines in culture and are subsequently reinfused into the patient. this type of cellular immunotherapy is individualized for each patient, with his/her car t cells ultimately destroying the tumor. since the first generation of cars appeared, namely, a chimeric receptor composed of an anti-cd -specific single-chain variable fragment linked to a transmembrane domain and intracellular signaling domain of the t cell receptor (cd ζ chain), researchers started to modify the original design. new generations of cars, including the cd ζ subunit together with other signaling domains, such as cd , cd , cd ( - bb), cd , and icos, or combinations (cd ζ, cd , and cd ) , have been developed in the second and third generations of cars to improve several aspects, such as the activation, proliferation and survival of car t cells. the fourth generation of cars show improved the antitumoral effects by carrying additional molecules (such as cytokines or drugs), improvements to the safety of car tcell therapy through the use of suicide genes and many new designs, such as dual cars or the so-called split universal and programmable (supra) car system . in addition to t cells, other types of cells, such as nk cells, are now being explored for use in antitumoral responses . in an effort to avoid using personalized treatment, researchers are now working on universal cars that may be used on many different patients without inducing the problem of rejection [ ] [ ] [ ] [ ] . the encouraging results obtained with this therapy have led to interest from companies, and some commercialized examples are available, although many more "in-house" or academia-produced cars are in clinical trials. car t-cell therapy was initially designed for use against hematological cancers (leukemia and lymphomas), but many new opportunities have been opened for its use against solid tumors , infectious diseases (such as hiv) , allotransplantation, autoimmune diseases and severe allergies . china and the usa are the leading countries in producing car t-cell therapy, and numerous clinical trials are underway. immunotherapy for covid- patients coronavirus disease (covid- ), which is produced by severe acute respiratory syndrome coronavirus (sars-cov- ), affects millions of people in many countries. most of the infected patients ( - %) are asymptomatic or have mild symptoms, but the disease in some patients progresses to a moderate or severe illness that requires hospitalization in intensive care units because of respiratory distress, multiorgan failure, and/or other pathologies, and more than one-half million fatal cases have been human immunology and immunotherapy: main achievements and challenges j varadé et al. reported worldwide. the most vulnerable population includes aging patients and those with comorbidities such as hypertension, diabetes and cardiovascular diseases. there are several aspects of the covid- pathogeny that suggest an overreaction of the immune system in severely ill patients, with increased levels of inflammatory cytokines such as il- , il- and others (creating the so-called "cytokine storm"), together with blood lymphopenia and cd t cell and nk cell exhaustion. special therapies have not yet been identified to cure these patients, and preventive vaccines are not yet available, but some immunotherapies have been proposed as adjunct therapies, and some of these are currently in different phases of clinical trials . the immunotherapeutic strategies include the following: future challenges in immunotherapy immunotherapy has been used for centuries, but only in recent years has this area expanded rapidly in several respects, mostly by the use of soluble elements (monoclonal antibodies and cytokines) and, more recently, with immune cells (cellular immunotherapy). there are many fields in which immunotherapy faces a range of challenges: vaccines. . researchers are working on reducing the number of injections by employing a combination of vaccines. several current vaccines contain components from - pathogens in a single injection, and these are able to provide adequate protection against all of these pathogens . . researchers are developing more stable and durable vaccines. improvements in the half-lives of vaccines, for example, by lyophilization, while maintaining immunogenicity is expected to reduce current problems, especially those involved in the transportation of vaccines to remote areas . in this respect, nanotechnology can help in the design of more stable vaccines that lead to slow antigen release and improved immunogenicity . . researchers are working on vaccines that confer protection against all serotypes of a specific pathogen (universal). this outcome is especially important for pathogens with high variability (such as the influenza virus). researchers are designing vaccines that can protect against several variants by using common regions that can induce protective immune responses to all or most of the variants . . researchers are developing alternative routes of administration (e.g., oral, inhaled, intranasal, skin, rectum, vagina) as substitutes for intramuscular or subcutaneous injections. one of the problems to be solved is the immune tolerance developed to elements delivered by the oral route, but some vaccines are already effectively administered by this route (such as the oral polio, cholera, typhoid fever and rotavirus vaccines). the intranasal route has also proven effective for some vaccines (nasal influenza vaccine), and vaccines administered through other routes are under investigation. . researchers are seeking the early protection of newborns . newborns are very susceptible to infections due to their immature immune system . moreover, the protection exerted by maternal antibodies transferred through the placenta during pregnancy against some pathogens interferes with the development of the newborn's own immune response. greater knowledge on ways to activate the immature immune system early will enable the development of vaccines for newborns. moreover, immunization of pregnant women may help to enhance neonatal protection against several pathogens . . researchers are developing new and more effective vaccines. this effort is crucial for very prevalent pathogens such as mycobacteria tuberculosis, hiv virus or plasmodium falciparum. although there are treatments against these pathogens, most are not curative-as in the case of hiv; prevention is the best way to stop their spread. . researchers are working to address emerging pandemics. in the case of new pathogens, such as sars-cov- , which has produced a recent global outbreak, effective vaccines are urgently required . new technologies for vaccine formulations and routes of administration, the identification of immune-related factors of protection and modifications to the governmental regulatory and approval process for vaccines for emerging pathogens are challenges that must be faced to achieve a rapid vaccination procedure for outbreaks. hundreds of vaccines against sars-cov- (using different strategies such as live attenuated or inactivated pathogens, viral vector-based, viral rna, dna, recombinant proteins, peptides, etc.) are now under development, and some are in clinical trials. however, the need to develop a new vaccine in a short period of time should not negate the main principles of vaccination use: safety and immune protection. . researchers are working on genetic (rna, dna) vaccines because they have great advantages, including no requirement for growing a pathogen. genetic vaccines can be obtained in a much shorter time, with much faster and safer production processes, and can be transported much more easily. however, the immunogenicity of these vaccines must be improved, and other problems need be avoided, such as the potential deleterious effects of integrating vaccine sequences into cells . . researchers are developing safer and more powerful adjuvants. many years ago, the only adjuvant authorized for vaccines was aluminum hydroxide (alum), but currently, several adjuvants are on the market . the use of ligands that activate the innate immune response, such as those linked to tlrs or nanostructures with adjuvant effects, is currently under study. . researchers are boosting trained immunity, a new concept related to the innate immune memory-like described for nk cells (expansion) and macrophages (epigenetic modifications). knowledge of how to handle trained immunity will enable better vaccine design and more effective secondary responses . . researchers are seeking to eradicate diseases from the earth. the greatest challenge, eradicating disease is possible in the short term for some pathogens, such as poliovirus. very few cases of polio have been recently reported, and these reports came from only three countries; therefore, it is feasible that this disease can be eradicated in the near future. human immunology and immunotherapy: main achievements and challenges j varadé et al. . advances are challenged by the anti-vaccine movement. paradoxically, there are people who doubt the beneficial effects of vaccines, and they are putting the health of their own children and society in general at risk . the effectiveness of community protection conferred through vaccinated people is disrupted by decreased numbers of immunized persons. this lesser coverage enables pathogens to infect the most susceptible people, such as small children, elderly patients and those who cannot be vaccinated due to certain pathologies or because they are undergoing immunosuppression treatment. thus, news about the return of illnesses that were nearly forgotten, such as tetanus in italy (the first case in years), the death of a child in catalonia from diphtheria, or the exponential increase of measles cases (already counted in the thousands) worldwide , should make parents think carefully about the risks of not protecting children by vaccines. the world health organization (https://www.who.int/topics/vaccines/en/) argues that anti-vaccine movements can roll back all the achievements thus far in this field and have cited this issue as one of the main challenges to be resolved. addressing the antivaccine movement requires a coordinated effort of professionals to inform parents adequately and perhaps other types of coercive measures that some countries are already applying (financial fines, denial of access to public assistance in childcare units, removal of authorization to travel/live in some countries, new laws, and so on). antibodies and cytokines. immunotherapy with monoclonal antibodies has been a true revolution for many pathologies, as has the use of certain cytokines and recombinant fusion proteins. it is therefore predicted that these approaches may have a bright future, and regulatory agencies are expected to authorize many more mab-based therapies in the coming years, especially given the good results obtained in clinical trials. complete antibodies or those modified to increase their functionality or decrease their immunogenicity, combinations of antibodies and cytokines, antibody fragments, etc., are only some of the many possibilities for this type of product, which will expand the range of therapeutic options. one of the main problems regarding the use of antibodies in therapy, especially in cancer, is based on their often unpredictable efficacy. large variability in terms of remission and durable clinical benefits between patients is observed (for example, in the antitumoral responses by antibodies directed to the checkpoint inhibitors). thus, the main challenge is to understand the situations in which an antibody will have the desired effect. it is crucial to find validated biomarkers (with predictive and/or prognostic value) that can help to stratify or select patients for the best immunotherapy. a better understanding is also required for tumor heterogeneity, resistance to some drugs and immunosuppressive microenvironments . an in-depth immunological study, together with a personalized approach, is certainly the way to improve the success of these types of therapies. in combination with conventional therapies (radiotherapy, chemotherapy, and surgery), other immunotherapeutic drugs or cellular immunotherapies can also help to maximize the efficacy of this immunotherapy, but increases in toxicity will be another challenge to face. pathogens. the use of oncolytic viruses (ovs), bacteriophages that selectively infect bacteria, modified pathogens for vaccines or for antitumor immuno-activation, and the manipulation/ modification of the microbiota are some of the therapies that are being considered. ovs are designed to kill tumor cells and to activate the immune system against those cells. however, many of ovs have shown limited therapeutic effects when applied in monotherapy; therefore, much more work is required to improve their systemic antitumor effects and avoid the attenuation of the virus, which limits the viral replication. several obstacles, such as low viral delivery and spread, resistance to therapy and antiviral immunity, have been observed . thus, the main challenges with oncolytic viruses are addressed by improving their antitumoral efficacy, including the optimization of viral delivery, the development of ovs engineered to activate the immune system (e.g., by releasing cytokines), and their use as adjuvant therapies or in combination with other immunotherapeutic agents, such as immunomodulators . regarding gut microbiota manipulation as a therapeutic approach, fecal microbiota transplantation is an effective therapy for recurrent clostridium difficile infection and is now being investigated for other indications, such as inflammatory bowel disease and cancer. some of the challenges facing microbiome transplantation are the lack of precise knowledge about the complete microbiome and the mechanisms of action involved in its therapeutic capacity, the large variability of its effectiveness and the external factors that affect it. more studies are centered on understanding how to manipulate bacterial colonies, the discovery of therapeutic molecules, nutrient competitions, etc., that are required for successful application. the best type of therapy (either individual or the combination of bacteria) is also under debate, along with how to reach the market by translating this individualized therapy into commercial scale products. the safety and potential adverse long-term effects are also being assessed. other components (nanomaterials and small molecules). nanomaterials. to obtain approval for the use of other elements from incipient fields, such as the use of different types of nanostructures, either alone or in combination with other immunotherapies, it is important to resolve certain issues. in the case of nanoparticle use, a better understanding of the interaction between nanomaterials and biological media; nanoparticle biodistribution, metabolism and biocompatibility; and the reproducibility of the synthesis and scaled up production of nanomaterials are among the issues to address. small molecules. a greater knowledge of several molecules involved in the immune system has led to the development of new therapeutic agents, which have been synthesized by traditional chemistry and block or activate intracellular signaling. the low cost of production of these molecules, along with the scaling and reproducibility of small-molecule batches, has attracted the attention of pharmaceutical companies interested in a whole set of new immunomodulatory drugs. a better understanding of the mechanism of action of small-moleculebased drugs and proof that they offer higher efficacy than existing therapies, either in monotherapy or in combination therapy, are challenges that face those seeking to engineer new types of targeting molecules. cellular immunotherapy. to date, cellular immunotherapy has been an individualized therapy with high production costs, and it requires the involvement of multidisciplinary groups in hospitals. a real challenge in the field of cellular immunotherapy is the acquisition of universal off-the-shelf cell therapies to replace those currently made to order in a very personalized manner. the development of universal cells, for example, in the case of car tcell therapy, would increase the number of patients who could benefit from this treatment at thus reduce the costs. other challenging aspects of cellular immunotherapy are the life-threatening toxicity of induced and their lack of effect on solid tumors, which is mostly due to the immunosuppressive tumor microenvironment. this approach requires new strategies to overcome these difficulties. in addition to cancer, cellular immunotherapy has a long history of use against autoimmunity, infectious diseases, allergies and transplantation rejection. it is also important to find biomarkers for prognosis/prediction that can help to optimize this method. other therapies that involve the use of activated nk cells, tumor-infiltrating lymphocytes, vaccination with dendritic cells, etc., are having partial clinical success. similar to other treatments, these approaches require further study, but it is feasible that they may become reality in the near future. greater knowledge of the immune system, especially concerning the variety of cellular and humoral components and the close regulation among them, the interaction with other systems or with elements such as the microbiota, will allow the development of new types of therapies that may be safer, more effective and specific but with much lower toxicity than found in current therapies. this long journey has been possible due to the efforts of numerous researchers (throughout the centuries), who have contributed with their work, creativity, successes and failures to advance our knowledge of the immune system, cellular components, membrane markers, interactions, signaling pathways and many more aspects. this great combined effort has paved 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study and conceived the project, and all the authors participated in writing the paper. competing interests: a.g-f is a copromoter of the spin-off nanoimmunotech. there are no competing financial interests in relation to the work described. key: cord- - vcy f i authors: bagheri, vahid; nejatollahi, foroogh; esmaeili, seyed alireza; momtazi, amir abbas; motamedifar, mohamad; sahebkar, amirhossein title: neutralizing human recombinant antibodies against herpes simplex virus type glycoproteins b from a phage-displayed scfv antibody library date: - - journal: life sciences doi: . /j.lfs. . . sha: doc_id: cord_uid: vcy f i abstract the hsv- envelope glycoprotein b (gb) plays a critical role in virus entry into host cells. neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. our present study focused on the selection of neutralizing single-chain fv (scfv) antibodies of a phage-displayed nonimmune human scfv antibody library against gb of hsv- . to enrich specific scfvs, two phage antibodies were isolated against amino acid residues – derived from the n-terminal part of gb using panning technique. two scfvs, scfv-gb and scfv-gb , with frequencies of % and % were obtained from scfv clones after performing pcr and mvai fingerprinting. in phage elisa analysis, both gb and gb scfvs demonstrated high reactivity with the gb peptide. in the neutralization assay, scfv-gb and scfv-gb represented neutralizing effects of % and %, respectively. upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of hsv- infections. herpes simplex virus (hsv) type i remains a significant pathogen and many people ( to % of the adult population) around the world have been reported to be infected with this virus [ ] . hsv- tends to the nerves and establishes a long-term latent infection in the nervous system [ ] . in certain people, recurrent infections occur in the trigeminal ganglia following periodic reactivation of latent virus [ , ] . the virus causes a variety of diseases with a spectrum of severities, including cold sores (fever blisters), conjunctival and corneal lesions, pneumonia and meningoencephalitis in the immunocompromised individuals [ , , ] . antiviral drugs are able to reduce viral infection and are considered as efficacious treatments for hsv- . however, virus resistance to these agents is increasing [ ] . several glycoproteins play a critical role in virus entry into mammalian cells and cell-to-cell spread, including gb, gd and gh/gl. the first stage to establish an infection is low-affinity and reversible attachment of the virus to the cell surface that is mediated by gb binding to surface heparin sulphate proteoglycan (hspgs) [ ] . in the next stage, gd interacts with one of the multiple potential entry receptors, nectin , -o-sulfated heparan sulphate or herpesvirus entry mediator (hvem). following gd attachment to one of its receptors, it undergoes conformational changes and a ph-independent fusion of an active multi-glycoprotein complex involving gb, gd, gh and gl will occur [ , ] . formation of neutralizing antibodies can reduce the severity of hsv- infection. high levels of neutralizing antibodies may inhibit hsv- spread and viremia [ ] . among glycoproteins of hsv- , gb has the most conserved entry glycoprotein that can act as fusion proteins. four functional regions on gb have been identified by monoclonal antibodies. these regions include (i) initial residues of the n-terminal part, (ii) residues to , to , and a less-defined zone within domain ii, (iii) residues to of domain v and residues within domain i, and (iv) residues of domain iv nearby to domain iii [ ] . gb elicits a remarkable amount of neutralizing antibodies. almost all neutralization activity is accounted for by epitopes in the n-terminal -amino acid portion of gb [ ] . isolation of specific antibodies by antibody phage display has become a popular method in recent years. in general, these libraries life sciences ( ) [ ] [ ] [ ] [ ] [ ] consist of either single chain fragment variable (scfv) or fab fragments [ ] . the smallest antibody fragments are scfv molecules ( - kda) [ ] . scfv antibodies have several advantages in terms of pharmacokinetic characteristics including lower retention in non-target tissues, higher penetration into target tissues, and lack of inducing human anti-mouse antibody responses [ , ] . therefore, human scfv antibodies perform considerably better than conventional antibody molecules used for therapeutic applications. thus far, several neutralizing scfv antibodies have been produced against a number of viruses including scfv antibodies against human immunodeficiency virus type (hiv- ) [ ] , scfvs to human cytomegalovirus [ ] and scfv against influenza a virus h n subtype [ ] that can inhibit viral infections in vitro. nejatollahi et al. described a neutralizing scfv-phage antibody against glycoprotein d of hsv- with neutralizing effect of %, which was capable of neutralizing hsv- and inhibiting virus entry to host cell [ ] . here, we selected two neutralizing anti-gb scfv antibodies to inhibit cytopathic effects in vero cells infected with hsv- . a phage scfv display library in the form of frozen tg e. coli cells was previously provided as described [ ] . briefly, μl of the expanded e. coli scfv library glycerol stock were grown overnight at °c on tyg agar/ampicillin plates ( . % tryptone, % yeast extract, . % glucose, . % nacl, . % agar, . % ampicillin in deionized water). all grown cells were scraped in ml ty broth and incubated at °c for h. after reaching an optical density of more than one, μl of m ko helper phage was added to medium and mixed. then, it was incubated at °c for min without shaking followed by shaking at °c for min. bacteria suspension was transferred to a ml conical tube and centrifuged at rpm for min. the bacterial pellet was transferred to ml ty broth containing ampicillin ( μg/ml) and kanamycin ( μg/ml) and cultured overnight with shaking at °c. afterwards, the culture was centrifuged at rpm for min. the supernatant containing phage was filtered and stored at °c. the procedure of panning was performed using of the human phage display library against synthetic epitope (apsspgtpgvaaa) of gb (isogen, netherland) as follows. in each round panning, the peptide [ μg/ml in phosphate buffered saline (pbs)] were coated on immunotube (nunc, roskilde, denmark) overnight at °c. the tubes were washed four times with × pbs and blocked h at °c with % fetal bovine serum (fbs) and % skimmed milk in pbs. then, the tubes were washed four times with pbs-tween ( . %) and four times with pbs. for dilution of phage library supernatant ( pfu per ml), an equal volume of blocking solution was added and incubated for h at room temperature. following washing, bound phages were eluted with log-phase e. coli tg and incubated at °c for h. after rescuing with phage m ko , other three rounds of panning were performed against the peptide. to analyze the diversity of the selected scfv antibodies, restriction fragment length polymorphism (rflp) fingerprinting was performed on pcr-amplified scfv dna as described. briefly, dna fingerprinting was carried out on colonies after the fourth round panning. the scfvs inserts in phagemids were amplified using forward (ccatgattacgccaagctttggagcc) and reverse (cgatctaaagttttgtcgtctttcc) primers. mvai enzyme (roche diagnostic gmbh, mannheim, germany) digested each product pcr at °c for h and the dna product run on % agarose gel electrophoresis. for measuring of phage concentration of each phage rescued supernatant, μl of phage antibody supernatant was added to ml log-phase tg e. coli and incubated with shaking at °c for h. serial dilution of bacteria was prepared and cultured onto ty/ampicillin plates. after counting of number of colonies per each dilution, scfv colonies concentration titer per milliliter was calculated. the reactivity of isolated scfv clones to the peptide was determined using phage elisa. μl of peptide ( μg/ml in pbs) was coated on the well elisa plates overnight at °c. after washing the wells with three times with pbs/tween ( . %) and three times with pbs (three times), the wells blocked by μl of blocking solution ( % w/v skimmed milk in pbs) and incubated at °c for two h. following washing, for dilution of phage rescue supernatant ( pfu ml − ), blocking solution was added to it ( : ). diluted phage supernatant incubated in wells at room temperature for h. after washing wells, μl of : dilution of anti-fd bacteriophage antibody (sigma, uk) in pbs was added to their and incubated at room temperature for . h. the plate was washed and μl of the hrp conjugated goat anti-rabbit igg ( : in pbs) added and incubated at room temperature for h. the wells were washed and stained using . mg/ml abts (sigma chemical, pool, usa) in citrate buffer (ph ) containing μl hydrogen peroxide in the dark at room temperature for min. the absorbance of each well was read at nm using an elisa reader and calculated for each scfv antibody. patients that had lip lesions were selected and hsv- was isolated. neutralization test confirmed virus genus using guinea-pig anti-hsv- serum (nih, usa) and monoclonal (glycoprotein d and g) anti hsv- antibodies [ ] . vero cells (atcc ccl- ) were cultured in dmem medium and supplemented with % fetal calf serum (gibco, australia), . % (v/v) sodium bicarbonate, units/ml penicillin, μg/ml streptomycin sulphate, and . μg/ml amphotericin b (gibco, germany). cells were grown in the humidified air with % co at ∘ c about - h to be confluent. the anti-hsv- activities of scfvs were evaluated using plaque reduction assay as following described. hsv- was diluted in dmem (sigma-aldrich, germany) to pfu/ml. equal amounts of virus and each scfv phage rescue supernatant were mixed and incubated at °c for h. a ml of each mixture was added into each well of -well plates containing confluent vero cells (performed in triplicate), which were then rocked gently min at °c. after removing mixture, dmem containing ml of % w/v carboxymethyl cellulose (cmc) supplemented with % fetal calf serum, . % (v/v) sodium bicarbonate, unit/ml penicillin, μg/ml streptomycin sulphate, . μg/ml amphotericin b and . n naoh added. the number of micro plaques was counted during five days and compared with the number of plaques seen in the virus control wells which were without scfv. after four rounds of panning against peptides of gb, clones were randomly analyzed by pcr to confirm the presence of the inserted segment. the predictable band ( bp) was observed in all clones (fig. ) . dna fingerprinting of scfv clones against epitope of gb after four rounds of panning is shown in fig. . two patterns, including pattern one (lanes , , , , , , , and ) and two ( , , and ) with the frequencies of % (scfv-gb ) and % (scfv-gb ) were obtained. the patterns of scfvs were used as predominant patterns for further investigation. the reactivity of scfvs-phage antibodies (scfv-gb and scfv-gb ) with the gb peptide produced positive elisa readings and was significantly higher than those of controls (unrelated peptide, unrelated scfv-phage antibody and without peptide) at nm (fig. ) . the neutralization capacity of the anti-hsv- scfv antibodies was evaluated using percentages of plaque reduction, and was % for scfv-gb and % for scfv-gb ( table ) . the first stage of hiv- infection is associated with the specific interaction of viral surface proteins with cellular proteins, lipids, or carbohydrates, and can be blocked by neutralizing monoclonal antibodies [ ] . in this study, monoclonal antibodies (mab) were mapped to an identified domain (domain i to vi) in the crystal structure of gb and classified as groups to . monoclonal antibody h that belonged to a subgroup bound functional region (fr ) in domain vi of gb. fr containing the first residues of the n terminus formed the epitope of mab h . mab h had high neutralizing effect and reduced the plaque number on vero cells by n % [ ] . the antibody recognized a single peptide spanning residues to as previously reported. pereira et al. mapped neutralizing epitopes in continuous regions of the aminoterminal half of gb [ ] . neutralizing antibodies h [ ] and h [ ] recognized residues - . precise peptide mapping of mab revealed that the epitope for mab h was residues to and constituted fr [ ] . therefore, we chose peptide fragment apsspgtpgvaaa (amino acids residues - ) as an immunodominant epitope that was recognized by neutralizing monoclonal antibodies. antibody display on phages is a powerful tool that provides ligand specificity for targets of interest through binding to pure antigens or synthetic peptides coated on a solid phase in repetitive panning rounds [ ] [ ] [ ] . in this study, phages expressing the scfv were selected on gb epitope after the fourth round of panning. scfv-gb and scfv-gb were chosen because of more frequency compared with other scfv-phage antibodies (fig. ) . antibody library with antigen can be screened in several ways, such as direct phage elisa and indirect or capture phage elisa [ ] . the resultant phage clones can be tested using phage elisa to determine whether scfvs displayed on phages are specific to the peptides. several studies have previously used this method to evaluate the specificity of antibodies for the targeted epitopes or antigens. for instance, specificity of scfvs against helicobacter pylori [ ] , hepatitis b virus surface antigen [ ] , staphylococcal enterotoxin b (seb) [ ] , and mycobacterium avium subsp. paratuberculosis surface proteins [ ] have been certified using phage elisa. if optical density (od) of specific scfvs against interest targets at nm are two folds higher than negative controls, a positive phage elisa is confirmed [ ] . our results revealed that scfv-gb and scfv-gb are specific for the epitope of gb and the mean od of both antibodies at nm were two folds higher than control ods (fig. ) . plaque reduction assay was used to test the neutralizing activity of scfv antibodies. hitherto, neutralizing scfv antibodies have been produced against many viral proteins, but surface glycoproteins have been the most appealing target owing to their important role in virus attachment to receptors of host cells; examples of such antibodies include scfvs against ha (haemagglutinin) of influenza a virus h n subtype [ ] , gb and gh cytomegalovirus [ ] , respiratory syncytial virus [ ] , rabies virus [ ] , and enteric coronavirus [ ] . in a previous study, one recombinant fab-phage antibody (fab - ) was selected against the gb of hsv- and hsv- using panning. this fab isolate was not capable of neutralizing either strain when used alone [ ] . nejatollahi et al. isolated one scfv-phage antibody (scfv-gd ) against glycoprotein d with a neutralizing effect of % [ ] . in this study, two scfv-phage antibodies (scfv-gb and scfv-gb ) could neutralize virus with neutralizing effects of % and %, respectively, and significantly decreased cytopathic effects in vero cells. moreover, both scfvs could inhibit the virus in vitro when used alone (table ) , and did not require dimerization to exert neutralizing effects. owing to their several advantages, single chain antibodies could serve as useful tools for antibody-based therapies. better penetration to target tissues because of the small size [ , ] has made scfvs more effective than antibodies in therapeutic applications especially when viral antigens are the targets [ , ] . development of the selected neutralizing antibodies with the human origin is essential for hsv- because these antibodies play a major role as therapeutic agents for the central nervous system infection and pneumonia induced by hsv- in immunocompromised individuals. in addition, about % of neonatal infections are caused by this virus [ ] . risk of acquisition of neonatal infection is increased with low maternal hsv- antibody avidity. thus, hsv- neutralizing antibodies can find potential application in preventing neonatal herpes infection [ ] . in conclusion, the present study aimed at selecting scfv antibodies of a phage-displayed nonimmune human scfv antibody library against gb of hsv- and two scfvs, scfv-gb and scfv-gb , with frequencies of % and % were obtained from scfv clones, and showed neutralizing effects of % and %, respectively. these results encourage further attempts (mainly through genetic engineering) to promote the development of scfv antibodies with preserved binding capacity but enhanced neutralizing effects. also, future studies are warranted to test the neutralizing effect of the identified scfv antibodies in a purified and phage-free form to obtain a better assessment on their neutralizing effect and also test the dose-response association for the neutralizing effects of scfvs. optimized scfv antibodies with enhanced neutralizing effects could also be relevant for testing in clinical trials. the authors declare that they have no conflict of interest. table percentage of plaque reduction in three plaque reduction assay experiments. the scfvs-gb and scfvs-gb antibodies showed reduction of % and % in plaque numbers, respectively. control well contained no antibody. number of plaques mean % reduction experiment experiment experiment scfv-gb fig. . phage elisa of positive scfv phage clones against the gb peptide. scfv-gb (a) and scfv-gb (b). od ( nm) of both scfvs were significantly higher than controls (unrelated peptide, unrelated scfv, m ko helper phage and without peptide and reacted specifically with the gb peptide. the data are presented as the mean ± standard error. p b . . herpes simplex virus infections herpes simplex virus latent infection in the nervous system vaccination prevents latent hsv infection of mouse brain experimental investigation of herpes simplex virus 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simplex virus glycoprotein b: neutralizing epitopes map in regions of continuous and discontinuous residues a subset of type-specific epitopes map in the amino terminus of herpes simplex virus type glycoprotein b an efficient method for isolating antibody fragments against small peptides by antibody phage display isolation of high affinity human antibodies directly from large synthetic repertoires overview of antibody phage-display technology and its applications antibodies from phage antibody libraries identification of single chain fv antibody fragment against helicobacter pylori screening and evaluation of human single-chain fragment variable antibody against hepatitis b virus surface antigen selection of staphylococcal enterotoxin b (seb)-binding peptide using phage display technology isolation of high-affinity single-chain antibodies against mycobacterium avium subsp. paratuberculosis surface proteins from sheep with johne's disease human monoclonal single chain antibodies (huscfv) that bind to the polymerase proteins of influenza a virus, asian pac cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus phage-displayed and soluble mouse scfv fragments neutralize rabies virus neutralization of enteric coronaviruses with escherichia coli cells expressing single-chain fv-autotransporter fusions directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries single-chain fragment variable antibody piezoimmunosensors a novel disulfide-stabilized single-chain variable antibody fragment against rabies virus g protein with enhanced in vivo neutralizing potency intracellular-delivery of a single-chain antibody against hepatitis b core protein via cell-penetrating peptide inhibits hepatitis b virus replication in vitro effect of serologic status and cesarean delivery on transmission rates of herpes simplex virus from mother to infant hsv neutralizing antibodies further refinement in preventing neonatal herpes infection the present article was extracted from the thesis written by vahid bagheri and was financially supported by the shiraz university of medical sciences (grant no: - ). the authors would like to thank mr. p. talezadeh shirazi for the technical assistance. key: cord- -grsv j authors: flego, michela; ascione, alessandro; cianfriglia, maurizio; vella, stefano title: clinical development of monoclonal antibody-based drugs in hiv and hcv diseases date: - - journal: bmc med doi: . / - - - sha: doc_id: cord_uid: grsv j today there are many licensed antiviral drugs, but the emergence of drug resistant strains sometimes invalidates the effects of the current therapies used in the treatment of infectious diseases. compared to conventional antiviral drugs, monoclonal antibodies (mabs) used as pharmacological molecules have particular physical characteristics and modes of action, and, therefore, they should be considered as a distinct therapeutic class. despite being historically validated, antibodies may represent a novel tool for combatting infectious diseases. the current high cost of mabs' production, storage and administration (by injection only) and the consequent obstacles to development are outweighed by mabs' clinical advantages. these are related to a low toxicity combined with high specificity and versatility, which allows a specific antibody to mediate various biological effects, ranging from the virus neutralization mechanisms to the modulation of immune responses. this review briefly summarizes the recent technological advances in the field of immunoglobulin research, and the current status of mab-based drugs in clinical trials for hiv and hcv diseases. for each clinical trial the available data are reported and the emerging conceptual problems of the employed mabs are highlighted. this overview helps to give a clear picture of the efficacy and challenges of the mabs in the field of these two infectious diseases which have such a global impact. the innate immune response is the first-line defense in determining the outcome of an infection. infectious agents contain conserved motifs on their surface that react with conserved pattern recognition toll-like receptors of the host. this interaction initiates a powerful innate immune response. moreover, the infectious agent's surface proteins and carbohydrates come into contact with b-cell receptors, membrane-bound immunoglobulin of isotype m (igm) or d (igd), and often induce potent antibody responses, which take some weeks to fully develop [ ] . when a vertebrate organism encounters a pathogen, such as a virus or bacteria, it generates a polyclonal antibody response against numerous epitopes on different antigens during infection; therefore, polyclonal serum contains a large and diverse population of antibodies, which also include neutralizing antibodies (nabs). thus, polyclonal serum-derived biotherapeutic products can contain various nabs against multiple and distinct epitopes; these nabs provide strong protective activity due to additive or even synergistic effects on neutralization. however, in this type of product the vast majority of their constituent specific antibodies are non-neutralizing, since they are directed against misfolded protein or against epitopes on native surface proteins for which antibody binding is not protective [ , ] . furthermore, for some viral and bacterial infections, no correlates of protection have been established; therefore, the significance of antibody titers, apart from indicating past exposure, is not clear. mechanisms of immunological escape can explain why total antibody titers are not always protective. many infectious organisms, including viruses, can constantly mutate surface proteins and exploit glycans to shield important epitopes, diverting the antibody response away from functionally important epitopes in favor of immunogenic irrelevant epitopes [ ] . thanks to their protective properties, the administration of hyperimmune sera from immunized animals or immune human donors, named 'serum therapy', was the first effective treatment of infectious diseases. later, the advent of antibiotic therapy with the advances in vaccine design has meant that serum therapy was almost abandoned for many infectious diseases. nevertheless, hyperimmune human sera immunoglobulin preparations are still used to treat different bacterial toxins and virus related diseases, including those caused by cytomegalovirus (cmv), respiratory syncytial virus (rsv), hepatitis a virus (hav), hepatitis b virus (hbv), rabies, vaccinia, vesicular stomatitis virus (vsv) and measles, underscoring the fact that antibody therapy remains an effective means of treatment [ , ] . today, the ability to rapidly generate and manipulate antibodies with a defined epitope recognition, named "monoclonal antibodies" (mabs) (figure ), has opened a new window of opportunity for a rematch of antibodies in clinical practice. this achievement has been possible thanks to advances in cellular biology and biotechnology (figure ) , and also to improved purification techniques which have made these therapeutics safer, less immunogenic and more effective. mab preparations have many advantages over immune sera-derived preparations which can vary due to both time and the source of origin, since different hosts mount different antibody responses. one advantage is that mabs, by virtue of the fact that they are chemically defined reagents, exhibit relatively low lot-tolot variability and low risk of pathogen transmission. another advantage for mab preparations is the much figure schematic structure of a mab. all immunoglobulins are composed of two identical light (l) chains and two identical heavy (h) chains, linked by disulphide bonds (black dashed bars). the heavy chains contain one variable domain (vh) and three or four constant domains (ch , ch , ch and ch ) depending on antibody isotype. by contrast, the light chains contain only one variable domain (vl) and a single constant domain (cl). within the fab region, at the end of the two arms of the y-shaped molecule, the variable domain of a heavy chain pairs with the light chain variable domain to form the antigen-binding site. in more detail, within the matched v regions, three short polypeptide segments on the heavy chain and three on the light chain form the complementarity-determining regions (cdrs), which dictate the precise antigen-binding characteristics of the antibody. on the other end, the fc domain, which includes the sites for interaction with the complement system and fc receptors, mediates effector functions determining the fate of the bound antigen. greater activity per mass of protein since all the ig molecules are specific for the desired target. this phenomenon is illustrated by the report that two . mg doses of two mabs provided the same protection against tetanus toxin as to mg of tetanus immunoglobulins [ ] . neither does mab therapy have the immunological complications associated with the use of heterologous sera in humans, such as serum sickness and immediate hypersensitivity, which significantly limited the latter's usefulness [ ] . in recent years, mabs have emerged as a new class of biological drugs in oncology as well as in immune and inflammatory diseases, albeit their development in infectious diseases has been slower. to date, the only mab approved in this field is palivizumab, an anti-rsv mab licensed for prevention of severe respiratory disease in high-risk infants and immunocompromised adults. now the scenario is gradually changing and there are many antibodies against viruses and bacteria in various stages of clinical development. this trend has also been influenced by the development of different scientific disciplines, which makes it possible to study and dissect the function of individual microbial structures supporting figure evolution of mabs linked to the need to decrease their immunogenicity. different methods to obtain mabs are depicted. mouse mabs, the 'hybridoma' cells derived from the stable fusion of immortalized mouse myeloma cells with lymphocytes from immunized mice, are screened to identify individual clones producing identical antibody to a single antigenic determinant [ ] . chimeric mabs, the murine constant regions of both heavy and light antibody chains (mch and mcl), are replaced with human counterparts (hch , hch , hch and hcl ), leaving intact the murine variable portions (mvh and mvl) [ ] . humanized mabs, only the cdrs of the murine mabs (mcdrs) from both the mvh and the mvl, are 'grafted' into a human backbone antibody [ , ] . human mabs, )human memory b-cells isolated from patients are immortalized by epstein barr virus (ebv) and cpg oligodeoxynucleotide, and then screened for specific antibody production [ ] . ) transgenic mice, obtained by a genetic replacement of the mouse immunoglobulin genes with human counterparts, are used to obtain fully human mabs by traditional hybridoma technology [ ] . ) antibody libraries, constructed by in vitro combinatorial assembly of human immunoglobulin variable-region gene (v genes) and cloned to provide the display onto phage surfaces, are subjected to a panning against an antigen in order to select specific clones [ ] . the first mabs of each category approved for clinical use are shown. palivizumab is the first and, so far, only mab approved for infectious diseases. the endings used to name the different types of mabs are also indicated. the development of more targeted drugs. there are excellent reviews about this topic [ , ] . in this review we focus on the mab-therapies now underway in clinical trials (table ) designed for human immunodeficiency virus (hiv) and hepatitis c virus (hcv) infectious diseases. both these worldwide epidemics require new strategies due to the lack of a definitive cure and effective vaccines, to the continuous emergence of drug resistant variants, to the toxicities of licensed drugs and to the need to ensure a treatment for all patients. in this context, antibodies represent an intriguing alternative as therapeutics; in their favor are their different resistance mechanisms and a more favorable toxicity profile when compared to other available drug classes, fitting them for use in conjunction with the current chemotherapy by slowing the onset of resistance and possibly enhancing therapeutic efficacy. the antibody structure comprises a pair of identical heavy and light chains linked by disulphide bonds held in a yshaped arrangement (figure ). the fragment antigen-binding (fab) portion, the region that binds the antigen, is composed of one variable and one constant domain of both the heavy and the light chain. the remaining constant sections of the longer heavy chains form the tail of the y, termed the crystallizable fragment (fc) region, which provides the signal for effector functions. antibodies can provide protective effects through various mechanisms [ ] . viral neutralization is generally meant as the ability of an antibody to provide sufficient steric interference to disrupt the interaction between a microbic antigen and its ligand in experimental conditions in vitro. this activity is clearly associated with protection, thanks to their fab domain alone, both in natural infection and after immunization. virus infection includes sequential steps beginning with attachment to cell-surface receptors and ending with delivery of the viral genetic material into the cytoplasm [ ] . fusion of viral and cellular membranes is a basic entry mode for enveloped viruses, such as hiv and hcv [ ] , which still differ in specific aspects of viral entry and assembly, thus offering unique therapeutic opportunities. the cell surface is certainly more directly accessible for the action of the antibodies; therefore, the phase of virus entry is one of the most important targets in preventing viral infection at the origin, and many known nabs act at this step. for the same reason, inhibition of the release of progeny virus is another possible mechanism of neutralization, as demonstrated by antibodies directed against influenza a virion surface neuraminidase [ ] . in hiv and hcv fields, no virus release inhibiting antibodies have been identified to date. the interaction of hiv envelope surface protein gp with its host receptor, cd , on human t cells triggers conformational changes in the envelope, resulting in exposure of a transient binding site for co-receptor ccr or cxcr . this in turn promotes additional conformational changes in virus gp protein which allow it to insert its fusion peptide into the target cell membrane to initiate membrane fusion and viral entry into host cells. nabs can inhibit viral infection by several different mechanisms in parallel with the steps that allow the viruses to enter into cells ( figure ). they can directly block virus attachment to target cells by interfering with virus-receptor interactions, as in the case of nabs against the cd -binding site on hiv gp [ ] . this same goal can also be achieved by directing the antibodies to the virus receptor and/or co-receptor on host cells. mabs can also block fusion at the cell membrane at the post-binding/pre-fusion stage, as exemplified by anti-cd [ ] and/or anti-ccr /cxcr (ccmotif receptor /cxc-motif receptor ) mabs, under development [ ] . again, mabs directed to the external proximal membrane region of hiv gp can interfere with conformational changes needed for membrane fusion [ ] . unlike hiv, hcv entry into target cells occurs via clathrin-mediated endocytosis of the viral particle [ ] . subsequent release of the viral genome into the cytosol requires the ph-dependent fusion of viral and cellular membranes. current models suggest that hcv circulate as lipoviral-particles (lvps) in the vascular system, these consisting of lipoproteins in complex with virus particles. following localization to the surface of hepatocytes through interactions of lvps with glycosaminoglycans and the low density lipoprotein receptor, specific binding of the e and e virus surface glycoproteins with the host sr-b scavenger receptor and cd occur [ ] . subsequently, viral particles are translocated to regions of the membrane possessing tight junction proteins occludin and claudins; the binding to these receptors results in clathrin-mediated endocytosis. as for hiv, mabs directed against spike viral proteins, as well as against host receptors, may act at an early stage of infection by preventing the binding of the virus on the cell surface. for example, antibodies recognizing the cd -binding site within the envelope glycoprotein e have been shown to block viral entry, as have a number of anti-receptor antibodies targeting cd [ , ] and sr-bi [ ] . some antibodies may act by blocking conformational changes and/or the requisite interactions between the viral and endosomal membranes required for fusion; although as yet no fusion determinant within the envelope glycoproteins has been defined. host protection in vivo is more complex and involves the interaction of antibodies with cells and molecules of the innate immune system. the antibody can exert protective actions through an fc region-mediated recruitment of other components of the immune system, including antibody-dependent cell-mediated cytotoxicity (adcc), complement-dependent cytotoxicity (cdc) and antibody-dependent cellular phagocytosis. receptors for the fc segment of igg (fcy receptors; fcγrs) are expressed on the surface of different types of cells, including natural killer cells (nk), monocytes, macrophages, dendritic cells and neutrophils. with the exception of γδt cells, fcγrs are not normally found on t lymphocytes. similarly, the receptor for fc segment of iga, the fcαr, involved in phagocytosis and induction of microbe killing, is expressed on monocytes, macrophages and neutrophils [ ] . the adcc process is triggered by the interaction between the fc region of an antibody bound to a nonself antigen exposed on host cells, and the fc receptors on immune effector cells. the subsequent release of cytokines and cytotoxic granules containing perforins and granzymes promotes the death of the target cell. cdc is initiated by complement component c q binding to the fc region of igg, which is in turn bound to the foreign antigen on the cell surface. this triggers a proteolytic cascade to activate the complement, so leading to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. the fc region can also mediate complement binding to and deposition on free virions, which can cause a direct virotoxic effect or inhibit virus binding to cells. moreover, the so-called opsonization process, consisting of the binding of antibody fab portion to the antigen following by the interaction of fc domain to an fc receptor on phagocytes, is a powerful mechanism to enhance the phagocytosis [ ] . with respect to hiv, a potential role for adcc in modulating the course of hiv infection was first proposed on the basis of studies showing an inverse association between adcc antibody levels and the clinical stage of disease. the strongest evidence for a role for adcc antibody in disease progression comes from a study by baum et al. of the multicenter aids cohort study [ ] . in that study, rapid progressors had significantly lower adcc antibody titers against cem. nkr cells coated with gp than did non-rapid progressors at corresponding visits or non-progressors at any visit. morever, hiv-infected individuals with spontaneously undetectable viremia were shown to have higher adcc antibody levels than viremic subjects [ ] . in the context of hcv infection, fc-mediated effector functions, although less well understood, can still have an important role. sera from both the acute and chronic phase of infection can mediate adcc via binding to viral protein e expressed at the cell surface [ ] , while several e -specific mabs are able to induce cdc of e -expressing cells [ ] . optimizing non-nab effector functions, such as adcc, cdc and fagogocytosis, may prove critical in the design of new effective anti-hcv therapeutic antibodies [ ] . ( ), as well as by binding to the viral receptor or co-receptor on host cell surface ( ) . some antibodies, can neutralize viral infection through interfering with conformational changes required for membrane fusion and subsequent release of the viral core into the target-cell cytoplasm; this postbinding neutralization may occur at the cell surface ( ), or inside the endosomes for the viruses (for example, hcv) whose entry into the cell requires an endocytosis step ( ). antibodies recognizing viral or host proteins expressed on infected cell surface can exert protective actions through the fc-mediated effector functions (for example, cdc, adcc) ( ). again, mabs may prevent the release of progeny virions ( ) . at the bottom the antibody neutralizing effects on the viruses before cell binding, including the direct virolysis by cdc and the mab-mediated enhanced phagocitosis, are shown ( ) . in panel b, the possible mab-mediated immunomodulary therapies are depicted. in some chronic viral infections, virus-specific immune cells may persist in a 'non-functional' state, because of an imbalance of immunoregulatory signals involving multiple inhibitory and activating receptors, triggered by soluble factors and/or cell surface ligands. therapeutic approaches using specific mabs to block host immunosuppressive molecules (antagonism) or to trigger activating receptors (agonism) may be a valid strategy to restore immune cell function and treat various chronic viral infections. system is the exhaustion of virus-specific t cells. exhaustion consists of a progressive dysfunction characterized by the inability to proliferate and to produce key antiviral and immune stimulating cytokines (for example, interleukin (il)- , tumor necrosis factor (tnf)-a, interferon (ifn)-γ), or to lyse infected cells [ ] . a feature of functional exhaustion is that it affects many antiviral properties of both mouse and human cd + t cells. loss of effector functions proceeds in a hierarchical manner starting with defects in il- production and proliferation, followed by the decrease of tnf production. cytotoxic activity is also lacking in exhausted human cd + t cells. at a severe stage of exhaustion, ifn-γ production is eventually compromised, with exhausted t cells ending up deleted if the high antigenic load persists [ ] . exhaustion can also occur in cd + t cells in both mice [ ] and humans [ ] . probably the best explanation for this progressive dysfunction and loss of effector t cells is the continuous triggering of virus-specific t cell receptors owing to a high antigenic load in persistently infected hosts without a critical rest period. the current consensus is that functional exhaustion is a way of limiting the magnitude of effector t cell responses. although this may safeguard against autoimmune responses, it may also compromise effective immunity against persistent infectious agents and tumors [ ] . exhausted t cells are subject to complex layers of negative regulation. this involves signaling through multiple inhibitory receptors that inhibit functional and proliferative responses. the cd family member programmed cell death (pd- ) has been shown to be the most highly expressed inhibitory receptor on cd + t cells during chronic infection, and to have a major role in regulating t cell exhaustion during infection [ , ] . increased expression of pd- by t cells also occurs during hbv and hcv infections [ ] [ ] [ ] . several other inhibitory receptors have also been shown to induce t cell unresponsiveness during chronic infections. these receptors include cytotoxic t lymphocyte antigen (ctla- ) [ , , ] , t cell immunoglobulin domain and mucin domain protein (tim ) [ , ] , and lymphocyte activation gene (lag- ) [ ] . in addition, certain cytokines, such as il- and transforming growth factor-β (tgfβ) as well as regulatory t cells, may also contribute to the lack of t cell functionality during situations of high antigenic burden [ ] . there is intriguing evidence that blockade of the inhibitory receptor could restore antigen t cell responses. for example, blockade of the pd- signaling pathway improves antigen-specific t cell proliferation and cytokine secretion in lymphocytic choriomeningitis virus (lcmv)-infected mice [ , ] and in humans with chronic hiv [ , , ] , hbv [ ] and hcv [ ] infections. this effect was synergistically improved in lcmv infected mouse following the simultaneous blockade of the t cell inhibitory receptors pd- and lag- , thanks to which a diminished viral load in vivo was observed, although blocking lag- pathway alone had little effect on the severity of exhaustion [ ] . moreover, mabmediated blocking of ctla- pathway in vitro augments hiv-specific cd + t-cell function suggesting that the immune modulation of this target may also provide a clinical benefit in infected individuals [ ] . another example is the manipulation of signals mediated by glucocorticoid-induced tnf receptor (gitr), a recently identified member of the tnf receptor superfamily, preferentially expressed on subset cd +cd + regulatory t cells. gitr signals break the suppressive activity of this subset. in fact, an agonistic anti-gitr mab immediately injected after viral infection significantly increased the number of activated cd + and cd + t cells secreting ifn-γ [ ] . one must remember that the manipulation of immunological responses could have detrimental effects on the host, as highlighted by the recent tragic human trial of tgn . this is a mab against human t cell costimulatory molecule cd developed by tegenero to treat b-cell chronic lymphocytic leukaemia, autoimmune and inflammatory diseases, on the basis of its capability of inducing preferential activation and expansion of immunosuppressive regulatory t (treg) cells, as observed in rodent models. tgn has been termed a 'superagonist' because it binds to cd and activates t cells without the need for prior t cell antigen receptor (tcr) signaling. in a phase i clinical trial (in march ), following administration of tgn , six healthy young men suffered a life-threatening cytokine-release syndrome (crs) involving multi-organ failure, something unpredicted by the preclinical studies. it is now clear that in the presence of tgn , activated cd + effector memory t (t em ) cells were the source of the cytokines that mediated the crs observed in the volunteers. treg cells were not able to prevent systemic inflammation, probably because the balance between activated treg cell and t em cell numbers is disadvantageous for humans compared with laboratory rodents. furthermore, in macaques, but not in humans, cd + t cells lose cd expression during their differentiation into t em cells; this detail, however, had gone unnoticed despite many years of primate testing. in conclusion, this model failed to prevent the disastrous case above [ ] . in view of these events, such a risk needs to be carefully assessed if the modulation of immune inhibitory or activating receptors is used for increasing the functional activity of virus-specific t cells in order to avoid nonspecific inflammation. these therapeutic approaches are being carefully evaluated for cancer as well as for hiv and hcv chronic viral disease. given the potential antiviral effect of the antibodies, viruses have evolved multiple mechanisms to protect themselves from antibody binding. one of these, the viral receptor glycosylation, is widely shared among different viruses. carbohydrates are poorly immunogenic and, therefore, do not stimulate the response of type b lymphocytes and simultaneously hide the underlying protein structures. hcv e protein contains up to potential nlinked glycosylation sites. specific glycans mask the cd binding site and, therefore, nab epitopes [ ] . lipid shielding may represent an additional strategy used by hcv to evade the antibody response. current data suggest that key neutralizing epitopes are less accessible on lvps. more recently, hcv has been found capable of direct cellto-cell transmission, which is largely resistant to antibody neutralization [ , ] . hiv envelope protein is also glycosilated and changes occur in the frequency and position of glycans hiv gp ; these 'evolving glycan shields' have been shown to decrease sensitivity to antibody neutralization [ ] . other factors of antibody escape for hiv are: trimerization of the gp and gp that can shield vulnerable epitopes better exposed on the individual monomeric subunits; kinetic and spatial constraints that impede antibodies from accessing potentially vulnerable sites during receptor binding and membrane fusion process; the variable loops of gp that are a prime target for nabs, which usually have a very narrow breadth of reactivity [ ] . finally, the high mutation rate of many viruses, including hiv and hcv, which undergo rapid antigenic variation, allows them to escape neutralization, constituting a significant hurdle for nabs development. all these problems may be counter-balanced by selecting nabs which target conserved and more accessible areas of viral particles, and/or by using mixtures of nabs which target various key epitopes. in fact, it has been demonstrated that combination therapy with mab cocktails prevents escape variants for many viruses, including influenza [ ] , coronavirus [ ] and lcmv [ ] , and that broad neutralization in the sera of most of some individual hiv infected donors can be associated with single or four to five principal specificities [ ] . recent studies have indicated that nabs play a critical role in hcv disease outcome. viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection with some evidence that these antibodies are broadly reactive [ , ] . in contrast, chronic hcv infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection, despite the induction of crossneutralizing antibodies in the later phase of infection. current understanding of the nab response raised against hcv suggests that e is the major target, and that multiple epitopes within e may be targeted by both linear-and conformation-dependent antibodies. predominantly, these neutralization epitopes overlap with cd binding sites and clearly demonstrate a role in inhibition of entry. currently, one of these mabs, mbl-hcv , is being investigated in clinical trials in the prevention of liver re-infection after transplantation, for which novel antiviral preventive and therapeutic strategies are urgently needed. in fact, re-infection of the graft is universal, being characterized by accelerated progression of liver disease; ifn-based therapies exhibit enhanced adverse effects and limited efficacy in these patients [ , ] . mbl-hcv is a fully human monoclonal antibody isolated from transgenic mice and directed to a highly conserved linear epitope of hcv e glycoprotein. it is able to neutralize pseudoviruses from multiple hcv genotypes and has demonstrated efficacy in preventing hcv genotype- infection in hcv naïve chimpanzees. a phase i open-labeled, dose escalation study was performed in healthy adult volunteers starting with mg/kg and escalating to , , and mg/kg after a -day post-infusion safety review. mbl-hcv was well-tolerated without any seriously adverse effect event. based on the favorable safety, tolerability and pharmacokinetics data, a phase ii study of mbl-hcv in chronically infected hcv patients undergoing liver transplantation has been planned [ ] . in the context of hiv disease, despite intensive study over two decades, only a small number of broadly neutralizing mabs have been identified from infected patients and little is known about their activity in vivo. these antibodies are able to inhibit viral entry of most primary hiv isolates in vitro [ , [ ] [ ] [ ] and the exceptionally high level of mutation found in their genes may reflect chronic immune responses to hiv and persistent hypermutation and selection [ ] . a number of trials evaluating different formulations of anti-hiv monoclonal antibodies are now in progress. the first trial assessed a chimeric monoclonal antibody cgp , to the v loop of the hiv- envelope gp over weeks [ , ] . subsequent studies evaluated the kinetics of monoclonal antibody f directed to the cd -binding site of gp [ , ] , a humanized antibody binding to the v epitope gpgraf [ ] . finally, a humanized mab, kd- is under evaluation in clinical trials. its epitope was mapped to aa, igpgra, at the tip of the v loop of envelope protein and demonstrates crossneutralizing activity against hiv- isolates in clade b [ ] . a drug based on the mab cocktail mode is also currently in clinical development. in this regard it has already been observed that in hiv neutralization assays the effectiveness of a mix of broadly neutralizing antibodies increased synergistically compared to the effect of the individual antibody. the synergy effect was relatively weak, with a maximum of two-to four-fold enhancement, between antibody pairs, thereby increasing neutralization titers about -fold in triple and quadruple antibody combinations [ ] . however, the use of antibodies in the cocktail mode, as an approach to improve their effectiveness, is already recognized for other pathogens or toxins. in the case of tetanus toxin, it has been reported that combining the action of three out of four antibodies increased the neutralizing activity up to times [ ] . in the case of botulinum toxin, neutralizing activity has been reported up to , times higher when using a mixture of three monoclonal antibodies [ ] . instead, other studies have demonstrated that the combination of two potent neutralizing mabs against hiv, vrc and pg , although not synergistic, can mediate additive neutralization viral activity and provides an improved neutralization coverage of % to % of viral strains by combining independent epitope targeting [ ] . in a proof-of-concept passive immunization trial with humans, it has been demonstrated that a cocktail of the three broadly neutralizing mabs - g , e and f was able to delay viral rebound in patients whose infections were fully suppressed by antiretroviral treatment before administration of the antibodies [ ] . interestingly, the main antiviral effect observed was primarily attributable to the g antibody, a mab that binds to a noncontinuous epitope composed of glycosylation residues distributed over the envelope protein gp [ ] , whereas the other two mabs, e [ ] and f [ ] , recognize two adjacent highly conserved epitopes on the membrane-proximal ectodomain of the hiv- envelope protein gp . in earlier phase i clinical trials, safety and tolerability were demonstrated [ , ] . during a longterm multiple dose phase ii clinical trial, high doses of the three neutralizing antibodies were given in combination to hiv- -infected individuals at weekly intervals over three months. pharmacokinetic analysis revealed that repeated infusions at high dose levels were well tolerated by the patients and did not elicit an endogenous immune response against the monoclonal antibodies. the antibodies showed distribution and elimination kinetics similar to those seen for other human-like antibodies, though monoclonal antibody g had a significantly longer elimination half-life ( . +/- . days) than monoclonal antibodies e ( . +/- . days) and f ( . +/- . days) [ ] . furthermore, analyses of the emergence of mutations conferring resistance to these three mabs were performed. sequence analysis of the g epitope relevant n-glycosylation sites of viruses derived from patients demonstrated that mutations in these sites are associated with resistance. in vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape g pressure. importantly, in vitro selection with f and e demonstrated that resistance to these nabs can be difficult to achieve and can lead to selection of variants with impaired infectivity [ ] . moreover, generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication; some generated triple-resistant viruses seemed to be impaired in their replicative fitness, and none of the patients developed detectable viruses that escaped neutralization by all three mabs within the day observation period [ ] . as is true with all mabs designed for infectious disease, the development of a successful vaccine would reduce the need for them. however, given the scarcity of drugs in the field of virology and given the slow progress on the hiv vaccine front, the development and use of microbicides, compounds that could be applied topically to prevent hiv transmission, is one of the possible strategies to counter the spread of hiv. in this regard, mabs could be proposed as suitable components of microbicides to fight hiv entry at mucosal surface. a safety study of p g mab administered vaginally in healthy women has been completed. p g is the broadly neutralizing g mab manufactured from tobacco plants [ ] . most mabs in clinical trials have been produced using a system called chinese hamster ovary cell (cho-cell) fermentation [ ] , including g used along with f and e antibodies as a cocktail. the cho-cell fermentation production method is very expensive and cannot produce enough mabs on a scale required for the global market; therefore, plant manufacture of such mabs may hopefully offer some solutions to lower production costs and improve output. the process yields five grams of purified antibody from kg of tobacco and production costs could be to times lower than when using conventional bioreactors. this study has been designed to confirm the safety of a vaginally delivered mab p g derived from plants and manufactured to good manufacturing practice (a quality standard used for the manufacture of medicinal products). the medicine is the first plant-produced antibody to be greenlit for clinical testing by britain's medicines and healthcare products agency (mhra). it took about a year to get that agency's stamp of approval because it required assurances that the drugs did not contain allergenic plant sugars or pesticides. no matter how it is produced, p g antibody has not been shown to actually prevent hiv- infection in clinical trials; thus a version made from tobacco plants would not see approval any time soon. p g would also likely be just one ingredient in a cocktail of plant-produced antibodies [ ] . to eliminate or reduce the development of escape variants it has been proposed that targeting the conserved cellular receptors of the virus may open new avenues for a viable antibody therapy for hiv infection. hiv entry into cd + t cells requires the presence of a co-receptor, either ccr or cxcr , on the target cell. thus, based on this hypothesis, mabs directed against cd and against the co-receptor ccr , have been developed and are being analyzed in clinical trials. cd functions as a co-receptor, physically associating with the tcr during ag recognition by binding to a nonpolymorphic component of the major histocompatibility complex (mhc) class ii molecules on the surface of the antigen-presenting cell. ibalizumab, a humanized mab, binds cd on t cell surface away from the binding site for mhc class ii molecules. it does not inhibit gp binding to cd but appears to exert its antiviral property by post-binding conformational effects that prevent cd bound gp from interacting with ccr or cxcr [ , ] . by contrast, other monoclonal antibodies, that competitively inhibit gp binding, interfere with mhc class ii immune function [ , ] . the reported human experience with ibalizumab consists of three clinical trials. during phase i study, it was observed that peak mean reductions in viral load occurred later in the higher dose cohorts, whereas the extent and duration of viral suppression correlated with the degree of cd + cell coating by ibalizumab, which was maintained longer in the higher dose cohorts, with a duration of to days. peak increases in cd counts at one day after infusion, well before the peak declines in viral load; this suggests that the increase may have been due to redistribution of cd + cells from lymphoid tissue rather than regeneration of cd + cells in the setting of viral suppression. a multidose study demonstrated continued safety over an extended treatment period and provided data on the development of ibalizumab resistance. resistance testing showed reduced susceptibility relative to baseline. resistant isolates remained dependent on cd for viral entry, suggesting that resistance did not develop through the use of alternative receptors. genotypic analysis was unable to identify mutations, diagnostic of ibalizumab resistance. consistent with the allosteric mechanism of ibalizumab's anti-hiv- effect, the development of resistance is associated with a reduction in the maximum percentage inhibition rather than the shift in the ic characteristic of competitive inhibitors [ , ] . the half-life of iggs under normal physiological circumstances is two to three weeks [ ] . in contrast, the average half-life of ibalizumab is to . days [ ] . this is consistent with observations of other anti-cd antibodies, in which internalization or shedding of the receptor results in more rapid antibody degradation. a randomized, double-blind, placebo controlled, phase iia study has evaluated the ibalizumab efficacy, the results showing a considerable viral load reduction with respect to the placebo arm [ ] . ccr is a chemokine receptor that mediates activation and migration of t cells and other leukocytes. ccr using (r ) viruses typically mediate transmission and then predominate through the progression to symptomatic disease. viruses can use an alternative chemokine receptor, cxcr , either exclusively or in addition to ccr . the cxcr -using virus can be present initially, but tends to result in an increasing proportion of subjects in the later stages of the disease [ ] . ccr co-receptor antagonists represent an emerging antiretroviral treatment class and the first to target a host molecule. currently, two anti-ccr mabs are being investigated. one of these is ccr mab , a fully human igg monoclonal antibody with robust activity against a diverse panel of hiv- isolates; it synergizes in vitro with other arv classes and appears safe and effective in reducing hiv viral load. high levels of receptor occupancy were observed for to days with the highest dose cohorts, suggesting the potential for weekly, fortnightly or even monthly dosing [ ] . the other anti-ccr mab is pro , a humanized mab that also synergizes with small-molecule ccr antagonists in laboratory studies [ ] . pro is being investigated in two modes of administration: the classical intravenous (iv) form, and subcutaneous (sc) form. the trial involving sc administration is the first to bear the proof of concept for a mab administered subcutaneously in hiv- infected subjects as a potent and longacting antiretroviral agent. an iv form of pro tested as monotherapy in hiv- subjects with only r virus detectable [ ] demonstrated potent and prolonged antiviral activity, with a . log mean reduction in hiv- rna and safety relative to placebo. the successive randomized, double-blind, placebo-controlled iia trial examined the antiviral activity, tolerability and pharmacokinetics of single intravenous infusions of up to -mg/kg of mabs. all pro -treated subjects treated with mg/kg experienced a -log -unit reduction in hiv- rna level, there being just one exception; a post-study analysis using the enhanced-sensitivity trofile assay determined that this subject had dual/mixed virus at screening. there was no change in co-receptor tropism or emergence of pro -resistant virus during the course of this study, supporting the view that pro broadly inhibits r hiv- with a high barrier to resistance. the maximum tolerated dose of iv pro has not been determined, suggesting a sizeable margin of safety for pro sc administration study [ ] . the study involving pro sc administration showed virologic suppression between successive doses and no changes in r viral susceptibility to pro following three weeks of monotherapy, indicating no adaptation of virus to use ccr in the presence of drug. pharmacokinetic data suggest the possibility of a drug regimen administered fortnightly for hiv infected individuals. proteins and other macromolecules drain from sc sites into both blood capillaries and the lymphatic system. in animals, proteins with molecular weights of greater than , daltons have been observed to drain primarily into the lymphatic system following sc administration [ ] . such proteins transit through lymph fluid and typically are not absorbed significantly into the blood until they reach the thoracic duct. since the molecular weight of pro is approximately , daltons, a substantial amount of sc pro can be expected to drain into the lymphatic system and potentially encounter ccr + cells in lymphoid tissues prior to reaching the bloodstream. for these reasons, serum concentrations may not provide a full picture of the overall exposure following sc dosing of pro . sc infusion is currently used by individuals with primary immunodeficiency to self-administer at home significantly larger amounts (approximately grams) and volumes (approximately ml total, up to ml/site) of the weekly sc-administered immunoglobulin [ ] . self-administration of mg sc pro would be much simpler in comparison. therefore, sc pro offers the potential for significant dose-dependent hiv- rna suppression and may offer greater convenience for many patients in terms of patient selfadministration [ ] . the sc injection mode was chosen in order to evaluate pro safety and efficacy as an adjunct to an oral antiretroviral regimen in hiv-infected injection drug users with viral rebound and documented poor adherence to the previous antiretroviral regimen. therefore, a phase iib, national, multicenter, randomized, doubleblind, placebo-controlled study was initiated and is currently recruiting participants. given the complications that arise from the occurrence of drug resistances, the use of antibodies together with combined therapy increases the drug number and, therefore, the therapeutic opportunities. in particular, in the case of ccr inhibitors, one report has demonstrated that resistance to ccr inhibitors may increase the sensitivity of the resistant virus to certain neutralizing antibodies [ ] . compared to ccr , cxcr -based blocking agents as therapy against hiv are less attractive due to the crucial role of cxcr in many biological processes, and the absence to date of known naturally occurring mutations leading to the inactivation of cxcr gene in humans. moreover, one major problem is linked to the fact that, whereas r viruses are found on their own in % or more of patients, viruses that using cxcr co-receptor (x ) usually are present mixed together with r viruses; therefore, the use of cxcr specific mabs could result in only little or transient effect on the overall viremia, also complicating the evaluation of pharmacological activity. however, antibodies against cxcr might still provide some benefits for some hiv positive patients when co-administrated with ccr antagonists, if the safety of such combinations is established [ ] . there is a pressing need for antiviral agents that are effective against multiple classes of viruses. broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or on viral envelopes. phosphatidylserine (ps), the most abundant anionic phospholipid of the plasma membrane, is segregated at the inner leaflet of the plasma membrane of resting mammalian cells. loss of ps asymmetry occurs during apoptosis, cell injury, cell activation and malignant transformation, and results from inhibition of the translocases or activation of ps exporters, or lipid scrambling enzymes, such as scramblases. after enveloped viruses replicate within the host cell, they create their 'envelope' by carrying along part of the host cell's membrane upon exiting. as a result, the target phospholipid becomes exposed on the surface of the virus as well as on the infected host cell [ ] . bavituximab is the first in a new class of patented antibody therapeutics that target and, preferentially bind, to these exposed phospholipids. it has demonstrated broad therapeutic potential across multiple oncology indications and represents a new approach to treating viral disease, too. bavituximab is currently being evaluated in randomized phase ii clinical trials for non-small cell lung cancer and pancreatic cancer, for therapy of chronic hcv infection and for hiv/hcv co-infection. the therapeutic effect of bavituximab appears to be due to adcc of tumor and virus-infected cells. since ps exposure is an early event during virus infection, adcc may limit virus spread. furthermore, in the infectious disease setting, bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. three completed phase i hcv clinical trials have shown that bavituximab is generally safe and well-tolerated. reductions in serum hcv rna levels were also observed. a randomized phase ii clinical trial with previously untreated hcv genotype- infected patients was designed to determine the early virologic response (evr) rate after weeks of therapy with bavituximab in combination with the antiviral drug ribavirin and safety profile versus pegylated ifn-α- a and ribavirin. the results show that the combination of bavituximab with ribavirin has a better safety profile than an ifn-containing regimen. however, the evr development in the bavituximab-containing arm was later than the ifn-containing group; therefore, a longer-term evaluation is needed to adequately compare their effectiveness. in addition, the lower dose level appears to be more active in hcv patients than the high dose does. such results suggest that future studies evaluating longer bavituximab treatment durations at or around the lower dose level in combination with ribavirin and potentially direct acting antivirals in certain patient populations may hold promise as ifn-free hcv therapeutic regimens [ ] . targeting ps on cells infected with multiple different viruses and on virions themselves is a promising antiviral strategy. although resistance has developed in monotherapy trials with ibalizumab (an anti-cd antibody), host-derived antigen, such as anionic phospholipids, on virus-infected cells are independent of the viral genome and as a consequence the acquisition of drug resistance should be theoretically less problematic than with agents that target virus-encoded components. since the discovery of pd- as an inhibitory receptor associated with t-cell dysfunction, the roles of various inhibitory receptors on virus-specific cd + t cells have been extensively studied in human chronic viral infections, such as hcv, hbv and hiv infections. as blocking the inhibitory receptors in vitro restores the functions of virus-specific t cells, novel hiv and hcv treatments based on blockade of several immune checkpoint molecules are being investigated. in particular, mabs interfering with two major inhibitory networks of the b :cd family, namely the pd- and ctla- pathways [ ] , are currently being studied in clinical trials, to evaluate their safety and efficacy. these mabs recognize the pd- or ctla- receptor and neutralize the binding with their respective ligands. the pd- :pd-l pathway delivers inhibitory signals which regulate t cell activation. as a result it performs a key role in various processes, namely in multiple tolerance checkpoints that prevent autoimmunity, in the suppressive tumor microenvironment, in the immune-mediated tissue damage, in host defenses aimed at eradicating microbial pathogens and tumors and finally, in t cell exhaustion that contributes to both lack of viral control during chronic infections and to t cell unresponsiveness [ ] . in cancers, a strong correlation between increased pd-l expression on tumors and a negative survival prognosis in patients has already been observed. various studies indicate that mabs targeting the pd- signaling pathway reinvigorate antigen-specific t-cell responses and promote an immune response to fight tumors [ ] . in hcv infection the relationship between the pd- expression and the outcome of the acute hcv infection was questioned; subsequently, recent studies have shown that the progression of acute hcv infection to the chronic stage is associated with a high level of pd- on hcv-specific cd + t cells, whereas the clearance of hcv infection is associated with lower levels of pd- expression [ ] . given these premises, mdx- , a fully human antibody also known as ono- , and ct- , a humanized antibody, both interacting with pd- receptor, are being developed as a treatment for cancer disease and for therapy of chronic hcv infection [ ] . to date, most clinical experience with pd- blockade has been gained with mdx- in the tumor setting. drug-related grade or toxic effects occurred in % of patients, in whom there were drug-related adverse events of special interest, those with potential immune-related causes; they included pneumonitis, vitiligo, colitis, hepatitis, hypophysitis and thyroiditis. pneumonitis ( %) ranged from isolated radiographic abnormalities to progressive, diffuse infiltrates associated with clinical symptoms in a small number of patients. although three deaths occurred, mild-to-moderate pneumonitis was managed successfully with either observation or glucocorticoids. however, objective responses were observed in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; overall, an adverse-event profile does not appear to preclude its use [ ] . besides these studies, an ongoing phase i safety trial with active hepatitis c genotype infected patients has been designed to assess the safety and tolerability profile of mdx- [ ] . clinical studies to evaluate the use of ct- in hcv disease have also been initiated [ ] . ctla- is up-regulated on activated t cells and inhibits t cell activation by reducing the production of il- and arresting cell cycle progression. ctla- has also been shown to have an impact on t cell responses in animal tumor models and humans [ , ] . human trials that used a blocking anti-ctla- mab demonstrated a reduction in tumor mass and clinical benefit in a substantial minority of treated subjects. studies of the role for ctla- in chronic infections have produced mixed results. in chronic hiv infection, many studies indicate that impaired cd + t cell function is associated with viral persistence [ ] , although the function of ctla- in causing hiv persistence by suppressing t cell function remains unclear [ ] . on the other hand, ctla- 's role in chronic hcv infection seems to be more defined. the hcv-specific cd + t cells found in the livers of chronic hcv patients overexpressed not only pd- , but also ctla- . co-expression of pd- and ctla- was observed in liverinfiltrating lymphocytes, but not in peripheral blood lymphocytes [ ] , suggesting the phenotypic differences of virus-specific cd + t cells in different in vivo compartments. pd- and ctla- expressing hcv-specific t cells were profoundly dysfunctional [ ] . tremelimumab is a fully human igg mab directed against ctla- . while a phase ii study for hiv disease with this drug has been withdrawn prior to enrollment, clinical trials for hcv disease are still underway. tremelimumab binds to activated t lymphocytes and results in inhibition of b -ctla- -mediated down-regulation of tcell activation. it also acts as an il- stimulant. it was generated, using xenomouse technology (figure ) , as an anticancer agent and is currently in worldwide phase iii development for malignant melanoma, phase ii development for colorectal cancer, gastrointestinal cancer, gynecological cancer and non-small cell lung cancer in the us and other countries. it is also being investigated for prostate, breast and pancreatic cancer in various countries. as for anti-pd- antibodies, immune-related adverse effects of tremelimumab are of special interest because of its presumed mechanism of action. most of the experience in identifying and managing ctla- treatment-related side effects has derived from studies in cancer, particularly in melanoma. these effects mainly include colitis/diarrhea, dermatitis, hepatitis and endocrinopathies; uveitis, nephritis and inflammatory myopathy also have been occasionally reported. these unique side effects are likely a direct result of breaking immune tolerance upon ctla- blockade; they are generally mild, reversible and manageable, following specific treatment guidelines that include symptomatic therapies or systemic corticosteroids [ ] . in december , pfizer initiated a phase ii trial in patients with latestage unresectable liver cancer who also have hepatitis c infections. the primary endpoint of this single-armed study is the ability of tremelimumab to produce tumor responses among hcv-infected patients with hepatocellular carcinoma and to produce changes in hepatitis c viral load. the first results indicate that tremelimumab demonstrated an excellent safety profile, with a promising antitumor efficacy against hcc in patients, as well as an intense antiviral activity. in fact, a significant and progressive decline in serum hcv viral load was observed, this being associated with an increase in anti-hcv immune response in % of patients [ ] . since there are multiple levels of immunoregulation, a synergistic use of antibodies against different checkpoint molecules might represent the next stage in immunotherapy for chronic infectious diseases, as evidenced from ex vivo studies about the combined pd- /ctla- blockade in hcv disease [ ] . furthermore, because the host mechanisms that inhibit t cell activity are common and conserved aside from specific virus-encoded immune evasion strategies, the antibodies targeting inhibitory receptors may prove extremely versatile drugs potentially effective against multiple classes of viruses. the need to treat hiv and hcv infectious diseases, two epidemics of global impact, has reawakened interest in mab-based therapy, supporting a variety of clinical studies. the results that are emerging, will help to create models for the further development of such drugs and extend their use against other viruses as well. although the mab production costs are high, increasing advances of biotechnology and production systems will make them more competitive on the market, and new approaches, such as using mab cocktails or combining mabs with available drugs, will improve effectiveness. treatment with mabs as part of a drug regimen is the most likely future for mabs that block hcv and hiv infection in order to avoid viral escape, while chronic treatment could attract further investments from pharmaceutical companies. furthermore, broad spectrum mabs, such as bavituximab and immunomodulatory mabs, could be useful against a whole range of diseases, thus extending marketability and profit margins. this review has focused on the use of intact mabs as a novel emerging and versatile class of pharmaceuticals. it is important to note, however, that biotechnology also provides the opportunity to build various antibody formats whose improved pharmacokinetics and pharmacodynamic properties could be co-opted in the fight against infectious diseases. resistance to antiretroviral drugs; the mechanisms of immune reconstitution; and, finally, operational and implementation research in resource limited settings. he chaired or 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outbreak of hepatitis c treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus recurrence of hepatitis c virus after loss of virus-specific cd (+) t-cell response in acute hepatitis c safety and pharmacokinetics of a novel human monoclonal antibody directed against e glycoprotein of hepatitis c virus (mbl-hcv ) in healthy volunteers. easl the international liver congress™ crystal structure of a neutralizing human igg against hiv- : a template for vaccine design a conserved neutralizing epitope on gp of human immunodeficiency virus type human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals a phase i/iia clinical study with a chimeric mouse-human monoclonal antibody to the v loop of human immunodeficiency virus type gp pharmacokinetics of an hiv- gp -specific chimeric antibody in patients with hiv- disease phase i study of a human monoclonal antibody directed against the cd -binding site of hiv type glycoprotein pharmacokinetics of f , a human monoclonal antibody, in persons infected with human immunodeficiency virus type monoclonal antibody hnm in hivinfected patients: a phase i study sequential immunization with v peptides from primary human immunodeficiency virus type produces cross-neutralizing antibodies against primary flego et al isolates with a matching narrow-neutralization sequence motif neutralization synergy of human immunodeficiency virus type primary isolates by cocktails of broadly neutralizing antibodies neutralization of tetanus toxin by distinct monoclonal antibodies binding to multiple epitopes on the toxin molecule potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody mascola neutralization coverage is improved by combining monoclonal antibodies that target independent epitopes hiv- delay of hiv- 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anti-cd monoclonal antibody with anti-hiv- activity in infected patients immunosuppression of cynomolgus renal allograft recipients with humanized okt a monoclonal antibodies functional epitope analysis of the human cd molecule. the mhc class ii-dependent activation of resting t cells is inhibited by monoclonal antibodies to cd regardless whether or not they recognize epitopes involved in the binding of mhc class ii or hiv gp safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly tnx- ), an anti-cd monoclonal antibody, in human immuno-deficiency virus type -infected adults antiretroviral activity of the anti-cd monoclonal antibody tnx- in patients infected with hiv type mouse/human chimeric monoclonal antibody in man: kinetics and immune response phase efficacy and safety of the novel entry inhibitor, tnx- the biology of ccr and cxcr a phase , doseescalation, placebo-controlled study of a fully human monoclonal antibody (ccr mab ) against ccr in patients with ccr -tropic hiv- infection potent antiviral synergy between monoclonal antibody and small-molecule ccr inhibitors of human immunodeficiency virus type a phase i study to explore the activity and safety of sch , a small molecule chemokine receptor- antagonist in hiv type- -infected patients phase a study of the ccr monoclonal antibody pro administered intravenously to hiv-infected adults transport and absorption of drugs via the lymphatic system safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency diseases anti-hiv- activity of weekly or biweekly treatment with subcutaneous pro , a ccr monoclonal antibody neutralizing antibody and antiretroviral drug sensitivities of hiv- isolates resistant to small molecule ccr inhibitors targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases advancing clinical trials in cancer and viral infection: bavituximab antiviral the pd- pathway in tolerance and autoimmunity tumor b -h is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up targeting the pd- /b -h (pd-l ) pathway to activate anti tumor immunity sznol m: safety, activity, and immune correlates of anti-pd- antibody in cancer clinical trial.gov a service of the u.s. national institutes of health: a study of mdx- to treat patients with hepatitis c infection national institutes of health: safety and tolerability study of the monoclonal antibody ct- in patients with chronic hepatitis c genotype infection biologic activity of cytotoxic t lymphocyte-associated antigen antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients cancer regression and autoimmunity induced by cytotoxic t lymphocyteassociated antigen blockade in patients with metastatic melanoma progress in defining cd helper cell responses in chronic viral infections pd- and ctla- inhibitory cosignaling pathways in hiv infection and the potential for therapeutic intervention functional restoration of hcv-specific cd t cells by pd- blockade is defined by pd- expression and compartmentalization the emerging toxicity profiles of anti-ctla- antibodies across clinical indications antiviral and antitumoral effects of the anti-ctla agent tremelimumab in patients with hepatocellular carcinoma (hcc) and chronic hepatitis c virus (hcv) infection: results from a phase ii clinical trial aacr annual meeting continuous cultures of fused cells secreting antibody of predefined specificity chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains replacing the complementarity-determining regions in a human antibody with those from a mouse humanized antibodies as potential therapeutic drugs an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus antibody engineering via genetic engineering of the mouse: xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies selecting and screening recombinant antibody libraries pre-publication history the pre-publication history for this paper can be accessed here clinical development of monoclonal antibody-based drugs in hiv and hcv diseases authors' contributions mf and aa contributed equally to writing and editing the manuscript. mc and sv critically reviewed the manuscript and made final changes. all authors have read and approved the final version of the manuscript.authors' information mf, after her doctorate (immunology, , university of rome, tor vergata, italy), was enrolled as a researcher at the department of therapeutic research and medicine evaluation at the istituto superiore di sanità, rome, italy (iss). she has acquired extensive expertise in biotechnologies, such as the isolation, characterization and genetic manipulation of human mabs in single chain format for the development of biological constructs designed for clinical use. the authors declare that they have no competing interests. mc is the author of a patent about a mab against p-glycoprotein (patent number: ), but this mab is not suitable for clinical applications in hiv and/or hcv diseases submit your next manuscript to biomed central and take full advantage of: key: cord- -ipoelk h authors: crouch, c. f. title: vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity date: - - journal: vaccine doi: . /s - x( ) - sha: doc_id: cord_uid: ipoelk h passive immunity against enteric viral infections is dependent upon the continual presence in the gut lumen of a protective level of specific antibodies. this article examines methods currently used to enhance the titre and duration of specific antibody in the mammary secretions of cows and pigs with particular reference to rotavirus and coronavirus infections. in addition, some of the potential problems to be found in attempting to produce vaccines against these viral infections are outlined neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination ~- . in domestic animals economic losses are suffered, as a result of mortality (ranging between and %), and also veterinary costs and decreased productivity of the survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been found to be primary pathogens in calves ,~ and piglets - . these viruses are most frequently isolated during the period from birth to weaning, and animals of this age have been the most intensively studied because of the frequency and severity of these infections. animals of all ages are, however, susceptible, with subclinical infections apparently common in both adult cows and pigs ,'°. in neonatal calves the incidence of rotavirus and coronavirus associated diarrhoea is similar varying between and % '~'-~ . the situation in neonatal piglets is less clear, rotavirus infections are apparently common .t -tt, w.hilst transmissible gastroenteritis virus (tgev), the prototype enteric coronavirus in swine, is an example of a seasonal cold-weather disease, probably related to both the thermal sensitivity of the virus ~ and the effect of cold-stress on converting subclinical to clinical infections ~ . the pathogenesis of enteric rotavirus and coronavirus diseases of swine and cattle are similar. in contrast to tgev, however, rotaviruses appear to be confined to the alimentary tract, predominantly the small intestine, although there is some evidence in both lambs and piglets for infection of the large intestinal , °. the infections are characterized by diarrhoea and dehydration caused by the functional and anatomical loss of the absorbtive cells of the intestine. the principal site of virus replication has been shown to be the intestinal villus epithelium. the infected cells are lost from the tips of the villi and are replaced with immature crypt cells. generally, there is a dimunition in the number and size of the villi and a progressive replacement of the epithelium with squamous and cuboidal cells which lack a brush bordep - .~. °- . such immature cells have been shown to possess reduced levels of disaccharidases =~. . the loss of the absorptive cells of the intestine is assumed to result in the observed malabsorption syndrome. this is further exacerbated by the decreased ability to utilize dietary lactose, resulting in its accumulation in the large intestine, thereby preventing further absorption of water by exerting an osmotic effect. as a result of the severity of these enteric viral infections during the first few weeks of life, passively acquired antibody is the major source of protection. in calves and pigs there is no selective transfer of immunogiobulins from the maternal to the foetal circulation during the last third of the pregnancy. instead, during the period immediately following birth, maternal immunoglobulin is acquired from the colostrum ofthe dam z ' °. absorption of colostral immunoglobulins by the intestinal epithelial cells is a non-selective process t- lasting - h , s. factors present in colostrum may influence the absorption of immunoglobulins , or help prevent their proteolytic degredation . in addition to immunogiobulins, colostrum and milk have recently been shown to contain functional immunocompetent cells including macrophages and t and b lymphocytes '~°. in contrast to colostral absorption, highly specific mechanisms operate in the colostrum-forming m a m m a r y glands of cattle and pigs causing large amounts of igg (relative to iga and igm) to concentrate in the coiostrum ' ' q ' . lgg passively acquired by the neonate from colostrum persists in the serum for several weeks protecting against systemic infection. in tgev infection of pigs ' and rotavirus infection of calves 'g circulating antibody has been found to be of little value. resistance to these infections appears to be mediated instead by local immunity at the epithelial surface of the intestine. in cattle the selective transfer of igg~ from serum to milk continues throughout lactation, although at a reduced level when compared with colostrum formation. the concentration of all three classes ofimmunoglobulin is significantly reduced ( to -fold) in milk and in consequence igg~ remains the primary immunoglobulin in bovine milk ~. in contrasl in pigs the concentration of lgg~ decreases about -fold during the first week of lactation, whilst that of secretory lga declines only about three-fold, leaving it to become the predominant class of immunoglobulin in swine milld t,' . most adult cattle are seropositive for both rotavirus ,s° and coronavirus s~ antibodies. there is a dramatic decline in these colostral antibody titres during the transition to milld ' "s~- , reflecting this reduction in concentration of immunoglobulins. this partially explains the high incidence of rotavirus and coronavirus infections in calves older than five days, as the titres of passively derived protective antibody decline. despite the presence of one or more common antigens it has been demonstrated that rotaviruses isolated from different species can differ antigenically from each other s - . more recently it has been shown that different serotypes exist within isolates obtained from single species. the existence of at least two different serotypes of porcine rotavirus ° and at least three distinct bovine serotypes ~ have been described. bridger et al have suggested the occurrence ofintermediate bovine rotavirus types , although more work is essential to clarify this situation. some recent isolates possessing the distinctive morphology of rotaviruses have been found to lack the common group antigen. to date, these atypical rotaviruses have been isolated from humans, birds, calves, lambs and pigs ~ . in pigs, preliminary results using two previously characterized atypical isolates , ° have indicated that these are distinct and do not share a common group antigen g ,~'. these observations have been extended by snodgrass et al ga who suggest the occurrence of at least four distinct groups ofrotaviruses based on their group antigen. the significance of the serotypic differences observed between rotaviruses in vitro still needs to be fully assessed in vivo. orbiviruses (also members of the reoviridae) possess many serotypes and require the use of multivalent vaccines . many of the cross-protection studies carried out using different rotavirus serotypes are contradictory and the data inconclusive. for example, in utero vaccination of calves with a bovine rotavirus was found to protect against diarrhoea caused by challenge with human rotavirus serotype , although challenge virus was still shed v . in contrast, one out of three calves was protected against a bovine rotavirus challenge after vaccination with a human serotype or an equine rotavirus tm and this animal shed no detectable virus. furthermore, piglets vaccinated with human rotavirus and challenged with porcine rotavirus were protected against the clinical disease but enhancement of lactogenic immunity:. c f. crouch shed virus ~s. using a more defined challenge system. evidence has been obtained indicating that rotavirus isolates from different animal species and of different serotypes show poor cross-protective properties in vivo tm. this observation has been confirmed and extended by studies in gnotobiotic calves and piglets showing that cross-protection only occurred between rotaviruses of the same serotype, and that even a minor serotype difference could be sufficient to affect cross-protection °,g~. further evidence for a lack of cross-protection between rotavirus serotypes can be obtained from studies of sequential infections, where subsequent rotavirus infections were found to be associated with different serotypes ~ . the situation with coronaviruses is simpler. to date, the coronaviruses isolated from mammals and birds have been grouped into four antigenic classes, where little or no cross-reactivity can be demonstrated between classes tm. tgev is antigenically distinct from bovine enteric coronavirus tm as well as from another as yet unclassified coronavirus causing diarrhoea in pigs (cv ) °. two approaches have been used in an attempt to provide calves with protection against rotavirus and coronavirus infections. the first approach involves oral vaccination with live attenuated virus in order to stimulate active immunity in the calf(scourvax ii, norden laboratories). the incidence of diarrhoea in neonatal calves orally vaccinated with attenuated rotavirus was found to be reduced ~- , but the vaccine was not proven to be effective in blind field trials s - . there are a number of limitations associated with this approach. these include the potential of the vaccine to regain virulence: a high incidence of seropositive adult animals, leading to the possibility of interference with vaccine virus replication by maternally derived (milk) antibodies: and the relative immaturity of the neonate's immune system, the second approach utilizes passive protection produced through lactogenic immunity, stimulated by maternal vaccination. attempts to vaccinate dams using an attenuated live vaccine (calf guard, norden laboratories) have failed to significantly enhance milk antibody titres ", ~ (table ) , whilst in- activated, adjuvanted rotavirus preparations have been found to enhance levels of specific antibody in coiostrum and milk (tables and ) . a number of parameters need to be considered in attempting to optimize the enhancement of antibody production in mammary secretions. dose and form of vaccine. in considering inactivated vaccines, it ~s to be expected that relatively large amounts are necessary to achieve a satisfactory response. further. the process of inactivation may decrease the immunogenicity of some viral polypeptides. table shows that no significant differences in milk antibody titres were obtained following vaccination of cows with rotavirus preparations containing either or elisa units (after inactivation) emulsified in freund's incomplete adjuvant. in contrast, if the same preparations were used. but adjuvanted with aluminium phosphate, the higher dose resulted in a greater antibody response. a similar result using an oil adjuvanted rotavirus vaccine has been previously reported ss. formaldehyde inactivated rotavirus vaccines have been used to successfully enhance milk antibody titres as compared with controls s "- °. other workers have reported increased antibody responses using/~-propriolactone as the inactivating agentol although saif et al found that antibody titres in mammary secretions were at least tenfold greater from cows vaccinated with binary ethylenimine inactivated rotavirus compared with those vaccinated with , -propriolactone inactivated rotavirus . adjuvant snodgrass et a found that oil-based adjuvants were more effective than alhydrogel for the enhancement of rotavirus antibody titres in mammary secretions. this concurs with the data presented in table . most workers have demonstrated a satisfactory immune response following vaccination using oil-based adjuvants. generally freunds incomplete adjuvant (table ). route and timing of vaccination. to some extent the route and timing of vaccination are dependent upon the type ofcattle being farmed. thus the intramammary route used successfully by saifetal , whilst applicable to dairy cattle, may not be practical in beef cows. similarly. from an administrative viewpoint a single vaccination would be preferable to a regime utilizing several doses. the majority of studies have reported a significant increase in rotavirus antibody titres in mammary secretions using either subcutaneous or intramuscular injection of oiladjuvanted vaccines. all such vaccines have also proved to be effective when administered as either single or double doses injected prior to or at parturition . - ( table ) . the efficacy of immune milk as a mechanism for providing passive immunity against rotavirus challenge has been examined by a number of workers(table ). the alactogenic antibody originated from either vaccinated (vacc), control (cont) or normal cows (normal). bcalves were either suckled naturally (suckled) or fed a supplemented diet containing antibody (supp). ccalves were either challenged experimentally (exp) or naturally exposed under field conditions (field). d days after challenge edays after birth, r days after start of experiment. ~cows vaccinated with commercial vaccine nr, not reported results, however, are difficult to compare, due to variations in the feeding regime used for the immune milk and also the challenge systems used. the amount and the timing of the feeding oflactogenic antibody and the dose, virulence and serotype of the virus challenge strain used will all affect the apparent susceptibility of the calf to infection. further, in situations where a field challenge has been used. failure of protection may be due to infection by rotavirus serotypes other than those used in the vaccine, or possibly by other agents capable of causing diarrhoea. generally, these investigators reported either a reduced incidence of rotavirus shedding or diarrhoea or both. in only one study did the feeding oflactogenic antibody fail to significantly affect the incidence or onset of diarrhoea. the majority of animals receiving passive immunity appear to be capable of developing active immunity during this period " , consequently vaccination should lead to elimination of clinical disease rather than a delay in its onset investigation of the immunoglobulin isotypes associated with this protective antibody induced by vaccination in bovine milk and colostrum suggests that igg, plays the major role , . these observations are in agreement with those discussed earlier concerning passive immunity in the bovine. in contrast to the bovine system, evidence suggests that milk or colostral immunoglobulin of the iga isotype is more effective than those of the igg isotypes at protecting piglets against infection by tgev -~°°. high persisting levels of lgg may, however, provide some degree of vaccine, vol. , s e p t e m b e r enhancement of lactogenic immunity:. c e crouch protection against virus challeng~ ~. as a result of these observations` most studies have examined methods for optimising the stimulation of secretory iga antibodies in milk. the origin of tgev-specific iga found in mammary secretions remains somewhat obscure, although there is a good correlation with the presence of an infection in the intestinal tract ~, , °°,' . secretory iga in porcine milk is almost certainly locally produced in the mammary glan& °=-~° . in order to explain this phenomenon, it has been suggested that specificallysensitized iga-secreting lymphocytes may migrate to the mammary gland following initial sensitization in the intestine s~-~°°. such an inter-relationship between the intestinal and the mammary immune systems has also been proposed in rabbits =° and humans ~° . direct evidence for such migration, under the influence of pregnancy-associated hormones, has been obtained in micd ° . a summary of various investigations into the antibody response and efficacy of lactogenic immunity following different vaccination protocols is given in table . reduced immunogenicity in pigs of cell culture attenuated tgev has been described ~° . oral vaccination with a live, attenuated tge vaccine, whilst producing neutralizing antibody, did not stimulate good lactogenic immunity in suckling pigs ~°°,~° .~'°. intramuscular vaccination of sows with live, attenuated tgev leads to the enhancement of specific igg levels in colostrum and milld~,"l higher titres oftgev-specific igg have been achieved using intramammary injection, with an associated increase in the protection provided to suckling pigs ~. these results are supported by the observations of other workers "=-" . feline infectious peritonitis virus (fipv) is a member of the same antigenic class as tgev and consequently the two viruses are serologically related. good levels of cross-protection, associated with high titres of tgev-specific neutralizing antibody have been reported in sows vaccinated orally with fipw ~. in contrast, the results of a more recent study have shown that whilst tgev neutralizing antibodies of the lgg subclass are stimulated in milk and colostrum, the survival rate for suckling pigs was low i". it may be possible to boost the level oflga in mammary secretions. preliminary investigations have revealed that specific secretory lga levels in milk can be enhanced by the parenteral injection, at parturition, of tgev or rotavirus into naturally infected (orally primed) animals "s." . a similar approach also combining oral with parenteral antigen administration has been proposed as a means of providing lactogenic immunity against colibacillosis in pigs t=°. lgg can be induced readily in the mammary secretions of cattle, by intramuscular or subcutaneous injection of adjuvanted immunogen. in pigs however, whilst live, virulent virus is capable of inducing high levels in iga iri milk. it is apparent that the ideal candidate vaccine virus must be sufficiently attenuated to produce only mild or no disease in neonatal pigs, whilst retaining sufficient virulence to infect the intestinal tract of adult swine. more work is essential in the possible use of inactivated vaccines for the boosting of existing iga levels in mammary secretions. these may require prior natural infection of the sow, the incidence of which will vary between herds, with an associated affect upon the efficacy of such a vaccine. further investigation into the variety of strains and serotypes of rotaviruses is of obvious importance, as is the response to vaccination of cattle and swine by rotaviruses or coronaviruses. 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ofthe data obtained during my employment at vido. key: cord- -y ji k authors: connell, anna r.; connell, jeff; leahy, t. ronan; hassan, jaythoon title: mumps outbreaks in vaccinated populations—is it time to re-assess the clinical efficacy of vaccines? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: y ji k history illustrates the remarkable public health impact of mass vaccination, by dramatically improving life expectancy and reducing the burden of infectious diseases and co-morbidities worldwide. it has been perceived that if an individual adhered to the mmr vaccine schedule that immunity to mumps virus (muv) would be lifelong. recent mumps outbreaks in individuals who had received two doses of the measles mumps rubella (mmr) vaccine has challenged the efficacy of the mmr vaccine. however, clinical symptoms, complications, viral shedding and transmission associated with mumps infection has been shown to be reduced in vaccinated individuals, demonstrating a benefit of this vaccine. therefore, the question of what constitutes a good mumps vaccine and how its impact is assessed in this modern era remains to be addressed. epidemiology of the individuals most affected by the outbreaks (predominantly young adults) and variance in the circulating muv genotype have been well-described alluding to a collection of influences such as vaccine hesitancy, heterogeneous vaccine uptake, primary, and/or secondary vaccine failures. this review aims to discuss in detail the interplay of factors thought to be contributing to the current mumps outbreaks seen in highly vaccinated populations. in addition, how mumps diagnoses has progressed and impacted the understanding of mumps infection since a mumps vaccine was first developed, the limitations of current laboratory tests in confirming protection in vaccinated individuals and how vaccine effectiveness is quantified are also considered. by highlighting knowledge gaps within this area, this state-of-the-art review proposes a change of perspective regarding the impact of a vaccine in a highly vaccinated population from a clinical, diagnostic and public perspective, highlighting a need for a paradigm shift on what is considered vaccine immunity. muv is an enveloped, non-segmented, negative-sense, single stranded rna virus that varies between a spherical and pleiomorphic shape of ∼ nm ( - nm) ( , ) . muv is responsible for an acute viral infection, spread by respiratory droplets (via coughs, sneezes) and urine ( , ) . with an incubation period of - days, muv replicates in the nasopharynx and regional lymph nodes, with a secondary viremia occurring late in the incubation period ( , ) . muv can be detected from saliva up to days prior, and as late as days after clinical onset of parotitis ( ) . the muv genome of seven genes consists of , nucleotides, and encodes six structural proteins and at least two non-structural proteins; the nucleocapsid protein (np), v protein (v), phosphoprotein (p), matrix (m) protein, fusion (f) protein, small hydrophobic (sh) protein, hemagglutininneuraminidase (hn) protein, and large (l) protein. the role of the i protein is not known ( , , ) . the sh gene is the most variable region of the muv genome; a - % intravariation and - % inter-variation has been documented ( ) . this gene is used in molecular phylogeny for genotyping and to identify transmission patterns in populations ( ) . despite being serologically monotypic, muv genotypes (a to l) have been described to date (muv genotypes e and m are omitted, as the muv previously assigned to these groups were later re-assigned) ( , , ) . the geographic distributions of the muv genotypes varies worldwide but can co-circulate and thus drive temporal shifts in their distribution. genotype a was frequently isolated in europe until the 's. currently genotypes c, d, e, g, and h are prevalent in europe and the united states of america (usa) whereas genotypes b, f and i are more common in asian countries ( table ) ( , , , ) . since numerous mumps vaccines have been developed worldwide, varying in efficacy and safety profiles but primarily consisting of an attenuated live muv without an adjuvant ( , ( ) ( ) ( ) . currently in europe and for the majority of the g countries who have a mumps vaccine in their immunization schedule (table ) , the mumps vaccine is included as part of the trivalent measles, mumps rubella (mmr) vaccine, and is primarily administered in two doses ( , ) . the jeryl lynn (jl) vaccine, derived from the genotype a muv strain was first developed in the usa and has been used extensively in the united kingdom (uk), ireland and usa since it was licensed in ( ) . derived from a single clinical sample, and propagated in a chick embryo cell culture, two viral isolates (jl and jl ) are present, differing by ∼ nucleotides and amino acid changes ( ) ( ) ( ) . the rit mumps vaccine, developed from the dominant viral component (jl ) in the jl vaccine strain appears to have comparative safety and efficacy (seroconversion) profiles to the jl vaccine strain ( , ( ) ( ) ( ) . however, since no controlled clinical trials of efficacy have been published to compare the two doses of the two vaccines, the clinical significance of this observation is not known. despite the integration of the mmr vaccine into childhood immunization programs, cyclical outbreaks [defined as two or more cases linked by place and time ( ) ] of muv have been documented in several highly vaccinated populations such as ireland and the united kingdom ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . between august -and january , , mumps cases were notified in ireland, primarily affecting individuals between the ages of - years. of the % of cases that stated vaccination status, % had received two doses of the mmr vaccine ( ) . an upsurge of mumps cases has also occurred in states of the united states over the last decades, primarily affecting people between and years in close contact/shared settings ( ) . in indiana, . % of mumps cases ( . % of university affiliated and % of community cases) had documented evidence of mmr vaccination ( ) . this results in a significant resource burden for public health departments to control. several reviews, both observational and systematic have demonstrated the clinical benefit of a mumps vaccine ( , ) , the pathogenesis and genomic diversity of the muv ( , , ) and the epidemiology surrounding the outbreak ( , , ) . it is not clear why these mumps outbreaks occur, although it has been alluded to be due to a number of interrelated factors, such as sub-optimal vaccine uptake ( , , ) , primary or secondary vaccine failure or failure of the mumps vaccine to protect individuals from infection (vaccine efficacy) ( ) (figure ). history depicts the remarkable public health impact of mass vaccination. previously inevitable childhood diseases with potentially debilitating or deadly outcomes have seen their rates plummet worldwide or become successfully eradicated. immunizations of vaccine preventable diseases are estimated to prevent ∼ - million deaths per annum and increase life expectancy by ∼ years ( ) . more recently there has been a shift in the public and media perception of vaccines to their safety, which has facilitated outbreaks such as mumps ( ) . organized opposition to vaccinations has a long history; public outcry and resistance following the introduction of the smallpox vaccine in the nineteenth century led to the introduction in england of the vaccination act of ( ) . with one in eight children in the usa under the age of currently thought to be unvaccinated due to parental choice, the who now considers vaccine hesitancy as one of the ten threats to global health in ( ) . vaccine hesitancy, defined as a "delay in acceptance or refusal of vaccines despite availability of vaccination services" involves a multitude of social, political, cultural and emotional factors in highly vaccinated, western populations ( , ) . one of the main issues is the parental concerns regarding the perceived risk of a vaccine to their child (such as timing/schedules of vaccines, associated pain of administration, and potential adverse effects) vs. the disease morbidity and mortality associated with the vaccine preventable disease ( , ) . the retracted paper published in the lancet in ( ) and "anti-vaccination" opinions on social media have also contributed to the persistent and insistent misinformation ( ) , despite vast follow-up epidemiological studies showing no relationship between the mmr vaccine and autism, or differing cognitive development/intelligence ( ) ( ) ( ) . however, the resultant reaction of the public led to the uptake of the first mmr vaccine falling sharply from , with uptake falling to below % in ( , ) . the age demographic that are experiencing the most cases of mumps in ireland during the current ongoing outbreak would have been scheduled to have received the first mmr vaccine between and . nevertheless, no deductions can be made, due to the lack of vaccination status information provided with reported cases ( ) . frontiers in immunology | www.frontiersin.org heterogeneity of immunization coverage in specific populations or geographic locations of susceptibility is also becoming an important epidemiological issue in maintaining proficient population immunity for mumps ( , , ) . the who recommends a > % mmr vaccine coverage for herd immunity. maintenance of such coverage is well-demonstrated in finland, where a country-wide -dose mmr vaccination program initiated in the 's eliminated measles, mumps and rubella within years ( , ) . recent publications from around the world indicate that the level of mmr vaccine uptake is far lower than what is recommended [reviewed in ramanathan et al. ( ) ] ( , ( ) ( ) ( ) ( ) . of the g nations that implement a mumps vaccine within their vaccination schedule, only countries have maintained vaccine coverage levels of > % (table ) . however, poor uptake/incomplete vaccination alone may not be the only issue relating to mumps outbreaks. in the netherlands, mumps outbreaks still occurred with an overall herd immunity threshold of - %, and where and % received the first and second mmr at months and years, respectively ( , ) . the clinical presentation of mumps is pathognomic (bi-lateral parotitis); therefore supporting laboratory diagnosis was rarely employed in the past. as the classical symptoms of mumps are not always typical, there may have been a significant number of individuals in the past who may have been infected but were not identified as such. when mumps vaccination was introduced in , the criteria the vaccine had to meet was the proof that it was clinically effective, i.e., that it reduced the risk of disease in vaccinated individuals in real-world conditions over a set period. such an example was seen the usa; the reported cases (i.e., diagnosis of clinical symptoms) of mumps declined from > cases per , population before (pre-vaccine era) to cases per , population in , a reduction of % ( , , , ) . to note, clinical efficacy was probably based upon the reduction of the "classical bilateral presentation" rather than the milder mumps presentation. therefore, one could argue that the original vaccine efficacy for clinical manifestations was over estimated. currently the laboratory diagnosis of mumps infection in ireland is based upon two approaches: detection of mumps rna by reverse transcriptase pcr (rt-pcr) in a buccal swab containing saliva, throat swab or urine specimen, and serological detection of immunoglobulin m (igm) using a capture assay ( , ) . both approaches for diagnosis are impacted significantly by the quality and timing of sample collection post-onset of symptoms and also if the subject is mumps naïve or had received mumps containing vaccine ( , , , ) . there are challenges in using standard serological laboratory diagnostic methods to reliably confirm mumps re-infection of individuals who had been previously naturally infected or vaccinated ( , ) . briefly, vaccinated individuals re-infected with muv may only generate a weak or undetectable igm response ( ) . although a rise in igg titer may also not occur in vaccinated individuals ( , ) , numerous studies have documented a rapid, variable increase in mumps-specific igg levels, with neutralization antibody concentrations present up to months post-infection ( , , ) . therefore, reverse transcriptase-polymerase chain reaction (rt-pcr) is recommended ( , ) , and was formally introduced in as the principle diagnostic tool in ireland to detect mumps in oral fluids ( ) . rt-pcr can identify current mumps infection more effectively in vaccinated individuals than serological techniques alone as it identifies the presence of the muv vs. the immunological response (igg, igm), and has been previously shown to % correlate with viral culture results ( , ) . the case numbers of more recent mumps outbreaks should always be assessed with this question in mind; are the number of mumps cases increasing, or/and are we better at diagnosing an acute infection? the latter seems to be the most probable, as many individuals who are being tested do not present with classical symptoms. in addition to enhanced surveillance of mumps cases, further optimizations of technologies are also occurring; the utilization of next-generation sequencing demonstrated that by editing one -fold degenerate nucleotide in the forward primer and three -fold degenerate nucleotides in the probe sequence optimized the fluorescence intensity and clinical sensitivity of the real-time rt-pcr when compared to the cdc-developed and who-recommended rt-pcr target [(np) gene] leading to ∼ % increase in clinical sensitivity (i.e., ct values that were ∼ . cycles lower) ( ) . much is not known about the immunological response to the mumps vaccine strain. however, a number of young adults who were vaccinated as children over the last two decades have demonstrated an increased risk of muv infection with time, which is assumed to be related to a decline of antibodies to sub-protective levels of immunity ( , , , , ( ) ( ) ( ) ( ) . primary vaccine failure is defined as the lack of a sufficient initial antibody response to a vaccine in a recipient resulting in a lack of protective immune responses ( , ) . although this type of vaccine failure may be because of improper storage/handling or administration of the vaccine, impacting its efficacy, it may also be due to the initial immunological response of an individual to the vaccine, which is usually quantified by the presence of antibodies that should be detectable in the weeks following vaccination. primary vaccine failure was attributed to primaryschool outbreaks of both mumps and measles in ireland, which subsequently resulted in reducing the age for the second dose of mmr vaccine from - years in to - years of age ( ) . with the cyclical outbreaks occurring, it has been proposed that primary vaccine failure could again be a factor. how is a response to a vaccine determined? in pre-licensure studies of the jl and urabe mumps vaccines, high seroconversion and low failure rates were observed in children after the first vaccine dose (> and . %, respectively), demonstrating that the vaccine induced an antibody response ( ) ( ) ( ) ( ) ( ) ( ) . a more recent study by ong et al. demonstrated that a ≥ fold increase in mumps antibodies -days post-vaccination was considered to be an adequate response of immunity ( ) . vaccine effectiveness (i.e., seroconversion post-vaccination) of vaccine doses has only been conducted on the jl strain; studies provided a median vaccine efficacy of %. these studies have shown that doses of mmr were more effective (but not statistically significant) than a single mmr dose to combat the incidence of mumps infection ( , , , , , ) . mumps-specific antibodies have been detected - years postvaccination and without substantial decline for years after mumps vaccination, with the immunogenicity and efficacy of the mmr vaccine showing comparable immunogenicity levels to post-vaccination levels at years ( , ) . however, most studies of this vaccine (involving either a mumps-specific vaccine or a combined vaccine) only followed-up to - days postvaccination ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . despite few follow-up studies estimating post-vaccination antibody titers specific to the vaccine mumps strain, the evidence of seroconversion post-vaccination in a number of studies indicate that primary vaccine failure does not seem to be a significant contributor to the outbreaks that have been recently observed ( , , , , , ( ) ( ) ( ) ( ) . it has been noted that a small percentage of the population do not seroconvert post-vaccination; < % who received the mmr vaccine were seronegative - years after the first dose of mmr (n = ) ( ) . poor immune responses to primary vaccination has been shown to be a good indicator of infection susceptibility ( ) . this is in agreement with the correlation of pre-outbreak jl virus neutralization titres and elisa results being significantly lower in individuals who became infected compared to non-infected individuals ( ) . further studies of these individuals may provide insights of which immunological process are integral to develop immunity. the current methods used to determine immunity against mumps cannot discriminate between primary and secondary vaccine failure; only the timing of these tests can assess whether an individual ever mounted an immune response postvaccination or whether the response is detectable years postvaccination. primary vaccine failure encompasses the failure to mount an immune response to a dose of a vaccine, secondary vaccine failure refers to a more gradual loss of immunity after a successful initial response that occurs over a number of years post-vaccination ( ) . several factors have been proposed to be implicated with secondary vaccine failure, such as waning immunity, a lack of cross-neutralization, and natural boosting. waning immunity is defined as a decline in immunological protection proportional to time since vaccination. potential waning immunity has been documented in the current mumps outbreaks seen in europe and the usa, mostly affecting young adults within highly vaccinated populations attending tertiary education who have received two doses of the mmr vaccine in early childhood ( , , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . a number of studies from the usa, where a jl vaccine has been used since have demonstrated waning immunity within the population. the risk of developing clinical mumps was shown to increase by - % for every year post-mmr vaccination ( ) , with the rates of mumps infection rising from . cases per , in those who received the second dose of the vaccine within years of the outbreak, to . cases per , in those who received it over years prior. using a mathematical model with analytical limitations, a recent metaanalysis of six studies estimated that vaccine-derived immune protection to muv wanes about years post-vaccination ( ) . kennedy et al. ( ) also demonstrated a decrease of ∼ % in mumps neutralizing antibody titers over years. in contrast, other studies appear to contradict, these findings, showing no link between mumps protection and time elapsed following administration of mumps vaccine ( , , , , ) . lebaron et al. ( ) and gothefors et al. ( ) demonstrated that - % of individuals still had detectable anti-mumps antibodies ∼ years after initial vaccination. cohen et al. ( ) also demonstrated minimal antibody level decline after two mmr doses - years after second vaccination. neutralizing antibodies against the jl- vaccine strain has also been detected in ∼ % for age groups - years, % for age group - years; and % for age group + years ( ) . implementation of a third dose of the mmr vaccine has been shown to be effective as a stop gap measure in limiting disease spread in outbreak settings situations ( ) . individuals vaccinated for the third time had a % lower risk of contracting mumps, with a decreased attack rate of . vs. . cases per , when compared to those who received a second dose. more than % of those who received a third dose of the mmr vaccine showed a -fold increase in mumps antibody titers ( , , , ) . an increase in mumps igg humoral immunity was also observed post-vaccine administration. however, this immunity boost has been shown to be a transient effect, with mumps antibody titers returning to pre-third dose of mumps-vaccination levels year after vaccination. therefore, as waning immunity is thought to be an important factor facilitating mumps outbreaks, the emphasis placed on the quantity/quality of mumps-specific antibodies may need to be re-assessed. it is yet undetermined if the total loss of detectable antibodies correlates to a loss of clinical protection, as the minimal level of neutralizing antibody required for protection against mumps has not yet been defined ( ). antigenic variation and thus reduced cross-neutralization between the vaccine and circulating strains of different muv genotypes have been cited as possible explanations for mumps outbreaks in highly vaccinated populations ( , , ( ) ( ) ( ) . recent outbreaks in europe and northern america (including ireland) have shown the circulating muv during the current outbreaks to be genotype g ( , , , ) . this muv genotype was first identified in , and has demonstrated intra-genotype diversity of up to % (table ) ( , ) . the jl vaccine strain (genotype a), differs phylogenetically to the circulating muv (genotype g) ( ) . in vitro studies of the genotypic distribution and temporal shift of muv suggest that cross neutralization between wild type and vaccine genotypes may be approximately half the concentration measured against the vaccine strain ( ) . pre-infection neutralization titers in mumps positive cases were also significantly lower against genotype g vs. mumps vaccine strain, potentially due to amino acid differences in b-cell epitopes and/or n-linked glycosylation sites on the hn and also within the f protein ( ) . santak et al. ( , ) also demonstrated that conformational changes within the f protein may lead to immunological escape. despite the decline/scarcity of cross-neutralizing antibodies, different mumps vaccines used worldwide have been shown to prevent significant clinical mumps infection during outbreaks ( , ) . dependent on the strain, a - -fold variation of patient sample titers has been shown to be protective in in vitro plaque reduction neutralizations ( , , ) . although the sera of one of these studies, was collected only weeks after mmr vaccination, a time point that may not signify the concept of waning immunity and antigenic differences, several other groups have shown that the most divergent strains of muv can be neutralized in vitro with only slight variations in titers, supporting the concept that muv is serotypically monotypic ( , , , ) . epitopes of the muv that are presented to cd + t-cells have been shown to be present in not only the circulating strains of virus but also in a number of vaccine strains ( ) . in addition, lewnard et al. ( ) also found no evidence that recent mumps outbreaks were due to the emergence of muv strains escaping vaccine-driven immunological pressure. therefore, the limited data does not suggest that antigenic drift of the muv leading to diminished neutralization capacity of the vaccine strain could fully explain the recent outbreaks ( ) . further studies into the cross-neutralizing capacity of the mumps vaccine strain administered - years previously to the current circulating strain of muv in countries where outbreaks are being observed will allow better deductions to be made. it is possible that differences in the neutralization capacity of vaccine-induced antibodies against different muv strains may be more significant when levels of neutralizing antibody are low and become "overwhelmed" when the mumps viral load challenge is high ( ) . several prominent mmr/mumps vaccine studies were undertaken at a time when there was still a high prevalence of circulating wild type virus, which enabled sub-clinical boosting to occur in an individual. such natural boosting is illustrated in belarus, where a subpopulation of vaccinated individuals only had a small amount of their overall mumps igg antibody levels specific to the vaccine-strain ( ) . neutralization antibodies against iowa-g/usa (the circulating wild type virus) were also present in pre-infection plasma of all mumps cases during a recent outbreak in the us ( ) . this indicates that the mumps vaccine alone is not solely responsible for the high levels of mumps antibodies ( ) , and that longterm antibody persistence or protective efficacy data of the vaccines used may not truly reflect the current circumstance of viral transmission/circulating within a highly vaccinated population ( ) . herd immunity increases the chance for natural mumps boosting for an individual is at a minimum, reducing the potential of the frequency of mumps outbreaks ( , , ) . with less opportunity for subclinical boosting (asymptomatic response to the circulating virus), the impact of other elements of waning immunity may play an increasingly critical role in the re-emergence of mumps outbreaks ( , ) . additionally, as the heterogeneous uptake of vaccines in this modern era is leading to susceptible individuals within the community, future work will need to encompass genotyping of circulating muv to examine how impactful subclinical boosting was on early measures of vaccine efficacy in current populations. why do we consider antibodies to be the best measurement of vaccine efficacy? the evolution of an individual's immune response differs between natural infection and vaccination, in particular the difference in the affinity and specificity of an immunological marker such as antibodies ( ) . true correlates of mumps immunity after vaccination have been poorly characterized; to date, there are no reliable correlates of protection from either symptomatic mumps infection (clinical immunity), or individuals previously exposed to muv ( ) . therefore, a serological surrogate/ substitute is used ( ). mumps vaccine efficacy is quantified by a single measure, igg which may not suffice to evaluate the magnitude of the actual humoral response. borgmann et al. ( ) proposed an increase in mumps-specific igg titer in sera as a diagnostic criteria of mumps reinfection ( ) . it has been suggested that vaccinated individuals have modified b-cell responses to muv that allow for the rapid generation of igg antibodies and a blunted or absent igm response ( , ) . in addition, emerging data in simian immunodeficiency virus studies suggests that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles ( ) . despite its use, the correlation to mumps-specific igg concentrations and neutralization titers against the jl virus is poor, suggesting that igg concentrations do not adequately represent a sufficient surrogate correlate of protection ( ) . this is demonstrated in finland; only % of vaccinees had no detectable mumps antibodies after years ( , ) . data from the european sero-epidemiology network (esen ) project in reported that mmr immunization uptake in ireland in was % ( ), however it was also suggested that only - % of -to -year-olds in ireland had detectable antibodies to muv by either natural immunity or immunization ( ) . in , vaccine coverage of medical students in germany was reported to be . % ( ) . in children between the ages of - years, where . % had been vaccinated with the mmr vaccine at least once, only . % showed prevalence of antibodies ( ) . however, . % showed a prevalence of antibodies to measles and rubella in the absence of mumps-specific antibodies. therefore, previous measurement of anti-mumps-specific igg that represented immunity induced by the mumps vaccine appears to be overestimated ( , ) . antibody levels of other components of the mmr vaccine have seen similar trends. waning rubella antibody titers have been observed, despite the number of acute rubella and congenital rubella syndrome cases not increasing. it has also been shown that college students who received rubella vaccination during childhood and had low/no antibody response were able to mount a secondary response when challenged with rubella indicating that an individual's low antibody levels are not always indicative of susceptibility to infection ( ) . measles antibodies can also be detected for up to a decade post-vaccination, with > % of individuals still measles igg positive at - years of age ( , ) . however, as with mumps and rubella, waning measles antibody titers have been observed ( , ) . despite this, a recent longitudinal study of up to years demonstrates how effective the mmr vaccine has been in preventing diagnosed measles cases during the 's/ 's ( ) . similarly, three doses of the hepatitis b (hbv) vaccine in a cohort of alaskan natives showed > % seroconversion in children and young adult post-vaccination and provided long term and durable protection against chronic hbv infection. although no increase of hbv prevalence were observed % individuals had low to undetectable antibody levels after years. these observations suggest that an individual's antibody levels do not indicate susceptibility to infection, that either an antibody titer lower than recommended guidelines is still protective, or/and is an ineffective surrogate of protection. this is emphasized in a study by amanna et al.; ( ) responses to non-replicating protein antigens (tetanus and diphtheria) were shown to have approximate antibody half-lives of - years. in comparison, antibodies following wild type infection were shown to have half-lives of years or more which was thought until recently to confer a more prolonged lifelong protection ( , , ) . however, reinfections observed in individuals that were previously naturally infected have demonstrated that the quantitative measurement of antibodies do not indicate sterile immunity ( ) . it is also important to stress that seroconversion rates due to immunization/natural infection only reflects a change of antibody status from negative to positive, but not necessarily the intensity of antibody response. in addition, there is no consistency in the timing of sample collected post-vaccination to test vaccine efficacy, and between the serological tests utilized for detecting mumps antibodies. as a result, documented seroconversion rates of the mumps vaccines used vary widely (jl: - %, rit strain: - %, urabe am : - %, rubini: - %). this highlights that the assays used to detect immunity to muv may not always detect an adequate post-vaccination response. only a small number of serological commercial assays such as the detection of hepatitis b surface antibody (anti-hbs) ( ) and rubella igg ( ) have been designed using who reference material as a standard for quantification. however, even utilizing this reference standard demonstrates significant differences in the determined quantification of either anti-hbs or rubella igg depending on the assays used; although a value for anti-hbs of iu/ml is regarded as protective against significant hbv infection, the detection of this anti-hbs is significantly influenced by which anti-hbs assays is used ( ) ( ) ( ) ( ) ( ) . therefore, it is possible that the current assays/tests mechanisms utilized to measure mumps antibodies are too insensitive/inappropriate/crude to identify nuances in the immune response which could correlate with immunity against mumps. in addition, variation within neutralization epitopes i.e., the quality of the antibody present could be a more important correlate than quantity ( , ) . though labor-intensive, neutralizing antibodies are considered to be a better correlate of mumps immunity. antibodies against the haemagglutinin-neuraminidase protein (hn) and nucleoprotein (np) have been shown to neutralize muv, however, repeated attempts to define a titer that provides a protective threshold titer have been inconclusive ( , ) . in older studies, during field evaluations of the jl vaccine, neutralizing antibody titers of : - : in unvaccinated individuals was considered seropositive and protective from mumps infection ( , , ) . using a more contemporary wild-type isolate (iowa-g/usa ), a : neutralizing titer cut off was defined between case patients and exposed patients, despite the fact that no cut-off could fully discern between the two groups ( ) . however, that these results are dependent on the challenge virus strain used in the assay. rasheed et al. demonstrated a fold lower neutralization titer to the g-genotype when compared to the jl vaccine strain in - year olds ( ) . this has also been seen between mumps vaccine strains vs. circulating strains in india and china ( , ) . despite studies in more highly vaccinated populations demonstrating that hn-inhibiting titers after natural disease were : compared to : post-vaccination, neither appeared to prevent reinfection ( , ( ) ( ) ( ) ) . there is increasing evidence that the mumps-specific antibody response is broader than neutralization alone ( ) . avidity testing for virus-specific igg has been proposed ( , , ) . individuals who lack measurable mumps-specific antibody levels may be susceptible to infection but protected from significant illness as they may be protected by cell-mediated immune memory. prolonged t-cell responses are reported after other vaccinations; - years after a single dose of the rubella vaccine ra / , a t-cell proliferative response to neutralizing antibodyinducing peptides suggest t helper and b-cell interactions. this indicates that full vaccine effectiveness could be dependent on mounting both an antibody and cell-mediated immune response ( ) . although cell mediated immunity has not been as wellassessed in mumps infection, a lymphoproliferative response was induced in infants vaccinated at , , or months of age was induced ( ) with antigen-specific t-cells reported to appear within month of infection ( ) . lymphoproliferative responses to measles and mumps vaccine viruses were shown to persist in two thirds of the population at least years after immunization ( ), with t-and b-cell immunity persisting for years post-immunization ( ) . low levels of mumps-specific memory b-cells have also been documented suggesting that mumps infection or vaccination may not generate a robust b-cell memory ( , ) . two principal mechanisms for maintaining long-term humoral immunity have been proposed and reviewed by amanna et al. ( ) : associations between memory b-cell levels and antibody may reflect an epiphenomenon in which serum antibody levels and memory b-cells are equally stable but independently maintained. if memory b-cells and plasma cells are independently regulated, then multiple re-exposures to antigens may cause divergence between memory b-cell levels and antibody levels ( ) . antigens with the highest rates of boosting through vaccination or latent viral infection coincidentally showed the weakest association between memory b-cell titers and antibody titers ( ). although the role and efficacy of t-cell immunity to mumps infection is unclear, there is a possibility that certain muv strains may be capable of escaping vaccine induced t-cell responses, which may not be considered of significance until b-cell waning immunity comes into play ( ) . in individuals who did not respond to vaccination (i.e., had a ≤ -fold of mumps antibody titers days post-vaccination), several genes including those implicated in antigen presenting, processing, t-cell response and function showed significantly increased expression, with mhc class ii hla-drb and hla-dra, and cd induced when compared to responders day post-mmr vaccination. this may indicate that the stimulation of a rapid adaptive immune response limits antigenic presentation and hence prevent the differentiation of memory b-cells to antibody-producing plasma cells ( ) . differences in predicted b-cell and t-cell epitopes between jl vaccine strain and other vaccine strains may also be implicated in the outbreaks witnessed ( ) . although, it has also been shown that natural mumps infection or vaccination do not always induce both cellular and humoral immunity. de wit et al. ( , , ) has shown the presence of th -type cd + t-cells recognizing a muv epitope in a hlr-dr restricted manner. in addition, the response of ifn-γ and tnf producing cd + t-cells specific to muv epitopes are lower in vaccinated individuals when compared to individuals who were naturally infected ( , , ( ) ( ) ( ) . utilizing current knowledge and new technologies may help define a better surrogate correlate of protection and potentially determine a cut-off between the immunity of a vaccinated individual and a secondary mumps infection. this may potentially move the diagnostic preference from serological tests to more comprehensive functional assays. despite the large resurgence of mumps outbreaks, there is insurmountable evidence highlighting the benefit of the mumps vaccine ( table ) . routine childhood mmr vaccination has resulted in a dramatic decrease in the incidence of mumps cases, and has shifted the peak age-specific attack rates from a young children (manifesting between and years) to one that affects young adults, in particular those who have close interaction with other young adults ( - years) ( , ) . additionally, clinical manifestations and severity of disease in vaccinated vs. unvaccinated individuals differ ( , ) . although muv can be clinically asymptomatic in about - % of those who become infected, the vaccine against mumps confers protection in a dose response manner; unvaccinated individuals saw an attack rate of based on the reduction seen upon the introduction of a mumps vaccine, it has been proposed that mmr vaccination also prevents the transmission of the virus. there is limited knowledge regarding the shedding and transmission of muv, but it is thought that close contact and transmission of a certain viral load may induce clinical symptoms ( , , ) . modeling data suggests that infectious muv shedding decreases rapidly after the onset of symptoms, however - % are patients are thought to still be virally shedding days after the onset of symptoms ( ) . this could be the reason why the transmission of muv can be exacerbated by close social situations within a heterogeneously vaccinated population. outbreaks generally occur in situations of intense contact such as college dormitories, boarding schools, and youth summer camps ( ) , with up to a third reporting some contact with a mumps case ( ) . evidence of lower levels of viral replication also suggests a clinical benefit of the vaccine ( , ) . viral load and presence of the mumps vaccine genome in areas of viral replication was lower in vaccinated individuals vs. unvaccinated individuals ( ) . in addition, patients who contracted mumps but had two doses of mmr have been shown to shed less muv in their urine, with fewer experiencing bilateral parotitis or orchitis than unvaccinated individuals ( ), this suggests that immunity induced by mmr vaccination limits virus transmission and complications ( , ) . it should be noted also that individuals who received two doses of mmr, and had a positive correlation between viremia, salivary viral loads and systematic clinical mumps infection may have an increased risk of transmitting virus. these individuals also lacked mature functional responses, with low neutralizing antibody titers and avidity indexes ( ) . overall, evidence demonstrates a clinical advantage to receiving a mumps vaccine ( table ) . currently no global consensus exists for the measurement of mumps antibodies, mumps avidity or neutralizing titers that correlate to vaccine response and protection in healthy individuals. if a biomarker is discovered, it could be utilized as an international diagnostic reference standard to allow global harmonization and evaluation of the relative effectiveness of the different vaccination programs worldwide. such an attempt was conducted by andrews et al. ( ) , who reported on the european sero-epidemiology network project which was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps, and rubella in eight european countries. the study concluded that the development of an international standard for mumps would help in the standardization and comparability of mumps antibodies in the different enzyme immunoassays used in laboratories. however, to date, no international reference standard for mumps has been established. in response to infection, the human immune system launches a series of immunological responses with the goal of controlling or eliminating the pathogen. if the pathogen circumvents the frontline defense of the innate immune system, an adaptive immune response specific for the pathogen will become activated to respond, with the intention to generate humoral-and cell-mediated immunity. humoral immunity, represented by antibodies secreted by b-cells are not effective against pathogens that invade host cells. therefore, cell-mediated immunity instructed by the vaccination aims to stimulate the host immunological process and formation of cell-mediated immunological memory via the use of live-attenuated or of inactivated/subunit vaccine components to promote a cell-mediated immune response. extensive knowledge gaps significantly hinder improvements to the mumps vaccine and prospects for mumps eradication and maintaining proficient population immunity ( , , ) . few studies have collected data that examines different aspects of mumps immunity and are limited in their predictive value for future outbreaks ( ) . for example, the importance of t and b-cell responses in protective mumps immunity and how memory/plasma cell numbers are homeostatically maintained post-infection or vaccination is relatively unknown ( ) . it should be acknowledged that the mechanism of protection of infection may not be the same mechanism of recovery from infection, which may make the identification of a common correlate of protection and recovery difficult ( ) . therefore, if a correlate or surrogate correlate is unobtainable to define an individual's protection to mumps, should we re-consider and re-focus efforts on optimizing the vaccine using available historical clinical and trial data? it has been suggested that wild-type infection could confer a "better quality, " broader and prolonged immuno-activation than vaccine-induced immunity. this is reflected in mean neutralizing antibody titers detected post-mumps vaccination, which were over five times lower than those detected following wild type infection. similarly, hemagglutination-inhibiting titers after natural disease were : compared to : post-vaccination ( , , ) . the use of a live-attenuated virus vaccine is intended to mimic immunological reactions and responses between the host and wild type virus ( ) . the current liveattenuated mmr vaccine is intramuscularly injected, a route that significantly differs from the natural infection mode of transmission. however, emphasized by differing immunological kinetics between immunized and naturally infected individuals when subjected to wild type pathogens, injectable vaccines are considered not to be the best inducer of antigen-specific mucosal immune responses for mucosal pathogens, especially if the mode of administration is not the natural route (the respiratory tract) ( , ) . improvements on a broader range of antigen delivery systems will improve vaccination strategies and potentially prolong the effect of a vaccination by producing a localized immunological response in the relevant tissues ( , ) . mucosal vaccines such as intra-nasal vaccination have advantages over traditional injectable vaccines as they can induce an effective, more robust immune response without any physical discomfort and more closely replicate the natural route of infection for mumps ( , ) . b-cells induced by the mucosal response are also capable of secreting iga class of antibodies in the lumen, where the interaction and neutralization of specific antigens form iga-antigen complexes are easily able to be entrapped in the mucus and eliminated by cilial epithelial cells ( ) . activated mucosal lymphocytes can also reach other mucosal sites via the lymphatic system and have the capability to transfer immunity ( ) . such an example is the intranasal immunization of inactivated influenza. with a - % similar efficacy between the injectable and intranasal influenza in healthy individuals this intranasal vaccine can elicit the secretion of haemagglutinin and neuraminidase specific iga antibodies in the upper respiratory tract, and corresponding igg antibodies ( ) . live, cold adapted attenuated nasal influenza vaccine has been routinely used in russia for over years ( ) . other liquid live-attenuated intranasal vaccines are available; "nasovac r " in india, and "flumist r " in the us, uk and new zealand ( , , ) . inactivated vaccines consisting of heat/chemical or liveattenuating monovalent or multivalent pathogens in animals/cell lines were developed to protect against disease causing microorganisms ( ) . less emphasis was placed on understanding the mechanisms related to conferring immunological memory; the focus lay on the availability, mass production and administration of the vaccine to introduce herd immunity into populations ( ) . currently, the least expensive and time effective method to licensure is the comparison of serologic responses of the new vaccine to an existing licensed vaccine, which can lead to a bias on the development of novel vaccines ( ) . this methodology also does not account for the fact that each vaccine developed elicits its own immunological signature and may need to be considered on an individual basis ( ) . raymond et al. ( ) has suggested that embryonated chicken egg-based vaccines may induce antibodies that are more preferential to egg adapted strains better than wild type virus. amino acid substitutions/differences in key antigenic targets due to the passage of the growing virus within this environment may optimize the growth of the virus, but could lead to differences over time that could affect the immunogenicity or potency of the vaccine ( , , ) . the jl vaccine contains two isolates of the jl strain (jl and jl ) and whilst no immunological differences have been documented, jl grows to higher titers than jl in embryonic eggs and also demonstrates significant sequence variability ( , ) . zost et al. ( ) also demonstrated that an egg selected mutation within a glycosylation site in the - influenza vaccine strain led to the production of poorer neutralizing antibodies to the vaccine strain compared to wild type influenza virus. vaccine rit strain derived from one of the two distinct virus subtypes of the jl vaccine (jl ) showed comparable seroconversion rates despite inducing a significantly lower geometric mean antibody titer when compared to recipients of the jl vaccine, but does not have any longitudinal trials investigating its efficacy, even though there are populations who are currently receiving it ( , ) . the significant time gap between pathogen emergence and vaccine licensure, could potentially lead to antigenic drift. there is potential that modern biotechnologies could be utilized to design novel vaccine platforms ( , , ). clinically derived recombinant muv lacking the expression of the immunomodulatory v or sh protein are currently being investigated ( ) . in china, a vaccine consisting of the prevalent wildtype virus genotype (f) has recently been produced and is currently undergoing trials ( ) . in addition, despite being extremely pleomorphic, utilizing mhc epitopes as potential b-cell and t-cell vaccine candidates are also being investigated ( , , ) . vaccine design has involved the utilization and templating of epitopes that previously induced a b-or t-cell response during natural disease that are considered to be immunogenic enough to induce similar responses if administered in a vaccine. however, the appropriate b-cell and t-cell epitope/peptide candidates to induce a protective immunological response can be difficult to correctly identify and synthesize, as it may differ to the immunodominant epitope and host presentation of that antigen ( , ). prediction of mhc-peptide binding and cleavage has demonstrated mismatches in both vaccine tcell and b-cell epitopes in vaccinated individuals highlighting small number of distinguishing amino acid changes of the jl major strain ( ) . the importance of understanding tand b-cell responses and how antigen-specific memory cells numbers are homeostatically maintained post-infection is crucial to understand to ensure successful vaccine development ( , ) . since the 's, significant progress has also been made in developing flexible, amplifiable, scalable, inexpensive, and cold-chain free rna vaccines, such as synthetic mrna molecules encoding only the antigen of interest and selfamplifying rna (sa-rna) ( ) . such examples include an experimental mrna vaccine candidate (mrna- ) which encodes a stable form of the sars-cov- spike protein and has been accepted as a trial candidate for clinical trials in healthy male and female individuals ( , ) . in addition, sa-rna viruses as gene delivery and vaccine vectors have also demonstrated therapeutic efficacy in a number of preclinical studies. in the context of influenza, sa-rna vaccines have shown comparable results of protection at lower doses than mrna vaccines ( , , ) . exponential developments in the "omic" area has enabled further vaccine development and understanding of the immunological response and challenges surrounding this area ( ) . systems vaccinology, which includes immunoformatics, dna/rnaseq, microarrays, mass spectrometry proteomics, transcriptomics, and metabolomics have all shown huge potential in elucidating differences in vaccine strains, vaccine growth and individual response in depth and on an epigenetic level allowing the identification of new vaccine antigens with increased speed and sensitivity ( , , ( ) ( ) ( ) . adjuvants, a group of biological and chemical compounds could also be considered to enhance and improve the longevity of the immune response of a vaccine such as the mmr. adjuvants have been successful in significantly reducing overall antigen dose in vaccine formulations as well as alter and broaden the host response through epitope spreading and qualitatively shaping the effector function of antibodies through subclass selection ( , ) . the re-purposing of live-attenuated vaccines as tibv are also being investigated. trained immunity based vaccines (tibv) elicit heterologous protective effects by inducing a broader, lasting priming of innate immune cells, in addition to the intended specific immunological response and memory of conventional vaccines [reviewed in ( ) ]. mmr and bcg vaccines have been considered as potential tibv in the context of the current coronavirus disease (covid- ) pandemic ( ) , however further research is needed. the mumps component of a vaccine is an unpurified product whose potency is measured through a biological assay for the substance rather than through evaluation of integrity of physical form (quantitative pcr after cell culture) ( ) . a monovalent mumps vaccine lot is used to characterize the performance of the mumps potency assay with international reference standards. degradation products are neither identified nor quantified ( ) . currently, the minimum potency of the mumps vaccine used varies between brands used [summarized by su et al. ( ) ] ( ) . however, this potency measurement differs to other mmr vaccines strains previously used [reviewed in ( ) ]. in addition, the maximum required potency is not usually specified. atrasheuskaya et al. ( ) demonstrated that the four out of lots of vaccine associated with six cases of viral transmission postvaccination to previously vaccinated contacts were in fact twice as potent as the lots that were not associated with viral transmission post-vaccination ( , ) . this may impact the use and efficacy of specific vaccines. due to their neurovirulence and increased incidence of aseptic meningitis and mumps cases, the urabe am and rubini mumps vaccine strains were discontinued in many countries ( , , ) . comparing alternative culturing technologies and defining a viral potency range for vaccines could help reduce variability within the mmr vaccine ( ) . ensuring the use of a reference sample that had similar replication rate and composition as the virus to be tested will allow accurate determination of the quantity of virus present per lot of vaccine. investigating novel vaccine candidates shown to induce a similar quantity but qualitatively different antibodies will help segregate and reveal potential correlates of protection ( ) . incorporating more modern technologies such as microarray technology or antibody pattern/profiling (rather than single antibody measures) to investigate biomarkers of neutralizing antibody response and/or correlates of protective immunity, in addition to incorporating what has been accomplished in finland will allow further understanding of mumps immunity ( , , , , , ) . the efficacy of a vaccine is defined by disease prevention (sterile immunity, establishment of primary infection and shedding of mature virus particle), or complications associated with infection (orchitis, neurological issues etc.) ( ) . despite the well-documented success of the global immunization programs demonstrating how vaccines significantly attenuate disease and onward transmission of infection, they are rarely totally efficacious (demonstrated in pre-licensure clinical trials) or effective (determined by practical use) ( , , ) . therefore, does "immunity" refer to sterile immunity or solely to protection from symptomatic infection? what defines an effective vaccine, or what constitutes vaccine failure? does the medical profession and the "pro-vaccine" message contribute to the public skepticism regarding immunization? is it time to shift the medical and public perception paradigm from "protection of infection following vaccination" to "protection from serious clinical mumps manifestation"? the lack of definition leads to misinterpretation by health professionals and media of what is truly occurring. such an example is currently observed with influenza; individuals who have recently being vaccinated against influenza and subsequently become infected with influenza, assume that the vaccine has "failed" even though there is a reduction in symptoms. the current assertion that vaccines "protect against" or "eliminate" the risk of infection may contribute to the misperception about what level of protection a vaccine actually provides (vaccination efficacy) perpetuated by the witnessing of visible clinical disease and outbreaks despite vaccination ( , , ) . therefore, definition and consensus of what is termed a true "vaccine failure" is required to inform both the clinical and public perception of what the function of a vaccine is. deciding what the clinical endpoint of a vaccine is i.e., infection with mild clinical symptoms vs. natural infection/disease with its associated complications and assessing the impact of the vaccine in a heterogeneously vaccinated population will allow a better consensus of what is required. a paradigm shift in what is considered to be a good vaccine i.e. one that provides protection against serious clinical sequalae, in addition to identifying a reliable laboratory marker for this protection is required ( ) . by focusing on, and acknowledging that vaccines may not prevent infection but will attenuate the clinical complications/consequences that arise from infection in addition to reducing onward transmission will provide a more realistic view of the benefits of vaccination ( ) . immunity is therefore beneficial but does not necessarily mean protection. if we can decide whether the end point of a vaccine is either the prevention of infection or protection against serious sequalae of infection, its efficacy and impact can be determined and will have enormous implications on how vaccine failure can be studied, quantified and interpreted. this teasing out of the immunological response to muv will ultimately provide potential correlates with robust predictive power, suggest directions for further vaccine improvement, and enable the discovery of potential biomarkers to help create a more efficient diagnostic assay that can discern between different infectious diseases and vaccination vs. disease status. the identification and incorporation of a correlate into diagnostic protocols which can be widely accessible may potentially allow global harmonization of criteria defining immunological protection against mumps. the medical and scientific field needs to inform the public more accurately about what a good vaccine consists of, which may result in a more positive attitude toward vaccines. in the majority of individuals, a vaccine can prevent serious clinical sequalae and associated complications following wild type infections, but also significantly reduce onwards transmission in particular to the cohorts who are not vaccinated due to a contraindication to vaccination. this is the positive and realistic view of vaccination which should be presented rather than the current flawed message of "get the vaccine and be protected from infection." the public deserves, and will appreciate, a more accurate and informed message. ac, jc, and jh contributed to the conception and design of the review. ac wrote the first draft of the manuscript. jc, tl, and jh contributed to manuscript revision. all authors have read and approved the submitted version. this work was 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evaluation of medicines for human use quality of biotechnological products: viral safety evaluation of biotechnology products from cell lines of human or animal origin a framework for research on vaccine effectiveness the urabe am mumps vaccine is a mixture of viruses differing at amino acid of the hemagglutininneuraminidase gene with one form associated with disease studies on live mumps virus vaccine. v. development of a new mumps vaccine "am " by plaque cloning highly parallel characterization of igg fc binding interactions the concept of vaccination failure effective messages in vaccine promotion: a randomized trial it's not all about autism: the emerging landscape of anti-vaccination sentiment on facebook key: cord- -vtzpf wr authors: byrne, hannah; conroy, paul j.; whisstock, james c.; o’kennedy, richard j. title: a tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications date: - - journal: trends biotechnol doi: . /j.tibtech. . . sha: doc_id: cord_uid: vtzpf wr artificial manipulation of antibody genes has facilitated the production of several unique recombinant antibody formats, which have highly important therapeutic and biotechnological applications. although bispecific antibodies (bsabs) are not new, they are coming to the forefront as our knowledge of the potential efficacy of antibody-based therapeutics expands. the next generation of bsabs is developing due to significant improvements in recombinant antibody technologies. this review focuses on recent advances with a particular focus on improvements in format and design that are contributing to the resurgence of bsabs, and in particular, on innovative structures applicable to next generation point-of-care (poc) devices with applicability to low resource environments. artificial manipulation of antibody genes has facilitated the production of several unique recombinant antibody formats, which have highly important therapeutic and biotechnological applications. although bispecific antibodies (bsabs) are not new, they are coming to the forefront as our knowledge of the potential efficacy of antibody-based therapeutics expands. the next generation of bsabs is developing due to significant improvements in recombinant antibody technologies. this review focuses on recent advances with a particular focus on improvements in format and design that are contributing to the resurgence of bsabs, and in particular, on innovative structures applicable to next generation point-of-care (poc) devices with applicability to low resource environments. antibodies as bispecifics antibodies belong to a class of globular proteins, called immunoglobulins, that are produced by b lymphocytes, and are deployed by the immune system to identify and target foreign or 'non-self' molecules [ , ] . the structure of an antibody ( figure ) determines its binding specificity and biological activity [ ] . antibodies are the most diverse proteins found in nature with the greatest variability in amino acid sequence contained within the hypervariable region or complementarity-determining regions (cdrs; see glossary), located in the fv region [ ] . thus, the antibody cdrs determines the specificity for its cognate antigen. the fc region is essential for mediating effector functions including: antibody-dependent cell-mediated cytotoxicity (adcc), antibody-dependent cellular phagocytosis, antigen presentation to the immune system, degranulation, complement-mediated lysis, and regulation of cell activation and proliferation. nobel laureates ( ) köhler and milstein devised the first and what has become the most freely available and, hence, widely used method to generate monoclonal antibodies (mabs) [ ] . however, extensive antibody engineering and optimization were required before effective antibodies were produced. the first antitumor mab, rituximab [ ] , was approved for use worldwide in and since then > mabs have achieved regulatory approval for therapeutic use. despite significant positive clinical results, especially in the case of hematological malignancies, adverse clinical outcomes and animal studies have highlighted underlying limitations of mabs. accordingly, many strategies have been developed in order to improve the specificity and control the functions of antibodies. one such important approach is the development of bsabs and this review focuses on current and future avenues of research for bsabs. in addition, the applicability of bsabs for diagnostics is discussed and critically assessed. glossary bispecific antibody (bsab): antibody-derived proteins with the ability to bind to two different epitopes. bispecific t cell engager (bite): a class of bsabs that is capable of directing a host immune system. complementarity determining region (cdr): the hypervariable region of an antibody, which exhibits a high level of sequence diversity and is involved in antigen binding. bsabs bsabs are antibody-derived proteins with the ability to bind to two different epitopes on the same or different antigens. the first bsabs were produced by oxidative recombination of two univalent antibody preparations, however, it was not for another years that bsabs with therapeutic potential were produced [ ] . the establishment of hybridoma technology in was a major advance paving the way for bsab development [ ] . bsabs are produced by three main methods: (i) chemical conjugation, which involves chemical cross-linking; (ii) fusion of two different hybridoma cell lines; or (iii) genetic approaches involving recombinant dna technology [ ] . the fusion of two different hybridomas produces a hybrid-hybridoma (or quadroma, figure a ) secreting a heterogeneous antibody population including bispecific molecules [ ] . alternative approaches included chemical conjugation of two different mabs and/or smaller antibody fragments [ ] . oxidative reassociation strategies to link two different antibodies or antibody fragments were found to be inefficient due to the presence of side reactions during reoxidation of the multiple native disulfide bonds [ ] . current methods for chemical conjugation focus on the use of homo-or heterobifunctional crosslinking reagents [ , ] . recombinant dna technology has yielded the greatest range of bsabs, through artificial manipulation of genes and represents the most diverse approach for bsab generation ( formats in the past two decades) [ ] . several bsab formats can redirect cytotoxic effector cells against target cells that play key roles in disease processes. they can induce cytotoxicity, phagocytosis, and present antigens or directly suppress deregulated immune responses, depending on the nature of interaction between the bsab and its target. furthermore, they can deliver payloads including toxins, drugs, prodrugs and contrast agents [ ] . bsabs have proven to be of major therapeutic interest over the past years, due to an array of potential capabilities. they also possess several advantageous characteristics for therapeutic development including design flexibility, modularity, optimal selectivity for activatory or downregulating molecules, oligoreactivity, and the delivery of therapeutic molecules [ ] . the vast majority of bsabs were initially designed to retarget effector cells towards tumor cells, and a variety of constructs were designed to retarget cells of the immune system by binding to and triggering fc receptors on the surface of effector cells or by binding to t cell receptor (tcr) complexes [ ] . these first generation bsabs were predominantly generated using hybrid-hybridomas or by chemical crosslinking. despite some significant biological effects elicited by these antibodies, there was no ongoing significant impact on the clinical course of a disease state. issues associated with these first generation bsabs included difficulty in largescale production of homogeneous batches and a lack of efficacy of murine antibody fragments. human anti-mouse antibody (hama) responses were observed in the majority of treated patients in addition to fc-mediated side effects (cytokine-release syndrome, thrombocytopenia, and leukopenia). subsequently, research has focused on approaches to overcome these limitations through novel antibody formats. with the advent of recombinant dna technology, it is possible to ameliorate the shortcomings associated with traditional approaches for bsab production. a plethora of different recombinant bsab formats exist (figure ), ranging from whole igg-like molecules (figure a -e) to small recombinant formats ( figure f-n) , such as tandem single chain variable fragment molecules (tafvs), diabodies (dbs), single chain diabodies (scdbs), and various other derivatives of these. bispecific tetravalent molecules are produced using fc-mediated dimerization and possess two binding sites for each antigen, which impart increased avidity. a frequent approach to produce a tetravalent bispecific molecule is through the fusion of a single-chain fv fragment to the c terminus of an antibody heavy chain or by substituting the fab arm with a bispecific singlechain antibody fragment such as a tandem scfv or an scdb [ ] . other approaches fuse two different scfvs to the n trends in biotechnology november , vol. , no. terminus of constant heavy and light chain domains. it is also possible to fuse a second variable heavy (v h ) and variable light (v l ) domain to the heavy and light chains of an antibody, therefore leading to the production of a dual-variable-domain (dvd) antibody ( figure f ) [ ] . recombinant strategies can also be used to produce small bsab fragments. the most common approach for this is the fusion of two different scfv molecules. this strategy forms the basis of the bispecific t cell engager (bite) developed for cancer immunotherapy ( figure l -m). a further expansion of this strategy is the fusion of an additional scfv fragment molecule, leading to the formation of a trivalent or trispecific antibody [ , ] . in an alternative approach, two bispecific dbs (bs . and bs . h) and two bispecific trivalent proteins (bs and bs ) were expressed within the same cell, produced and tested as potential agents for pretargeted delivery of radiolabeled bivalent haptens to tumors expressing carcinoembryonic antigen. these chains assembled in an antiparallel manner to form heterodimeric molecules [ , ] . scfv fragments expressed in bacteria are known to exist in both monomeric and dimeric forms [ ] and this can be exploited to form dbs, which are generated by linking the v h domain of one antibody to the v l domain of another ( figure j ). the linker is deliberately short ( - amino acids in length), which induces the two domains to pair with the complementary domain of another chain, thus creating two different antigen-binding sites. scdbs are a derivative of the db approach ( figure k ) and are produced by introducing an additional peptide linker to join the two antibody fragments, hence, the domains are expressed as a single polypeptide chain [ ] . although recombinant production of scfv fragments may circumvent the shortcomings associated with hybrid-hybridomas, the approach faces significant challenges. certain forms of scfv have variable and unpredictable expression yields and the linker used can cause spontaneous aggregation of the recombinant material. introducing shortened linkers to produce a db does not always guarantee success, as the linker can induce deleterious conformational changes resulting in a reduction of antibody functionality [ ] . to overcome solubility-related issues, several unique approaches have been undertaken to stabilize rbsabs. single domain antibodies (sdabs) occur in the natural repertoire of both camelid and cartilaginous fish ( figure e ,f). these single v domain constructs, known as v hh in camelids and v-nar in sharks, are of minimal size ( kda). in addition, they demonstrate high expression levels, and exhibit high stability and solubility in vitro, which has made them attractive entities for bsab generation [ ] . sdabs can be produced in bacteria (or yeast) and their properties support facile conversion to bispecific formats through linkage of two sdabs directed against two different trends in biotechnology november , vol. , no. antigens. the resultant low molecular mass ( kda), although advantageous for biodistribution, can hamper therapeutic success due to rapid clearance in vivo by renal filtration and degradation. the positive attributes associated with sdabs have made them a key point of therapeutic interest [ ] [ ] [ ] . the 'dock-and-lock' construction method involves homo-and heterodimerization of the dimerization and docking domain (ddd) of human camp-dependent protein kinase a (pka) with the anchoring domain (ad) from a-kinase anchor protein (akap) [ , ] . therefore, fusion of a fab fragment directed against the first antigen to ad and fusion of a fab fragment directed against the second antigen to the ddd domain (homodimer) and subsequent in vitro assembly of the two protein preparations results in a trivalent molecule composed of one fab-ad and two fab-ddd moieties ( figure g ) [ ] . furthermore, a disulfidestabilized scfv was fused to the c terminus of an igg light chain creating an igg-scfv bsab ( figure c ), expressed in mammalian cells and purified by one-step protein a chromatography. in this format, the bsab exhibits igg-like stability, and demonstrates igg-like tumor targeting and blood clearance in vivo. this format is suggested as a standardized platform for the construction of functional bsabs [ ] and several such igg-scfv formats are described in the literature ( figure b -e) [ ] . the monospecific nature of mabs for therapy may often be a limitation for their effective use in complex human diseases. cancer, hiv, inflammatory disease, infectious disease, and allergic disorders are multifaceted in nature and therapeutic resistance can result from engagement or upregulation of alternative receptor pathways. current combination mab therapies are undergoing clinical trials and show some efficacy in diseases, such as cancer, by simultaneous engagement of target antigens [ ] . although this approach shows promise, each mab in the combination therapy may be required to achieve regulatory approval. therefore, additional polyclonal strategies are under investigation for oligoreactive treatment of complex disease states [ , ] . rbsabs have significant potential for application in multifaceted disease therapy, because they can be engineered to block or engage multiple sites on a single target simultaneously, or sites on different targets, within a single therapeutic entity. in addition, payloads can be delivered specifically to the target cell, and fc effector engagement can recruit cells of the immune system. bsabs are of particular therapeutic interest as strategies in cancer therapy, which is the predominant disease state to which bsab are applied. most current applications have focused on redirecting the cytotoxic activity of lymphocytic effector cells against tumor targets through binding to the t cell co-receptor molecule cd . in addition to cd , cd is the major activating receptor on natural killer (nk) cells and mediates low-affinity interactions with igg. several antigen targets have undergone evaluation with the majority of them representing tumor-associated antigens (taas) ( table ) . despite the ongoing development of various increasingly complex bsab designs, only two formats, bites and triomabs, have made a substantial impact. the triomab format has proven to be the most successful due to its performance in clinical trials and the approval of catumaxomab by regulatory agencies. those formats currently in clinical trials are listed in table . bites combine a unique set of properties unreported for any other kind of bsab (box ). the bite format potentially overcomes several limiting factors relating to the biological activity of tumor-directed bsabs. bites combine the minimal binding domains (fv fragments) of two different mabs fused together by a short flexible linker that allows free rotation of the two arms, and thus facilitates optimal antibody:antigen interaction [ ] . they function by forming a link between t cells (cd or cd ) and taas, inducing t cell dependent cytotoxic activity by proteins including perforin and granzyme, independently of the presence of mhc i or co-stimulatory molecules [ ] , and these proteins enter tumor cells, initiating apoptosis. mt (blinatumomab, table ) a cd -specific bite is currently in clinical trials for the treatment of non-hodgkin's lymphoma and acute lymphoblastic leukemia [ ] . mt , an anti-human epithelial cell adhesion molecule (epcam)  anti-human cd tandem single chain fragment variable (tafv), was the second bite tested in clinical trials, and the first directed to a wide spectrum of solid tumors [ ] . in vitro testing of mt reconfirmed the results obtained with mt on tumor cell lines, thereby demonstrating the modularity of the bite format. mt is currently being tested in a phase i study with lung, colorectal, and gastrointestinal cancer. initial results suggest that the pharmacokinetic properties and the risk of systemic activation associated with mt might require further molecular development in order to develop a safe and feasible treatment option [ ] . trifunctional antibodies (triomabs) are intact igg molecules characterized by their unique ability to engage three different cells types, typically, tumor cells, t cells, and accessory cells, such as, macrophages, dendritic cells, nk cells, and other fc-receptor-expressing cells. trifunctional antibodies have two different antigen-binding specificities, most commonly cd and a tumor antigen [ ] . the presence of the intact fc region facilitates interaction with receptors triggering several immune defense reactions (box ). although trifunctional antibodies were initially perceived to be unfavorable for therapeutic development, due to retention of fc effector functions, several of these antibodies were assessed in clinical trials. trion pharma and fresenius biotech developed the first successful trifunctional antibodies, composed of whole hybrid mouse/rat igg molecules with specificity for cd and the tumorassociated antigens: her (ertumaxomab) and epcam (catumaxomab). catumaxomab was the first bsab to reach the pharmaceutical marketplace ( ) for the treatment of malignant ascites in patients with epcam-positive carcinomas, by simultaneous engagement of type i, iia, and iii fc receptors [ ] . at present, an ongoing phase ii clinical trial for catumaxomab is underway for the treatment of ovarian and gastric cancer. bi (lymphomun) trends in biotechnology november , vol. , no. targets cd , a b lymphocyte membrane antigen, which is a viable target for the treatment of b cell related malignancies. bi is generated by chemical heteroconjugation of the cd-specific chimeric mab, rituximab, and the cd specific murine mab okt- (muromonab). it differs from catumaxomab because it is composed of two whole immunoglobulins [ ] , and initial in vitro studies demonstrated that bi mediated efficient and specific lysis of b cell lines compared to unarmed t cells or cell lines treated with rituximab alone. in a pilot study, consisting of six patients with recurrent b cell malignancies, promising results indicate the therapeutic potential of bi . phase i clinical trials are currently underway to evaluate efficacy in patients with relapsed or refractory cd + non-hodgkin's lymphoma (nhl) [ ] , in addition to other indications [ ] [ ] [ ] [ ] . the ability of bsabs to bind simultaneously to a specific antigen and a given detection moiety enables them to function as excellent bifunctional immunoprobes in diagnostic assays (figure ). associated advantages of bsabs over traditional mabs include: design flexibility and onestep addition of reagents compared to traditional multistep procedures. obviating the requirement to label a reagent directly, such as a secondary antibody, reduces the deleterious effects of chemical modification of either the enzyme or the antibody. antibodies are extremely versatile and are incorporated into a variety of different immunodiagnostic assay platforms, such as: microtiter plate assay, swab, strip, filter disk, and 'spinning-disc-type' assays. bsabs are attractive in such assays because they simplify the detection steps and are currently used for the development of simple, rapid, and highly sensitive immunoassays for the detection of bacterial and viral infectious diseases and in cancer diagnostics. in vivo cancer diagnostic imaging in vivo cancer diagnostic imaging facilitates insight into the molecular and functional characteristics of cancerous tissue, which permits targeting, identifying, and assessing several different types of tumors. bsabs in cancer diagnostic imaging have proven effective, particularly when implementing a pretargeting strategy [ , , ] . the pretargeting strategy enhances the sensitivity of imaging by adapted from [ , ] . abbreviations: ad, adenovirus; met, hepatocyte growth factor receptor; pdgfr, platelet-derived growth factor receptor; ccr, chemokine cc receptor; cea, carcinoembryonic antigen; dota, , , , -tetraazacyclododecane-n,n ,n ,n -tetraacetic acid; egfr, epidermal growth factor receptor; fap, fibroblast activation protein; hmwmaa, high molecular weight melanoma-associated antigen; igfr, insulin-like growth factor receptor; ltbr, leukotriene b receptor; muc- , mucin ; psca, prostate stem cell antigen; psma, prostate specific membrane antigen; sap, secretory aspartyl proteinase; trail-r , tnf-related apoptosis inducing ligand receptor- ; vegfr, vascular endothelial growth factor receptor. trends in biotechnology november , vol. , no. elimination of background noise, as a consequence of untargeted radioactivity, and reduces unintended toxicity. in such a strategy, the bsab is designed to bind to the target antigen and a radiometal-chelate or hapten-peptide complex (effector molecule). the pretargeting approach is a two-step imaging process in which the bsab is injected initially, and once excess antibody has cleared from circulation, it is followed by injection of mono-divalent radiolabeled haptens [ ] . animal model experiments have shown that bsabs are capable of an equivalent tumor uptake compared to conventional radiolabeled antibodies and have minimum accretion within a matter of minutes rather than several hours or days. additionally, the radionucleotide is cleared rapidly from circulation and tissue retention in the kidneys and liver was low. positron emission tomography (pet) imaging approaches use i -labeled haptens after pretargeting with a bsab, however, the visualization of small malignant lesions remains problematic [ ] . to improve the ability to image such lesions, an alternative strategy combines pretargeting with highly specific, high-activity radiotracer-labeled polymers [ ] [ ] [ ] . bsab complexes were utilized to pretarget pc- human prostate cancer xenographs in scid mice. these complexes consisted of intact anti-diethylenetriaminepentaacetic acid (dtpa) antibody or fab-linked bombesin (bom) coupled via thioether bonds (bom-bscx or bom-bsfcx). results indicated that the radiolabeled polymers accumulated at the pretargeted sites and enabled visualization of cancerous lesions as small as - mm. additionally, metastatic melanoma lesions in the lungs of mice were imaged with a bsab and m tc-dspl (polylysine polymer conjugated to dtpa), succinylated, and labeled with m tc. the use of bsabs in cancer imaging has significantly enhanced the quality and reliability of this technique. furthermore, atherosclerotic lesions as small as mm were successfully imaged in apolipoprotein e (apoe) knockout mice by use of surrogate antigen-coated polystyrene beads pretargeted with a bsab f(ab )  f(ab ) [ ] . within animal models, bsabs have been proven to be superior to mabs in terms of specificity, sensitivity, and signal intensity while maintaining minimal signal background. infectious-disease-causative agents include bacteria, viruses, parasites, and fungi. in the present 'decade of the vaccine', the world health organization (who) attributes . million deaths annually to infectious diseases, which is a significant socioeconomic burden on society. global healthcare systems use surveillance measures to control the spread of infectious diseases, which are reliant on early detection. tuberculosis (tb) is caused by the highly resistant bacterium mycobacterium tuberculosis and particularly affects those with a weak immune system, for example, individuals infected with hiv. although most infections are asymptomatic and latent, one in ten infections eventually progresses to active disease, which if untreated, kills > % of those infected. the who reports that of the . million cases of tb in , there were . million deaths worldwide [ ] . a rapid, sensitive, specific, and inexpensive tb diagnostic for a poc setting is of considerable value and importance for tb control. current methods of detection including sputum smear microscopy (ssm) and bacterial culture do not facilitate early diagnosis. the bacterial culture method is considered to be the gold standard for tb diagnosis, however, - weeks are - -fold higher efficacy in tumor cell lysis relative to other cd -bispecific formats and monoclonal igg antibodies. induces target cell elimination by unstimulated peripheral t cells without the need for t cell co-stimuli or t cell preactivation regimens. strictly targets cell dependent, polyclonal activation of most cd + and cd + t cells. high protein stability and homogeneity. cytotoxic t cells, with their highly cytotoxic potential, abundance, and 'search-and-destroy' function, are the most potent killer cells of the human body. they are capable of effectively inducing tumor cell lysis and apoptosis. accessory cells target and eliminate tumor cells through processes such as phagocytosis or apoptosis. additionally, they have the ability to release cytokines, which further stimulate the action of t cells. dendritic cells are capable of inducing long-lasting immunity against cancerous tumors by processing and presenting tumor cells and tumor cell derived material to the immune system. trends in biotechnology november , vol. , no. required to obtain results. through the development of molecular assays including the interferon gamma release assay (igra) [ ] and nucleic acid amplification (naa) [ ] , early detection has improved. these approaches require significant expertise and infrastructure and as a consequence are not suited for resource-limited areas. lipoarabinomannan (lam) is an important non-protein antigen of the bacterial cell wall, which is present in different body fluids of tb-infected patients. a bispecific mab with specificity towards the lam antigen and the reporter molecule, horseradish peroxidase (hrpo), was developed through hybrid-hybridoma technology. this bsab was incorporated into a simple low cost immnuoswab-based assay to detect the lam antigen [ ] . the limit of detection of this assay for spiked synthetic lam was . ng/ml in bovine urine, . ng/ml in rabbit serum, and . ng/ml in saline, and for bacterial lam from m. tuberculosis h rv, it was found to be . ng/ml in rabbit serum [ ] . the assay was further evaluated using clinical serum samples collected from tb patients, of which were positive and seven were negative in terms of anti-lam antibody titer. the assay exhibited % specificity and % sensitivity ( % confidence interval) in a parallel comparison with laboratory culture. in addition to good specificity, visual results were obtained within h of sample collection. this immunoassay was also evaluated in another format to test the sensitivity of the assay using a biotinylated cs- mab. the transformation of the assay from a bispecific-based format to a monoclonal-based format reduced the sensitivity of the assay. the use of this bsab, the first of its kind to detect any tb antigen, in the immnuoswab assay provided enhanced sensitivity and specificity [ ] , and the reported assay is particularly suited to resource-limited areas, as a rapid tool for detecting tb in resource constrained laboratory settings. hepatitis b is a significant health concern and viral hepatitis is a leading cause of liver cancer and commonly results in liver transplantation. hepatitis b virus surface antigen (hbsag) is the key screening target for detecting infected individuals. current approaches for hbsag screening rely on laboratory-based elisa testing. a novel agglutination test for hepatitis b infection uses an anti-hbsag  antihuman erythrocyte bsab [ ] . the db format was developed by phage display, selecting scfv from red blood cells and hbsag-specific libraries. in human blood samples ( clinical specimens), agglutination was observed in samples containing hbsag ( % specificity) and the test showed . % sensitivity. the agglutination kinetics were hbsag concentration dependent with high viral loads leading to agglutination in - min. although the assay was specific and sensitive, it did not have equivalent limits of detection, compared to elisa ( -fold difference), but operated within the observed hbsag concentration range for actively infected individuals ( ng/ml to mg/ml) [ ] . as a primary poc screening tool, the simple agglutination test is a powerful approach to assist in the rapid screening for hepatitis-b-infected individuals. escherichia coli e. coli o :h is considered to be a serious human pathogen and is associated with bloody diarrhea (hemorrhagic colitis) and hemolytic uremic syndrome (hus). current methods of detection of e. coli in food and water focus on enrichment and microbial culture [ ] , which may take - h to identify the organism. other techniques focus on molecular methods such as pcr, which despite the sensitivity and rapid result retrieval capabilities, this technique is limited by the requirement to isolate dna from samples. a more sensitive, rapid, and reliable diagnostic technique for the identification of e. coli is essential for appropriate detection and management to prevent disease outbreaks. to develop a highly sensitive bsabbased assay for e. coli o :h in water samples, a hybridoma secreting a mab specific for e. coli o :h whole bacteria and e. coli o :h lipopolysaccharide (lps) was fused with an anti-hrpo mab-secreting hybridoma to generate a quadroma. the resulting bsab was incorporated into a sandwich elisa [ ] , allowing rapid, one-step detection of e. coli o :h . in this assay the anti-e. coli o :h mab was used as the capture reagent to enrich bacteria at the surface followed by bsab mediated detection. the detection sensitivities of this assay were determined to be , , and cfu/ml for tap water, lake water, and apple juice, respectively, in a microtiterplate-based assay. a marked improvement in sensitivity for e. coli o :h was achieved using both immunofilter and immunomagnetic elisa formats, with detection . diagrammatic representation of a generalized assay format for a bispecific antibody (bsab)-based immunoassay. the capture monoclonal antibody is immobilized on to a solid surface and binds to a specific epitope on its cognate antigen present in the test sample. upon addition of the corresponding bsab, one arm binds to a specific epitope on the antigen, while the other arm binds to a reporter molecule, such as horseradish peroxidase (hrpo), and converts the subsequently added substrate to a quantifiable signal or colored product. adapted from [ ] . trends in biotechnology november , vol. , no. limits of cfu/ml and cfu/ml, respectively. additionally, the specificity of the assay was confirmed, because it did not detect other bacterial strains such as salmonella, pseudomonas, and non-pathogenic e. coli. this novel, bsab-based assay had high specificity with low background and was both rapid and ultrasensitive. a major advantage of this assay, in comparison to current assays, was removal of the requirement for amplification steps. bordetella pertussis b. pertussis is the causative bacterium of pertussis (whooping cough), which affects individuals of adolescent age, and there were an estimated pertussis-related deaths in children ( ) . despite the availability of a vaccine, it has become a major health concern in recent years. estimates from the who suggest that in there were million cases of pertussis, of which % were in developing countries. similar to many other bacterial infections, early detection is critical in order to prevent the spread of infection and to ensure correct treatment of infected individuals. currently, there are two methods for detecting this bacterium. the first requires that clinical nasopharyngeal swab or aspirate samples are cultured for - days [ ] . the second method involves the use of a fluorescently labeled mab directed against an antigenic lps molecule, which is present on the outer membrane of b. pertussis. both assays have limitations regarding sampling, operator training, and sensitivity, which restrict their use in resource-constrained locations [ ] . a 'molecular velcro' sandwich assay for the detection of b. pertussis was found to be useful for the analysis of clinical samples, immunochemical structural studies, and for the serological characterization of b. pertussis lps [ ] . the bsab was produced by fusion of the anti-b. pertussis lps mab-secreting hybridoma with an anti-hrpo mab-secreting hybridoma. the anti-lps-specific mab was capable of binding to heat-killed b. pertussis bp in both buffer and sample matrix (spiked nasopharyngeal aspirates) [ ] . the assay proved highly sensitive with a lower limit of detection of $ cfu. interestingly, the binding of the b. pertussis to the anti-b. pertussis lps mab-coated solid phase was found to be irreversible, despite significant washing suggesting that the assay works as a form of 'velcro molecular' assay. this immunoassay achieved ultrasensitive detection of b. pertussis due to the availability of multiple lps molecules on the bacterial surface for bsab binding. additionally, to facilitate ease of use and rapid detection, the immunoassay was tested in an immunoswab format permitting poc detection of b. pertussis in a clinical setting. staphylococcus aureus thermonuclease (tnase) s. aureus is the most frequent cause of wound infection among hospitalized patients, with studies suggesting that it is present in % of infected leg ulcers [ ] . the major biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species have become major health concerns. a novel fluorescence-based immunoassay enabled qualitative detection of s. aureus tnase to confirm the presence of s. aureus in vitro [ ] . rhodamine and fluorescein-labeled hemocyanin from megathura crenulata (klh) were prepared as immunoconjugates containing a sensitive fluorescent reporter moiety. a bsab that both specifically quenched the fluorescence of the reporter conjugate and bound the tnase target antigen was produced using cell fusion techniques. in that study two klh-fluorophore/tnase-specific antibody series were generated, with each member of the series specific to an epitope on either the klh or the fluorescent moiety of the reporter conjugate. the decrease in fluorescence emission intensity at nm/ nm for the rhodamine and fluorescein conjugates, respectively, was analyzed with each bsab of the series, and quenching was observed upon antibody-antigen binding [ ] . increased fluorescence intensity observed upon tnase binding was demonstrated with antibodies that have a higher affinity for the bacterial antigen than the fluorescent reporter. the specificity of the assay for s. aureus was also tested using the pseudomonas aeruginosa lps antigen as a control, which demonstrated that variations in the fluorescence quenching effect did not exceed %. these preliminary investigations suggest a highly sensitive assay with potential applications in biological specimens with a limited risk of false positive results. severe acute respiratory syndrome (sars) sars is a serious form of pneumonia and affected > people worldwide, spreading to countries across five continents, in the - outbreak. sars coronavirus (sars-cov) is the responsible agent and was transmitted from wild animals to the human population [ , ] , and delayed identification of this virus aided contagion. the sars virus is detected in humans by rt-pcr [ , ] indirect fluorescence assay, which detects anti-sars-cov antibodies in body fluids [ ] and by isolation of sars-cov from clinical samples [ , ] . such viral culturing is time consuming, tedious and insensitive, therefore, pcr-and antibody-based detection methods are the predominant techniques adopted for surveillance. a highly sensitive and rapid bsab-based immunoswab assay was developed for the early detection of sars-cov. this assay was superior to the traditional monoclonal elisa-based assay [ ] and detected the viral antigen nucleoplasmid protein (np). three different mabs that recognize various epitopes on the np antigen and the anti-hrpo mab were utilized to generate the anti-sars-cov np  anti-hrpo bsabs. this immunoswab assay showed np detection limits of pg/ml in saline, - pg/ml in pig nasopharyngeal aspirates, and pg/ml in rabbit serum, thus, demonstrating the bispecific detection approach as superior in terms of sensitivity and specificity. the immunoswab assay facilitated rapid detection ($ min) [ ] and was robust, potentially permitting screening of numerous infected individuals within a short period of time, and hence, assisting to contain viral infection. furthermore, by targeting the sars-cov spike protein (s ) in both maband bsab-based assays, to identify sars infected individuals, it was also demonstrated that the bsab detection reagent could improve the sensitivity of the assay from to ng/ml [ ] . although these examples demonstrate the utility of bsabs for diagnostic applications, there are several shortcomings related to the use of quadromas including yield, format uniformity, and purification. the advent of recombinant formats is of considerable value to overcome these limitations. it is anticipated that bsab formats in the therapeutic arena can be meaningfully applied to diagnostics as the development of next generation diagnostic devices for poc, decentralized, and aged care applications demand superior diagnostic reagents. the bsab stable is large with big pharmaceutical companies investing over us$ . billion in bsabs since [ ] . the front-runners have yet to clearly emerge but the increasing confidence in bsabs over the past few years has resulted in a plethora of bsab formats. in combination with highly innovative molecular biological approaches, the momentum gained through clinical success is prompting the emergence of additional formats. in many cases these may prove ineffective for therapeutic applications, resulting in a high attrition rate. however, the therapeutic arena has seen significant advancements in bsab-based agents, which culminating in a single bsab achieving regulatory approval. this bsab, catumaxomab, does not extend far beyond the traditional igg format but to access truly unique functions many more original and groundbreaking approaches will be required. similar to the development of human mab therapies, the efficacy and safety of simultaneous engagement of multiple disease targets will take considerable research and potentially, considerable time, to show marketplace dominance [ ] . current clinical trials are in progress and larger numbers of entities are expected to enter clinical evaluation in the future. it is still unclear if such smart retargeting strategies will be successful but bsabs are backed as 'hot stock' at present due to their real potential to have a significant impact on human disease treatment and management [ ] . although the next big blockbuster therapy may well be a bsab, the application of such constructs to diagnostics is an attractive alternative to current strategies, which has real potential to pave the way for improved, nextgeneration diagnostics with applications in low-resource and poc settings. bsabs in simple immunodiagnostic tests are applicable to such settings and can assist in ensuring these tests meet the assured principles [ ] . regardless of the application there are some common issues that will need to be addressed (box ) including manufacturability, scale, and stability, with issues relating to immunogenicity, pharmacokinetics, and biodistribution being of particular concern for therapeutics [ ] . the bsab tale is moving beyond two specificities and into chapters that will vastly improve their therapeutic applications and will also significantly impact on current diagnostic challenges. will the multitude of bsabs formats provide comparable preclinical information? is the triomab or bite or sdab the way forward for human therapeutics? will the paucity of diagnostic bispecifics be improved by a single bispecific format coming to the fore in the therapeutic arena? can bispecific antibody deliver superior, simplified diagnostics with application in low resource settings, through simplification of the assay capture and/or detection steps? trends in biotechnology november , vol. , no. the human antibody repertoire to infectious agents: implications for disease pathogenesis antibody production antibody production, design and use for biosensor-based applications continuous cultures of fused cells secreting antibody of predefined specificity rituximab, an anti-cd monoclonal antibody: history and mechanism of action bispecific antibodies for diagnostic applications hybrid hybridomas and their use in immunohistochemistry chemical production of bispecific antibodies recombination of a mixture of univalent antibody fragments of different specificity dual targeting strategies with bispecific antibodies immunotherapeutic perspective for bispecific antibodies natural killer (nk)-and t-cell engaging antibody-derived therapeutics bispecific antibodies for cancer immunotherapy: current perspectives simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin a novel cd -directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells development of new multivalent-bispecific agents for pretargeting tumor localization and therapy human anti-self antibodies with high specificity from phage display libraries antigen-independent selection of stable intracellular single-chain antibodies camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs engineered antibody fragments and the rise of single domains bispecific antibodies for cancer therapy: the light at the end of the tunnel? stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting a novel bispecific, trivalent antibody construct for targeting pancreatic carcinoma a modular igg-scfv bispecific antibody topology recombinant bispecific antibodies for cancer therapy buy buy bispecific antibodies bites: bispecific antibody constructs with unique anti-tumor activity mt : a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors development and approval of the trifunctional antibody catumaxomab (anti-epcam  anti-cd ) as a targeted cancer immunotherapy immunotherapy of recurrent b-cell malignancies after allo-sct with bi (fbta ), a trifunctional anti-cd  anti-cd antibody and donor lymphocyte infusion ganglioside gd -specific trifunctional surrogate antibody surek demonstrates therapeutic activity in a mouse melanoma model trifunctional bispecific antibodies induce tumor-specific t cells and elicit a vaccination effect transient lymphocyte decrease due to adhesion and migration following catumaxomab (anti-epcam  anti-cd ) treatment in vivo cancer therapy with trifunctional antibodies: linking innate and adaptive immunity improved targeting of pancreatic cancer: experimental studies of a new bispecific antibody, pretargeting enhancement system for immunoscintigraphy antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy bispecific antibody pretargeting of radionuclides for immuno single-photon emission computed tomography and immuno positron emission tomography molecular imaging: an update bispecific antibody pretargeting pet (immunopet) with an i-labeled hapten-peptide imaging experimental atherosclerotic lesions in apoe knockout mice: enhanced targeting with z d -anti-dtpa bispecific antibody and mtc-labeled negatively charged polymers targeting very small model lesions pretargeted with bispecific antibody with mtc-labeled highspecific radioactivity polymers imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates pretargeted gamma imaging of murine metastatic melanoma lung lesions with bispecific antibody and radiolabeled polymer drug conjugates interferon-gamma release assays for the diagnosis of latent tuberculosis infection in hiv-infected individuals: a systematic review and meta-analysis nucleic acid amplification tests for diagnosis of smear-negative tb in a high hiv-prevalence setting: a prospective cohort study a bispecific antibody based assay shows potential for detecting tuberculosis in resource constrained laboratory settings rapid detection of hepatitis b virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis b virus surface antigen comparative evaluation of different chromogenic/fluorogenic media for detecting escherichia coli o :h in food monospecific and bispecific antibodies against e. coli o for diagnostics use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for bordetella pertussis detection of bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost bispecific antibody-mediated detection of the staphylococcus aureus thermonuclease isolation and characterization of viruses related to the sars coronavirus from animals in southern china review of bats and sars the aetiology, origins, and diagnosis of severe acute respiratory syndrome evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus immunofluorescence assay for serologic diagnosis of sars growth kinetics of sars-coronavirus in vero e cells susceptibility of human and rat neural cell lines to infection by sars-coronavirus a rapid point of care immunoswab assay for sars-cov detection quantitative and sensitive detection of the sars-cov spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay bispecific antibodies and adcs: once and future kings? diagnostic tests for infectious diseases in the developing world: two sides of the coin a critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data t cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human b cell lymphoma xenografts by a cd -/cd -bispecific single-chain antibody construct extremely potent, rapid and costimulationindependent cytotoxic t-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody efficient elimination of chronic lymphocytic leukaemia b cells by autologous t cells with a bispecific anti-cd / anti-cd single-chain antibody construct t-cell-mediated lysis of b cells induced by a cd  cd bispecific single-chain antibody is perforin dependent and death receptor independent redirected t-cell cytotoxicity to epithelial cell adhesion molecule-overexpressing adenocarcinomas by a novel recombinant antibody, e bi, in vitro and in an animal model bispecific single-chain antibodies as effective tools for eliminating epithelial cancer cells from human stem cell preparations by redirected cell cytotoxicity efficient tumor cell lysis by autologous, tumor-resident t lymphocytes in primary ovarian cancer samples by an ep-cam-/cd -bispecific antibody construction of a bispecific single chain antibody for recruitment of cytotoxic t cells to the tumour stroma associated antigen fibroblast activation protein a novel recombinant bispecific single-chain antibody, bscwue-  cd , induces t-cell-mediated cytotoxicity towards human multiple myeloma cells cloned transgenic farm animals produce a bispecific antibody for t cell-mediated tumor cell killing a recombinant bispecific single-chain fv antibody against hla class ii and fcgammariii (cd ) triggers effective lysis of lymphoma cells increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity targeting of adenoviral vectors through a bispecific single-chain antibody combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma gene therapy for meningioma: improved gene delivery with targeted adenoviruses efficient and selective gene transfer into primary human brain tumors by using single-chain antibodytargeted adenoviral vectors with native tropism abolished selective gene transfer into primary human gastric tumors using epithelial cell adhesion moleculetargeted adenoviral vectors with ablated native tropism cd -targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain fv antibody enhances cytotoxic t cell activation recombinant bispecific antibodies for the targeting of adenoviruses to cea-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scfv construction and expression of a bispecific single-chain antibody that penetrates mutant p colon cancer cells and binds p treatment of human b cell lymphoma xenografts with a cd  cd diabody and t cells synergistic antitumor effect of bispecific cd  cd and cd  cd diabodies in a preclinical model of non-hodgkin's lymphoma efficient inhibition of human b-cell lymphoma xenografts with an anti-cd  anti-cd bispecific diabody a highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and t-lak cells a mutated superantigen sea d a fusion diabody specific to muc and cd in targeted cancer immunotherapy for bile duct carcinoma efficient inhibition of multidrug-resistant human tumors with a recombinant bispecific anti-p-glycoprotein  anti-cd diabody combined effect of recombinant cd  cd diabody and thalidomide in a preclinical model of human b cell lymphoma anti-hla-dr/anti-dota diabody construction in a modular gene design platform: bispecific antibodies for pretargeted radioimmunotherapy the effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody durable complete responses from therapy with combined epratuzumab and rituximab: final results from an international multicenter, phase study in recurrent, indolent, non-hodgkin lymphoma a bispecific diabody directed against prostatespecific membrane antigen and cd induces t-cell mediated lysis of prostate cancer cells effect of domain order on the activity of bacterially produced bispecific single-chain fv antibodies bispecific single-chain diabody-mediated killing of endoglin-positive endothelial cells by cytotoxic t lymphocytes targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (cd ) retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scdb) adapter that binds to fiber knob and hmwmaa enzyme recruitment and tumor cell killing in vitro by a secreted bispecific single-chain diabody. tumor target bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics effect of tetravalent bispecific cd Âcd recombinant antibody construct and cd costimulation on lysis of malignant b cells from patients with chronic lymphocytic leukemia by autologous t cells bispecific minibodies targeting her /neu and cd exhibit improved tumor lysis when placed in a divalent tumor antigen binding format anti-tumor activity of stabilityengineered igg-like bispecific antibodies targeting trail-r and ltbetar simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody a stable igg-like bispecific antibody targeting the epidermal growth factor receptor and the type i insulin-like growth factor receptor demonstrates superior anti-tumor activity combination of two insulin-like growth factor-i receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response engineering a cd  cd bispecific scfv immunofusion for the treatment of leukemia and elimination of leukemia stem cells a series of anti-cea/anti-dota bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance a novel bispecific egfr/met antibody blocks tumor-promoting phenotypic effects induced by resistance to egfr inhibition and has potent antitumor activity the bs  anti-cd -cd bispecific antibody has more lymphomacidal activity than do the parent antibodies alone domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hex diabody molecular characterization of novel trispecific erbb-cmet-igf r antibodies and their antigen-binding properties single domain antibody-based and linker-free bispecific antibodies targeting fcgammariii induce potent antitumor activity without recruiting regulatory t cells novel humanized and highly efficient bispecific antibodies mediate killing of prostate stem cell antigenexpressing tumor cells by cd + and cd + t cells a dual-targeting pdgfrbeta/vegf-a molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo a novel bispecific antibody recruits t cells to eradicate tumors in the ''immunologically privileged'' central nervous system systemic administration of a bispecific antibody targeting egfrviii successfully treats intracerebral glioma development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits ige receptor signaling a bispecific antibody against human ige and human fcgammarii that inhibits antigen-induced histamine release by human mast cells and basophils reversal of airway inflammation and remodeling in asthma by a bispecific antibody fragment linking ccr to cd a development of tetravalent, bispecific ccr antibodies with antiviral activity against ccr monoclonal antibodyresistant hiv- strains heavy chain-only antibodies and tetravalent bispecific antibody neutralizing staphylococcus aureus leukotoxins t-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites human domain antibodies against virulence traits of candida albicans inhibit fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis engineering of stable bispecific antibodies targeting il- a and il- therapeutic control of b cell activation via recruitment of fcgamma receptor iib (cd b) inhibitory function with a novel bispecific antibody scaffold molecular construction and optimization of antihuman il- alpha/beta dual variable domain immunoglobulin (dvd-ig) molecules depletion of ccr -expressing cells with bispecific antibodies and chemokine toxins: a new strategy in the treatment of chronic inflammatory diseases and hiv a bispecific antibody against il- beta and il- a is beneficial for experimental rheumatoid arthritis a phase-i trial of the epidermal growth factor receptor directed bispecific antibody mdx- without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors antitumor activity of a novel bispecific antibody that targets the erbb /erbb oncogenic unit and inhibits heregulin-induced activation of erbb cd as an attractive target for antibody-based therapy cancer immunotherapy by retargeting of immune effector cells via recombinant bispecific antibody constructs clinical and pharmacologic aspects of blinatumomab in the treatment of b-cell acute lymphoblastic leukemia epcam/cd -bispecific t-cell engaging antibody mt eliminates primary human pancreatic cancer stem cells poster sessions rescue of impaired nk cell activity in hodgkin lymphoma with bispecific antibodies in vitro and in patients dual targeting of egfr and her with mehd a overcomes acquired resistance to egfr inhibitors and radiation clinical impact of serum proteins on drug delivery biologic properties of a bispecific single-chain antibody directed against - a (epcam) and cd : tumor celldependent t cell stimulation and cytotoxic activity a recombinant bispecific single-chain antibody, cd  cd , induces rapid and high lymphoma-directed cytotoxicity by unstimulated t lymphocytes key: cord- -j ye ed authors: loemba, h. d.; mounir, s.; mardassi, h.; archambault, d.; dea, s. title: kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: j ye ed the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally- and experimentally-exposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the - or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus ande. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. summary. the kinetics of appearance of antibodies directed to the major structural proteins n, m and e of porcine reproductive and respiratory syndrome virus (prrsv) was followed in pigs naturally-and experimentallyexposed to the virus. specific igm antibody titers were first detected by indirect immunofluorescence (iif) at the end of the first week of prrsv infection, peaked by day to post-inoculation (p.i.), then rapidly decreased to undetectable levels by day to p.i. on the other hand, specific igg antibody titers peaked by day to p.i. and remained unchanged to the end of the -or -week observation period; in addition, a persistent viremia was observed. virus neutralizing (vn) antibody titers > were not detected until to weeks p.i. taken together, the results obtained by western blotting analyses using purified virus and e. coli-expressed orfs to gene products, suggested that antibodies directed against the envelope e protein appear by day p.i., whereas antibodies directed against the nucleocapsid n and membrane m proteins can only be detected by the end of the second week p.i. no correlation could be demonstrated between vn and iif antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i. the porcine reproductive and respiratory syndrome virus (prrsv) is an enveloped rna virus which has been found to be responsible for reproductive failure in sows of any parities (late-term abortions, increased numbers of stillborn, mummified and weakborn pigs) and respiratory problems affecting pigs of all ages [ , ] . this new porcine virus has been provisionally classified h.d. loemba et al. within the genus arterivirus, according to its biochemical and morphological characteristics [ , ] . the viral genome is a positive single-stranded rna molecule approximately kb in length that contains eight open reading frames (orfs) similarly organized to that of the coronaviruses [ , ] . the virion contains three major structural proteins: a nucleocapsid protein n of kda, an unglycosylated membrane protein m (matrix protein) of - kda and a glycosylated membrane protein e (envelope associated glycoprotein) of - kda which are encoded by orfs, , and , respectively [ ] . clinical symptoms and production losses may vary widely among herds, but the vast majority of prrsv infections are subclinical [ , ] . presently some field and experimental evidence exists that indicates persistent lifelong viremia during prrsv infection [ , ] . an antibody-dependent enhancement mechanism has been reported to explain the persistent infection of porcine alveolar macrophages and circulating monocytes [ , ] . so far, little is known about the immunological status of prrsv-infected pigs. some investigators suggest that prrsv causes immunosuppression because of the recrudescence of secondary bacterial infections among the infected pigs [ ] . previous data indicated that antibodies to prrsv may be detected as soon as to weeks post-exposure using indirect immunofluorescence [ , ] and the immunoperoxidase monolayer assay [ ] . virus neutralizing antibodies were reported to appear later during the infection, usually when clinical signs are resolved [ , ] . the purpose of the present study was to evaluate the humoral immune response of prrsv-infected pigs. the kinetics of the appearance of specific igm and igg antibody titers during prrsv infection, in addition to viral structural protein specificities of the antibodies and duration of viremia have been investigated in pigs naturally-and experimentally-exposed to prrsv. the qu bec cytopathic strain iaf-klop of prrsv [ ] was propagated in marc- cells [ , a highly permissive cell line to prrsv kindly provided to us by j. kwang, u. s. meat animal research center, clay center, nebraska. serological identification of the cell culture-adapted iaf-klop strain was confirmed by indirect immunoftuorescence (iif) using monoclonal antibody (mab) sdowl [ ] , kindly provided by d. a. benfield and e. nelson, department of veterinary science, south dakota state university. this mab was found to be directed to an epitope of the n protein shared by both the north american and european isolates of prrsv [ , ] . homologous hyperimmune sera to the iaf-klop strain of prrsv were produced in pigs and rabbits, as previously described [ ] . in the first experiment, seven spf piglets were placed in a clinically prrsvinfected herd, whereas two similar piglets were introduced in a clinically-healthy neighboring barn [ ] . the spf pigs were selected from a farm free of atrophic rhinitis, mycoplasma hyopneumoniae, transmissible gastroenteritis virus, swine influenza virus, encephalomyocarditis virus, s. hyodysenteriae, sarcoptic mange, salmoneltosis, a. pleuropneumoniae and prrsv. in the second experiment, four five-week old spf piglets were intranasally inoculated with l s tcidso of the iaf-klop strain of prrsv. clinical signs of both the above piglets and a simultaneously mock-infected group of three pigs were monitored daily. blood samples were collected at days and p.i., then weekly during -day and day observation periods for sentinel piglets and experimentally-infected pigs, respectively. among the principal clinical signs observed in both naturally-and experimentally-infected pigs were intermittent raising of body temperature ( - °c), anorexia, lethargy, periocular oedema, conjunctivitis and blue discoloration of the ears. coughing and dyspnea (abdominal thumping) were usually noticed by the end of the first week of exposure [ , ] . no symptoms were observed in control piglets. for each group, one representative animal was necropsied at the end of the first week p.i. macroscopic and histopathological lesions of nonsuppurative interstitial pneumonitis were only demonstrated in the two infected pigs [ , ] . at the end of the -day observation period, no significant macroscopic and histologic lesions could be observed in pigs that had been infected experimentally. however, naturally-exposed sentinel pigs still manifested mild to moderate macroscopic and histologic lung lesions days p.i., as previously described [ ] . to attempt virus isolation, marc- cell monolayers were inoculated with sera samples collected from prrsv-infected pigs and monitored daily for the appearance of a cytopathic effect (cpe) or the presence of specific fluorescent foci [ ] . for all pigs tested (naturally-exposed and experimentally-infected) the virus could be isolated from sera samples collected by the end of the first week of exposure till the end of the observation periods (data not shown). at no time, could prrsv be recovered from serum samples collected from the control pigs. the iif test was carried out using acetone fixed prrsv-infected marc- cells, and fluorescein-conjugated anti-pig igm (kirkegaard & perry laboratories inc.) or anti-pig igg (sigma), as previously described [ ] . the iif antibody titers were expressed as the reciprocal of the highest serum dilution giving specific cytoplasmic fluorescence (table ). in both naturally-and experimentally-infected pigs, igm antibodies to prrsv were detected as early as days p.i. (iif titers of to ) and maximal titers were reached by day p.i. (iif titers of ). then, specific igm antibody titers drastically decreased until day or day p.i. specific igg antibodies were also detectable by the end of the first week of exposure, maximal titers being obtained around day to p.i. (iif titers of to ). thereafter, there was generally little change in igg antibody titers to prrsv of sera collected till the end of the -or -week observation period. the modified procedure described by yoon et al. [ ] was used for in vitro detection of virus neutralizing (vn) antibodies to prrsv. the vn antibody titers were expressed as the reciprocal of the highest dilution of sera that neutralized cpe induced in marc- cell monolayers by a constant dose of tcid o of virus. specific vn antibodies to prrsv could be detected in sera samples from only half of the infected piglets (data not shown). antibody titers > were not observed until - weeks p.i., with maximal titers ranging between ---. to study the reactivity of pig sera to prrsv specific proteins, immunoblotting experiments were performed using sucrose-gradient purified virus, as previously described [ ] . briefly, replicas of viral proteins, separated by sds-page and electrophoretically transferred onto nitrocellulose membranes, were incubated for h at °c in the presence of : dilution of the tested porcine sera. after washing in a . m tris-buffered saline (tbs) solution containing . % tween , the membranes were further incubated in the presence of : dilution of an alkaline phosphatase-conjugated anti-porcine igg (sigma). the immune complexes were revealed using a commercial alkaline phosphatase conjugate substrate kit (biorad, mississauga, ont.), containing nitroblue tetrazolium and -bromo- -chloro- -indolyl phosphate in tbs buffer. under reducing conditions, the three major prrsv structural proteins n ( kda), m ( kda) and e ( . kda) were consistently identified with the tissue cultureadapted iaf-klop strain of prrsv using homologous porcine hyperimmune serum [ ] . in the immunoblotting experiments with sera from both naturallyand experimentally-infected pigs (fig. ) , seroconversion to the m and n proteins of prrsv could not be demonstrated before the end of the first two weeks of infection. on the other hand, seroconversion to the e glycoprotein was generally obtained by the end of the third or fourth week p.i. viral protein specificity of the humoral immune response of infected pigs was further confirmed by western immunoblotting analyses using e.coli-expressed orfs to gene products. genomic rna was extracted from purified virus by the one-step guanidinium isothiocyanate-acid phenol method [ ] , then respective viral genes were amplified by rt-pcr, as previously described [ , ] . six oligonucleotide primers, containing ecori (sense primers) and bamhi (antisense primers) restriction sites at their '-end, were designed according to the sequence of the iaf-exp. strain of prrsv (embl/genebank accession number l ) [ ] . the pcr amplified products were purified using the geneclean ii as illustrated in fig. , with clinical sera samples no. and (naturallyinfected pigs), antibodies directed against the recombinant e protein could be detected by the end of the first week of infection, whereas antibodies directed against the recombinant n and m proteins could only be detected by the end of the second week of the prrsv infection. no reaction has been noticed with mbp protein (fig. c) and gst protein (data not shown) alone. in the present study, prrsv-infected piglets generally developed mild clinical signs of a respiratory disease within the first week post-exposure to the virus that lasted not more than to days. however, all the animals tested remained viremic till the end of the or -day observation period. our data are in agreement with previous observations by others who demonstrated that prrsv may persist for many weeks, even months in the infected pigs, despite their relatively high iif antibody titers [ , , ] . noteworthy, our results on the detection of igg antibodies to prrsv in infected pigs sera are also compatible with previous findings that specific igg antibodies can be detected by iif from - days p.i. up to several weeks (more than - months) after exposure to the virus [ , ] . in general, igg antibody titers detected by iif peaked by the end of the third or fourth week post-exposure. more recently, nelson et al. [ ] demonstrated that peaked igg antibody titers to prrsv (iif titers > ) may persist in experimentally-infected pigs for more than months, then decrease progressively to reach very low levels (iif titers < ) after more than days p.i. consequently, detection of igg antibodies in pig sera may indicate that the animals have been infected by prrsv in a recent or distant past. since no correlation could be demonstrated between the levels of igg antibody titers determined by iif and the viremic status of the animals, one cannot precisely pin point when the exposure to the virus had occurred or whether the pigs had been carrying the virus for a long time. interestingly, our results indicate that detection by iif of igm antibodies to prrsv may provide more precise information in the serological diagnosis of prrsv infection. indeed, the short-term persistence of circulating specific igm antibodies may be considered in the differentiation between acute and chronic prrsv infections in pigs. also in agreement with previous findings by other authors, antibodies with in vitro vn activity were relatively slow to appear and were not detected until - weeks p.i. [ , , ] . although the vn test has been reported to be less sensitive than the iif tests and immunoblotting [ ] , the long term persistence of prrsv in the experimentally-or naturally-infected animals despite high levels of antibodies to prrsv, raises the question as to what role the humoral immune response has in the protection to prrsv infection. also, it is possible for antibodies to enhance viral infection of fc-receptor-positive cells such as macrophages, by forming immune complexes that use the fc receptor to bind to the macrophages [ ] . thus, the putative protective role of neutralizing antibodies in prrsv infection needs to be further documented. western blotting analyses demonstrated that the immune response of experimentally-and naturally-infected pigs was directed to the three major viral structural proteins n, m and e, as previously reported in cases of pigs that have been experimentally-infected with the reference atcc-vr us strain [ ] . these preliminary observations were further confirmed by testing the reactivity of the porcine anti-prrsv sera against e. coli-expressed orfs to gene products. in light of our observations, it appears that antibody development to the various viral proteins in prrsv-infected pigs is chronological, initial detection of antibodies to the n, m and e proteins varied among pigs, ranging from to days p.i. these results are in agreement with previous findings by others [ , ] . however, it has been postulated that the stronger signal observed to the n and m proteins may reflect a higher molar ratio of these proteins in the virion relative to the e protein, or it may suggest a greater immunogenicity of these proteins in the pigs [ , ] . the results obtained using e. coil-expressed orfs to are in favor of the first hypothesis. indeed, antibodies to the recombinant mbp-e protein could be demonstrated as early as days pi, whereas antibodies to the n and m proteins could only be detected by the end of the second week p.i. this finding was not expected in case of the recombinant mbp-e protein since reactivity towards the viral envelope glycoprotein in immunoblotting experiments, using purified virus, could not be detected until the end of the third or fourth week p.i. this discrepancy may be due to the fact that when dealing with recombinant proteins, comparable amounts of the various proteins could be transferred onto the nitrocellulose membranes, whereas with purified viral preparations, the n and m proteins are expected to be present at a higher molar ratio comparative to the e glycoprotein [ , ] . indeed, in the case of equine arteritis virus it has been demonstrated that with the exception of gs (small envelope glycoprotein), the proteins are approximately equal in abundance, being present at a molecular ratio of (n): (m): (gi: large envelope glycoprotein) [ ] . the gs protein, which is expressed at a level similar to that of m in infected cells, is strikingly underrepresented in virus particles ( to %) [ ] . thus, it can be expected that for prrsv, the immunogenicity of the e glycoprotein in the pig is likely comparable to that of the n and m proteins. from results obtained with recombinant proteins, as well as with purified virus, there was no clear correlation between the appearance of circulating antibodies as detected by vn and iif tests, viremia and protein specificities of the circulating antibodies after various time p.i. the neutralization process may be the result of virus interaction with antibodies directed against epitopes located on different viral proteins. recently, we have been able to demonstrate that individually recombinant n, m and e proteins expressed by e. coli cells can induce the production of specific antibodies in rabbits and pigs; although iif antibody titers to iaf-klop strain ranged between to , the antisera are devoid of neutralization activity. expression of recombinant viral proteins in eucaryotic vectors should permit us to demonstrate if the conformation and glycosylation of viral envelope proteins are essential requirements for the expression of epitopes involved in virus neutralization. comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation antibody-dependent enhancement of sirs virus replication single-step method of rna isolation by acid guanidium thiocyanate-phenol-chloroform extraction molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group structural proteins of equine arteritis virus porcine reproductive and respiratory syndrome enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line antigenic comparison of canadian and u.s. isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to the nucleocapsid protein porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of quebec isolates associated to acute and chronic outbreaks of prrs detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between canadian and european strains by reverse transcription and pcr amplification molecular analysis of the orfs to genes of porcine reproductive and respiratory syndrome virus, quebec strain iaf-exp kinetics of humoral immune response to structural proteins of prrsv diagnosis of porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav characterization of proteins encoded by orfs to of lelystad virus differentiation of us and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus antibody production and blastogenic response in pigs experimentally infected with prrs virus mystery swine disease in the netherlands: the isolation of lelystad virus morrison rb (t ) an indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera persistent and contact infection in nursery pigs experimentally infected with porcine reproductive and respiratory syndrome virus characterization of the humoral immune response to porcine reproductive and respiratory syndrome (prrs) virus infection we thank louise wilson, micheline ch nard, and nicole sawyer tbr their technical assistance. this work was supported in part by grants received from the national research council of canada (strategic grant # strgp- ), the minist re de t'agriculture, des p~cheries et de l'alimentation du qu bec, la f d ration des producteurs de porcs du quebec, and vetrepharm research inc., london, ontario, canada. received august , key: cord- - pm l d authors: quinteros, daniela a.; bermúdez, josé m.; ravetti, soledad; cid, alicia; allemandi, daniel a.; palma, santiago d. title: therapeutic use of monoclonal antibodies: general aspects and challenges for drug delivery date: - - journal: nanostructures for drug delivery doi: . /b - - - - . - sha: doc_id: cord_uid: pm l d monoclonal antibodies are routinely used in several fields but the great challenge has been their use as therapeutic agents for the treatment of diseases, such as breast cancer, leukemia, asthma, macular degeneration, arthritis, crohn’s disease, and transplants, among others. monoclonal antibodies are protein molecules made in the laboratory from hybridoma cells by recombinant dna technology. important advances have been made over the past decade to improve some critical points, such as safety and efficacy of the first generation of therapeutic antibodies. this type of molecules presents a significant challenge from the pharmaceutical point of view due to their characteristics, such as molecular size, stability, and solubility. in this chapter we have attempted to identify the major issues associated with therapeutic approaches, formulating drawbacks and delivering antibody drugs, particularly focused on the challenges and opportunities that these present for the future. mabs are indispensable not only to health, but also to prevent food poisoning, and are used to investigate environmental pollution. however, despite their widespread distribution and significance, most people have never heard of mabs or how they have both transformed healthcare and spawned an entire new industry. produced in the laboratory, mabs are derived from the billions of tiny antibodies made every day by our immune systems to combat substances, known as antigens, which are regarded as foreign or potentially dangerous. millions of different types of antibodies can be found in the blood of humans and other mammals. made by white blood cells known as b lymphocytes, each antibody is highly specific-that is, it has the ability to bind to only one particular antigen, which may be derived from bacteria, viruses, fungi, parasites, pollen, or nonliving substances, such as toxins, chemicals, drugs, or foreign particles considered alien to the body. once antibodies have marked their antigen, they and other types of cells produced by the immune system can attack it. the field of genetically engineered therapeutic mabs has relied on many inventions during decades of research, but two key discoveries in the mid- s stand out as seminal events that laid the groundwork for this field to exist as we know it today. although mabs were first described in (kohler and milstein, ) , only when the original rodents forms were replaced by their human equivalents did their potential as therapeutic agents began to be properly appreciated (lonberg, ; winter et al., ) . the reasons for this are complex, but related to a combination of perceptions including patentability, immunogenicity, effector function, and a wish to avoid undesirable side effects. the increasing scientific interest on mabs can be clearly seen in fig. . , which shows the rising number of published articles using the keyword monoclonal antibodies in different databases (pubmed and scopus). athough fewer published articles, the number of publications is constant over the years to science direct and wiley. general introduction mabs are used not only as drugs for treating various diseases, but are also used as powerful tools for a wide range of medical applications. they are routinely used in hospitals for blood type and tissue, a vital process to ensure safe blood transfusion and organ transplantation. in other cases, they are employed as research probes to determine the pathological pathway and the cause of diseases, such as cancer, autoimmune diseases, and neurological disorders. on the diagnostic front, monoclonal antibodies are intrinsic components of test kits for the detection of ovulation, pregnancy, or menopause. they are also used for analyzing body fluids for medical diagnosis, and to determine whether there has been a heart attack. unlike polyclonal antibodies, mabs are identical antibodies because they are produced by one type of immune cell. using current hybridoma (mouse/human hybrid cells) technology originally developed by georges kohler, cesar milstein, and neils jerne, mabs can be produced to bind tightly to virtually any material or antigen, which is defined as a substance that prompts the generation of antibodies that specifically bind to it. antigens typically consist of proteins or polysaccharides. epitopes, also known as antigenic determinants, are the part of the antigens through which actual binding to antibodies occurs. the mabs technology allowed scientists to produce huge quantities of pure antibodies aimed at specific selected targets, leading to the design of new diagnostic tests and therapeutics. by injecting a payload of mabs into the bloodstream, the antibodies were headed straight to their disease target. today, the growth and profitability of mabs are outstripping those of earlier types of biotechnology drugs and more traditional pharmaceutical ones. indeed, their expansion is among the fastest in the global pharmaceutical world. in part, this reflects the sector's embrace of mabs as an answer to dwindling drugs in the pipeline and reduced revenue streams in the face of the expiration of key patents and the growth of generic medicines. drugs based on mabs technology have names ending in "mab." the first drug that received food and drug administration (fda) approval was rituxan (rituximab) (fig. . ) for the treatment of non-hodgkin's lymphoma, in . it was developed by idec, and it has since merged with biogen. rituximab is a mab against the protein cd , which is primarily found on the surface of immune system b cells. rituximab destroys b cells and is therefore used to treat diseases that are characterized by excessive numbers of b cells, overactive b cells, or dysfunctional b cells. this includes many lymphomas, leukemias, transplant rejection, and autoimmune disorders. idec teamed up with genentech to get fda approval and to comarket rituxan. in parallel, genentech developed herceptin (trastuzumab) for the treatment of breast cancer associated with the her /neu receptor. herceptin was approved by the fda in and is the first important example of personalized medicine requiring a diagnostic test to ensure efficacy, since only patients who express the her gene in their breast cancer tissue have a chance to benefit from treatment with this mab. today, six therapeutic mabs are among the best-selling drugs in the world. in it was estimated that the global sales of the more successful mabs were worth more than $ . billion. among these, adalimumab (humira) is used for the treatment of rheumatoid arthritis and other autoimmune diseases and was at the top of the selling drug list across the world in , reaching an annual income of $ . million. thus, mab sales are expected to be higher than those for lipitor, used for cholesterol reduction, which has been the largest prescribed drug historically (king, ) . since the s, the methodologies available for generating mabs have dramatically evolved from the hybridoma technology used to produce murine mabs (kohler and milstein, ) to sophisticated recombinant engineering technologies (brüggemann et al., a,b; steinitz et al., ) appropriate to design specific mabs of choice, thus producing humanized, chimeric, and completely (fully) human mabs (fig. . ) (brüggemann et al., a,b; jones et al., ; lonberg, ; riechmann et al., ) . these novel methods have had major consequences in the development of clinically applicable mabs against disease targets. the immunogenicity and safety of mabs, a substantial limitation of antibodies produced by the hybridoma technology, could be overcome, and mabs against any antigen specificity and with desirable physicochemical properties could be developed. high-scale production of mabs at the industrial level could further be achieved. this has sparked a race for the development of antibody-mediated therapeutics in the clinic with the investment of large amounts of money from the biopharmaceutical sector toward that end, which is already paying off. the major success of human mabs in immune and inflammatory conditions, such as rheumatoid arthritis (ra), crohn's disease, ulcerative colitis, spondyloarthropathies, juvenile arthritis, psoriasis, psoriatic arthritis, and others (andreakos et al., ) , marks merely the beginning of a rapidly evolving field with more than diverse mabs currently being evaluated in clinical trials or being candidates for approval by the fda in the usa. scaling up mabs production for therapeutics posed several challenges. the major issue was how to produce high quantities of drugs at a reasonable cost. most cell lines in the s yielded only half a gram of mabs per liter, so production was time consuming and expensive. the ideal was to develop figure . monoclonal antibody rituximab antibody-drug conjugates a hybrid cell line that could produce between and g of mabs per liter. this demanded several steps, and even more, each requiring skill and patience. first was the creation of a hybrid cell, after which a clone secreting mabs in high concentrations had to be selected. then a culture medium had to be developed to encourage the optimal growth of the hybridoma. scaling up such media was not easy in terms of quality control because they contained fifty or more ingredients, and it was important to determine how many nutrients to add. hybridomas stop secreting mabs, for example, if given too much glucose. antibody-drug conjugates (adcs) are mabs bearing cytotoxic drugs covalently bound via a chemical linker and can be defined as prodrugs. prodrugs are inactive or less active derivatives of drug molecules and undergo enzymatic or chemical transformation to regenerate the active forms. adcs are designed to be superior to either antibody therapeutics or chemotherapy alone by overcoming their limitations while preserving the merits from both. the antibody connected to the cytotoxic warhead (drug) via a linker serves as targeted delivery system to the tumor expressing the antigen/target recognized by the antibody. ideally, in blood, after systemic administration, this prodrug is nontoxic. upon binding of the antibody to the targeted tumor antigen and internalization of the complex into the cancer cell, the drug is then released in its active form and in sufficient quantity to kill the cell. on top of the careful choice of a target/antigen expressed in specific tumor indication, it requires finding the best combination between the antibody, the linker, and the drug, which, besides its own characteristics and constraints, are linked and impact each other (bander, ) . the combination of nanoparticles and antibodies can offer versatility together with specificity ( fig. . ) , allowing a huge potential market. the target/antigen is the starting point to build an adc. it first determines which tumor indication will be targeted by the adc and potentially impacts the choice of the conjugated drug. in addition, the target will also drive the criteria that will be defined for the selection of the targeted patient population within the tumor indication. mabs are typically susceptible to noncovalent aggregation upon storage. aggregation is driven by the minimization of free energy achieved when antibodies come into close contact-for instance, at relatively hydrophobic sites on the molecules. dimerization may be thought of as the first step in aggregation, and sometimes the process proceeds no further. dimers maybe reversible (moore et al., ) or not, but growth can lead to larger and larger oligomers and even potentially insoluble particulates, as time progresses (wang et al., ) . because adcs are decorated with one or more small molecule drug moieties, they have a different set of biophysical properties compared with the unconjugated antibody, leading to new or altered intermolecular interactions. an antibody that exhibits acceptable figure . conjugation of nanoparticles to antibodies and their advantages antibody-drug conjugates aggregation behavior in its unconjugated form may have different physical behavior in its conjugated form, either because of changes in surface properties, such as hydrophobicity due to drug attachment or because the drugs have altered the higher-order structure of the antibody such that new modes of antibody-antibody interactions are possible. these factors could contribute to a substantially different propensity for aggregation. this effect is most readily observed in a formulation screen designed to test both conjugated and unconjugated forms in parallel and subjected to the same assays. such a study does not automatically reveal the exact cause of the effect, only that it is related to the conjugated form or conjugation process. the degree to which the molecule has been physically altered by conjugation may be assessed by techniques such as differential scanning calorimetry (dsc) (wakankar et al., ) , with physical perturbations manifested as changes (likely decreases) in the onset of melting or melting temperature, tm, of the conjugated mab as compared to the unconjugated antibody. this decrease in thermodynamic stability may translate to a decreased colloidal stability of the conjugate compared to an unconjugated antibody. part of adc chemical stability is inherited from the unconjugated antibody. for instance, if the unconjugated antibody is susceptible to deamidation or isomerization with known ph dependence, this degradation mode is also likely to be present in the adc. these types of degradations may impact the product potency, especially if the affected residues are found in the complementarity-determining region. much has been learned through years of experience with unconjugated antibodies about the susceptibility of various amino acid residues to chemical degradations, allowing substantial insights to be gained from examination of the protein primary sequence alone, coupled to available information about local solvent exposure and flexibility (kosky et al., ; robinson and robinson, ) . it may be expected that most chemical modifications to the primary sequence of the unconjugated antibody will also occur in the adc form. fragmentation is another possible chemical degradation pathway for adcs, driven by the breakage of covalent bonds between chains or within the peptide backbone. the altered physical state of the molecule following addition of the small molecule drug may result in some differences in the susceptibility of the adc to fragmentation, but the fundamental susceptibility of the adc to fragmentation is likely derived mostly from the mab. a common unconjugated antibody degradation mode involves breaking the heavy chain peptide near the hinge region, leading to free fab and fab + fc products (cordoba et al., ) . this is still a feasible degradation pathway for adcs as well. comparison of fragmentation rates between conjugated and unconjugated antibodies can reveal whether the adc form has different stability toward fragmentation. the covalent bonds linking drugs to antibodies may be broken by a variety of mechanisms over the shelf life of the product and under stressed storage conditions. formulation development of adcs aims to ensure that stable, high-quality products are dosed to patients. though this goal is common to all pharmaceutical formulations, adcs present a unique set of physicochemical properties that can impact safety, quality, and efficacy as compared to traditional therapeutic proteins. although there is a body of literature that outlines which product attributes affect the safety, quality, and efficacy for conventional, unconjugated mabs (goetze et al., ) , an understanding of attributes important to adcs is only now emerging. the choice of formulation has the potential to affect all categories of adcs quality attributes. because conjugation of an adc requires an unconjugated antibody as an intermediate, the formulation development process for a conjugate necessarily involves formulating a mab at some stage, likely as an intermediate for manufacturing of the final adc form. the formulation optimization for the antibody portion of the adc follows a similar course to that of an unconjugated antibody and, therefore, can leverage the biotechnology industry's considerable experience developing this class of molecule. many examples of successful commercial antibody formulations, both liquid and lyophilized, have been reported (wang et al., ) . however, the complexity of the formulation-development process potentially increases when quality attributes of the small molecule drug and those of the conjugated form are taken into account. the antibody-conjugated nanoparticles can be used principally in two biomedical applications: diagnosis and therapy. in therapy, the development of targeted drug delivery represents, together with tissue repair, the main applications of antibody-conjugated nanoparticles. in diagnosis, the applications can be divided into those using in vivo and those using in vitro experimentation and include contrast agents for magnetic resonance imaging (mri), sensing, cell sorting, bioseparation, enzyme immobilization, immunoassays, transfection (gene delivery), and purification. today, one of the most interesting challenges posed by drug-delivery systems is the development of smart vectors, which are required to be safe, easily administered, and economic, and which allow simultaneous diagnosis and treatment. moreover, it is intended that the delivery is precisely controlled in term of dose and site of action to reduce adverse side effects. in this regard, there are many important site-selective drugs, such as highly toxic antitumor molecules, that must reach targeted cells or tissues without being released before. nanoscience and nanotechnology have found their way into the fields of biotechnology and medicine. nanoscience is the study of structures and materials on an atomic and molecular scale, in a level that ranges usually below nm. nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. antibodies are nanosize biological products that are part of the specific immune system. in addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc.), they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc.) and be used in several diagnostic procedures, or even in therapy to destroy a specific target. the conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. since the properties of materials differ significantly between atomic or subatomic level and larger scales, nanomaterials have attracted the attention of researchers for pharmaceutical application, especially in the area of drug delivery. this technology overcomes some of the drawbacks of large-size materials, such as poor bioavailability, in vivo stability, solubility, intestinal absorption, sustained and targeted delivery to site of action, and generalized side effects, among others. in this regard, nanostructures have been reported to protect drugs from the degradation in the gastrointestinal tract, and even more, to provide means of bypassing the liver, preventing the first pass metabolism. this technology also has the ability to target delivery of drugs to various areas of the body, and enables the delivery of drugs that are poorly water-soluble. moreover, nanotechnology improves drugs bioavailability and allows them to remain circulating in the blood for a long time, controlling their release. nanostructures can also be able to penetrate tissues and be easily taken up by cells, allowing for efficient delivery of drugs to target sites of action. nanostructures were reported to be taken up by the cells at rates - times greater than microparticles. nanotechnology improves performance and acceptability of dosage forms and even may enhance the performance of drugs that fail in clinical trial phases. nanotechnology will definitely revolutionize the science of drug delivery and help overcome the major challenges of conventional drugs used for the treatment and management of chronic diseases such as, cancer, asthma, hypertension, hiv, and diabetes. in this context, nanomedicine can be defined as the medical application of nanotechnology and involves research, and diagnostic and therapeutic applications related with nanotechnology. nanotechnology has been applied in medicine to develop strategies to manage, treat, or even cure virtually every type of human disease. mabs are not only successful drugs but also powerful tools for a wide range of medical applications. in this regard, over the last years several nanotechnological platforms for medical applications have converged upon a specialization toward diagnosis, therapy or even both, as in the case of magnetic nanoparticles. the study of nanoscience and the optimization of technologies, such as adcs and nanocarriers have driven the design of methodologies that are tailored to address specific diseases and medical anomalies. adcs, for example, represent excellent nanoscale-based drug delivery vehicles and thus tend to be focused on delivery to certain drugs, as in the treatment of cancer. the conjugation of different moieties to the nanoparticles widens their application fields and provides them with new or enhanced properties. a range of biomoieties can be conjugated to the nanoparticles including low molecular weight ligands (folic acid, thiamine, dimercaptosuccinic acid), peptides (rgd, lhrd, antigenic peptides, internalization peptides), proteins (bsa, transferrin, antibodies, lectins, cytokines, fibrinogen, thrombin), polysaccharides (hyaluronic acid, chitosan, dextran, oligosaccharides, heparin), polyunsaturated fatty acids (palmitic acid, phospholipids), dna, and plasmids. nanotemplates provide unique access to extremely high-throughput capabilities for rapidly defining the presence of a biomarker and thus lend themselves as excellent platforms for diagnostics applications. rapid progress is being made to drive these advances in nanomedicine, especially in the area of cancer treatment. although still in its infancy when compared to other medical technologies, nanomedicine is undoubtedly here to stay and will have an enormous impact on the future health and well-being of man. nanocarriers are nanometer-sized materials that have the capacity to deliver therapeutic agents at the disease site (arias, ; schroeder et al., ) . they are designed to possess unique physicochemical properties, aiming to improve the pharmacokinetic and biodistribution of a drug molecule (li and huang, ) and to deliver a significant amount of drug molecules. examples of some therapeutic nanocarriers are lipid-based particles (al-jamal and kostarelos, ), micelles (kedar et al., ) , nanoparticles (shi et al., ) , dendrimers (svenson, ) , and polymersomes (levine et al., ) . some of these have been proposed for the treatment of various diseases including cancer (dhar et al., ) , coronary artery diseases (chan et al., ) , and rheumatoid arthritis (thomas et al., ) . in the particular case of cancer, the unique anatomy-that is, the leakiness of the tumoral vasculatures-concedes a passive transport of the nanocarriers by enhanced permeability and retention (epr) effect (maeda et al., ) . however, the porosity of the tumor blood vessels may vary with the tumor type (bae, ) . even with a successful delivery by the epr effect, the nanocarriers must be able to internalize into the cancer cells (gradishar et al., ) . a new paradigm in drug delivery embroils a combination of active and passive targeting. targeting ligands, such as antibodies (mamot et al., ) , peptides (srinivasan et al., ) , small molecules (kukowska-latallo et al., ) , or aptamers (farokhzad et al., ) can be attached onto the surface of a nanocarrier. the carriers recognize and bind to the cell-surface receptors and are subsequently taken by the cells via receptor-mediated endocytosis for releasing the therapeutic payloads (shi et al., ) . the binding affinity of targeted nanocarriers can also be increased several orders of magnitude by the multivalent effect . among all the targeting ligands, antibodies are well known for their high binding affinity, specificity, and availability for a number of disease biomarkers (manjappa et al., ). an antibody can be simply absorbed on the surface of a nanocarrier via hydrophobic and/or electrostatic interaction (sokolov et al., ) . however, using this approach, the absorbed antibody may orient randomly on the surface and result in losing the binding affinity. furthermore, the antibody may exchange with other endogenous protein in vivo (nobs et al., ) . therefore, antibodies are generally preferred to attach to the nanocarriers covalently (arruebo et al., ). an antibody consists of a number of functional groups that provides many options for bioconjugation (manjappa et al., ) . bioconjugation can take place by means of adsorption (at the isoelectrical point of the antibody via electrostatic interaction), by direct covalent linkage between the surface of the nanoparticle and the antibody, or by using adapter molecules. the use of adapter molecules generally involves streptavidin and biotin for the formation of the complex. biotin-labeled polyclonal goat anti-escherichia coli antibodies have been attached to streptavidin-coated magnetic nanoparticles, and used for the separation and selective quantification of escherichia coli o :h in ground beef in the presence of other bacteria (varshney et al., ) . mabs are capable of specifically recognizing and binding to many molecules, mainly those of protein nature. this capability have been widely used in the field of diagnosis for the detection of hormones, vitamins, cytokines, allergens, numerous tumor markers, and a wide range of markers associated with many diseases, including microbial infections. for these properties, the mabs are the substances most commonly used in the field of clinical diagnosis and in identifying markers and therapeutic targets. besides applications in disease diagnosis and in drug discovery, therapeutic applications include mabs, such as vehicles or carriers of other drugs. current major therapeutic applications of mabs include cancer, chronic inflammatory disease, and infection, and they constitute the largest and fastest growing sector of the biological pharmaceutical industry. in addition to offering a host of new drugs to fight disease, mabs have provided the means to monitor a patient's response to therapy and helped lead the way in personalized medicine. mabs are now marketed not only for cancer and autoimmune disorders, but also for a range of other diseases, including allergic conditions, such as asthma, age-related macular degeneration (an eye disorder), multiple sclerosis (a neurological disorder), and osteoporosis (brittle bones). they are also being investigated for central nervous system disorders, such as alzheimer's disease (a degenerative brain disease), metabolic diseases such as diabetes, and the prevention of migraines. mabs used for therapeutic purposes may bind to a variety of antigens present on the surface of tumor and cancer cells. they may also be utilized to deliver a number of different types of payloads to destroy the targeted cells in question including radioactive ligands, cytokines, toxins, liposomes containing drugs, and specific killer cell types. these immunoconjugates, which are defined as antibodies linked to a second molecule, such as a toxin, radioisotope, or label may act at the cell surface as killing agents or be internalized to release payloads intracellularly. despite their high sensitivity and reproducibility, conventional diagnostic methods for a microbial infection require cumbersome sample preparation and long readout times (kaittanis et al., ) . unique electrical, magnetic, luminescent, and catalytic properties of nanomaterials enable fast, sensitive, and cost-effective diagnosis, as well as rapid determination of the susceptibility and resistance of antibacterial drugs (bruchez et al., ; edgar et al., ) . antibody-conjugated nanoparticles amplify the signals for bioanalysis and enumeration of highly pathogenic bacteria, such as e. coli o :h , resulting in highly selective, convenient, and rapid detection of single bacterium within min (look et al., ) . the rapid and sensitive determination of pathogenic bacteria is extremely important in biotechnology and medical diagnosis. current methods either lack ultrasensitivity or take a long time for analysis. the use of magnetic nanoparticles also could be a very sensitive and rapid strategy to detect microbial infection. magnetic nanoparticles, in particular superparamagnetic iron oxide nanoparticles (spion) with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications, such as magnetic resonance imaging contrast enhancement, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, drug delivery, in cell separation, and so forth (neuberger et al., ) . dextrancoated supermagnetic iron oxide nanoparticles were clustered by con-a treatment, or equipped with con a-conjugated nanosensors (zhao et al., ) . supermagnetic iron oxide nanoprobes greatly assisted the identification of mycobacterium avium spp. paratuberculosis, as well as the quick quantification of these bacteria in milk and blood with high sensitivity (basu et al., ) . a quick method for detecting infections in the urinary tract has also been developed using gold nano-wire arrays in conjunction with a linker arm attached to specific e. coli antibodies (de la escosura-muñiz and merkoçi, ). li and huang ( ) have shown that nanoparticles with specific raman spectroscopic fingerprints can distinguish antibiotic resistant bacteria, by detecting single-nucleotide polymorphisms in microarray-based systems . a bioassay based on bioconjugated nanoparticle was developed by zhao et al. ( ) for in situ pathogen quantification. the detection is carried out by a high fluorescent signal provided by the nanoparticle, which is easily attached to an antibody or other biorecognition molecule. these conjugated nanoparticles can be used in the specific identification of a wide range of bacteria, such as e. coli o :h , through antibody antigen interaction. these nanoparticles were able to assess the microbial metabolic activity and determine antimicrobial susceptibility in blood, by rapidly quantifying polysaccharides. the broad absorption spectra of quantum dots (qds) can be exploited to simultaneously excite qds emitting different colors using a single wavelength (cao et al., ) . these characters suggest that qds are a promising modality for the analysis of complex samples for histology, pathology and cytology, and can facilitate double or even triple immunostaining of bacterial cells (kaittanis et al., ) . studies have demonstrated that the use of nanotechnology feasibility of achieving fast and reliable pharmaceutical assays for microbial infections in opaque media (e.g., whole blood and milk), without any sample preparations (alper et al., ; tully et al., ) . the ability to track the distribution and differentiation of progenitor and stem cells by high resolution in vivo imaging techniques would have significant clinical and research implications. lewin et al. ( ) developed a cell labeling approach using short hiv-tat peptides to derivatize superparamagnetic nanoparticles. these particles were efficiently internalized into hematopoietic and neural progenitor cells in quantities up to - pg of superparamagnetic iron per cell. iron incorporation did not affect cell viability, differentiation, or proliferation of cd + cells. following intravenous injection into immunodeficient mice, % of magnetically cd + cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance imaging in tissue samples. in addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells (lewin et al., ) . nanomaterials with fluorescent properties or nanoparticles labeled/encapsulated with fluorescent dyes have also been applied for microbial detection. using antibody-conjugated silica nanoparticles, these materials can be used as a superior signaling element, to detect cells, proteins and bacteria in an immunoassay (grifantini et al., ; tan et al., ) . ultrasensitive methods for bioassays have been developed using fluorescent bioconjugated silica nanoparticles (santra et al., ; zhao et al., ) . thousands of fluorescent dye molecules are encapsulated in a protective silica matrix, resulting in a nanoparticle with an amplified and reproducible signal for fluorescence-based bioanalysis. whereas in conventional immunoassays only one or a few dye molecules are linked to an antibody molecule and then used to signal an antibody-antigen interaction event, the bioconjugated nanoparticles, which are attached to the antibody molecule, are enabled to carry many dye molecules inside, allowing a significant amplification of the analytical signal. furthermore, bacteria present many surface antigens available for antibody recognition, and, therefore, a greatly amplified signal can be achieved since thousands of nanoparticles can attach to each bacterium. these bioconjugated nanoparticles allow the detection of a bacterium cell per given sample just in min with a spectrofluorometer. santra and coworkers used covalent method to attach surface-modified, rubpy-doped silica nanoparticles to mouse antihuman cd antibody. this complex was then incubated with mononuclear lymphoid target cells. after washing away the unbound nanoparticles, target leukemia cells could be clearly detected. in comparison to control group, results of this experiment have shown that this technique was very effective to detect leukemia cells selectively (santra et al., ) . tan and coworkers developed an assay tool for in situ detection of single bacterium cells in less than min and developed multicolored fret (fluorescence resonance energy transfer) silica nanoparticles by coencapsulating three tandem dyes that emit unique colors upon excitation with a single wavelength . among therapeutic applications of antibody-conjugated nanoparticles, the use of gold nanoparticles was tested in killing staphyloccocus aureus (zharov et al., ) . the bacteria were killed by light-adsorbing gold nanoparticles conjugated to antiprotein a antibodies, using laser irradiation at nm. protein a was chosen because it interacts specifically with the fc fragment of the antibody. according to the authors, killing efficiency depends on the local overheating effects accompanied by bubble-formation phenomena. direct irradiation of bacteria with the laser did not damage the bacteria, because of low absorption by natural endogenous cytochromes. the diagnosis of cancer at early stages of growth is a critical factor for obtaining optimal results in therapy and for improving the chances of survival. several imaging techniques collaborate with physicians in the diagnosis, including magnetic resonance imaging (mri), positron emission tomography (pet), computed tomography (ct), ultrasound, radiography, photoacoustic imaging, fluoroscopy, and so forth. in some of these techniques, antibodyconjugated nanoparticles may offer increased selectivity and sensitivity. fluorescent semiconductor nanocrystal qds are a novel class of multifunctional inorganic fluorophores that are promising in biological imaging, including immunofluorescence imaging (wu, ) , and are useful for labeling molecules (lidke et al., ; wu, ) and cells in various materials (gao et al., ; jaiswal et al., ; kim, ) , even clinical human samples (tholouli et al., ) . the first generation of adcs presented limitations in relation to the linker instability because the early linkers were either too stable, resulting in low potency and reduced efficacy, or too unstable, resulting in poor targeting and high systemic toxicity. furthermore, cytotoxics without sufficient potency, inefficient internalization, limited expression of the target antigen and immunogenicity of mabs (alley et al., ; chari, ) . second-generation adcs were designed to deliver potent anticancer agents to tumors in a targeted manner to limit systemic exposure. the success of targeted adcs depends on antibody selection, potency of the cytotoxin and the method selected to link the antibody to the cytotoxin. nanotechnology is a disruptive technology that drives a new generation of cancer preventive diagnosis, and therapeutic products, resulting in dramatically improved cancer outcomes. nanotechnology in the field of cancer has the potential to improve the monitoring of therapeutic efficacy, provide novel methods for the detection and profiling of cancers at early stages, and allow surgeons to delineate tumor margins and sentinel lymph nodes. nanomaterials have unique features that are attractive and can be applied to biosensing. the development of various nanomaterials and nanotechnology has enabled detection of cancer biomarkers with great precision and sensitivity that could not be achieved before. many studies are being conducted on developing sensing mechanisms that will push down the detection limit as far down as possible (anajwala et al., ) . ligand-free nano-formulations generally possess passive targeting property with little tissue specificity. to address these limitations, research has been continued to advance active or specific targeting to enhance the efficacy of anticancer therapeutic agents, as well as to reduce the toxicity to nontargeted healthy tissues. nanoparticles containing the chemotherapeutic agents are specifically designed to target the cancerous cells either by ligand receptor interaction or antibody-antigen recognition. highly specific mabs are used to strengthen the immune response and to intensify the immune system's antitumor capacity. moreover, mabs are highly specific when attached to nanoparticles to aid in targeted delivery of various antitumor cytotoxic agents or function themselves as effective therapeutic agents (sutradhar and amin, ) . there are different ways of destroying cancer cells with mabs: directly by inducing apoptosis, blocking growth factor receptors or inducing the formation of antiidiotype, and indirectly by activating complement-mediated cellular cytotoxicity and antibody dependent cell-mediated cytotoxicity . the paramagnetic properties of iron-oxide nanoparticles have been harnessed for therapeutic and imaging applications (mccarthy et al., ) . chen et al. ( ) have reported that dextran-coated iron-oxide nanoparticles conjugated to radiolabeled (iodine isotope , i) anti-vegf mab significantly increased imaging resolution, as well as destruction of liver cancer in mice (chen et al., ) . srinivasan, lakshmikuttyamma, and shoyele (srinivasan et al., ) studied selective targeting of mabs to oncoproteins in cancer cells while avoiding their accumulation in normal cells. results of fluorescence microscopy, tem, and flow cytometry revealed that bevacizumab nanoparticles were internalized by a cells times more than by mrc- cells. macropinocytosis and energy-dependent pathways were elucidated to be involved in their uptake by a cells. this study presents the first evidence that uncoated mab nanoparticles can be selectively delivered to cancer cells while avoiding normal cells. mabs have been also employed as an effective active targeted therapy since these antibodies can specifically recognize the over-expressed her positive tumor cells and internalize through receptor mediated endocytosis. to treat her positive breast cancer, anti-her humanized mabs are commonly used, although advances can be made in targeted cellular localization via conjugation strategy through a nano-particulate system focusing on surface modified ligand/receptor-mediated nano-therapy to target the tumor cell at the molecular level. various nanoparticle systems, such as liposomes, micelles, and dendrimers, have been studied not only in vitro but also in vivo, concluding that the presence of receptor targeting antibody on the nanocarriers demonstrated a higher toxicity than the nano-carriers without an antibody after time dependent incubation (hamidreza montazeri et al., ; matsumura, ; yokoyama, ) . antibodies against cell surface biomarkers are the commonly used ligands for the development of targeted nanoparticles. although mouse mabs have been used for making targeted nanoparticles, strong cross-species immune responses limit their potential for future clinical translation. currently only a few types of humanized mabs, such as her- antibody (herceptin), are available for the production of targeted nanoparticles (slamon and pegram, ) . alternatively, high affinity recombinant antibody fragments have been developed as targeting ligands (yang et al., ) . for example, a human single chain antibody against the epidermal growth factor receptor (scfvegfr) that is highly expressed in the majority of epithelial tumors was conjugated to different types of nanoparticles. specificity of tumor imaging and targeted therapeutic effects of these nanoparticles have been demonstrated in several animal tumor models (yang et al., ) . the major advantages of using natural ligands for tumor targeting are their high binding affinity, specificity, and, most importantly, low immunogenicity. owen et al. ( ) reported higher antibody-conjugated lysosomal p (la-co-tmcc)-g-peg-furan micellar concentration in her positive cell lines with significant cellular distribution resulting in two-to threefold increase in lysosomal accumulation compared to using the antibody alone (owen et al., ) . in addition, nobs et al. ( ) reported that thiol functionalized anti-her mab can be conjugated with the maleimide containing liposomes. internalization of these antibody coupled immunoliposomes by her positive breast cancer cell line (sk-br- ) was observed with enhanced effects compared to the control liposomes (nobs et al., ) . many authors observed that the use of lisposomes was more effective in vitro than in vivo. to prove targeted drug-loaded liposome as an effective and safe treatment option for her positive breast cancer, more in vivo studies need to be conducted. when used, anti-her mab conjugated with dendrimers resulted in the effective delivery to target the overexpressed her receptors of tumor cells in animal models. more recently, miyano et al. ( ) developed biocompatible sixth generation (g ) anionic lysine (amino acid) dendrimer, on which the surface was modified with glutamate and, thereafter the glutamate modified g lysine dendrimer was coupled with trastuzumab mab and a fluorescent label. following this, the whole conjugated formulation was evaluated in both her positive and negative cell lines to assess the targeting efficiency and cellular internalization compared to the free antibody application. the results of this study revealed high binding affinity with low cytotoxicity; and cellular internalization or lysosomal trafficking in her positive cells with a dose-dependent profile compared to the her negative cells (miyano et al., ) . in subsequent studies by chen et al. ( ) and gao et al. ( ) , a model protein toxin (pe kdel) was used to develop anti-her antibody functionalized pe kdel loaded plga nanoparticles utilizing a two phase carbodiimide process. modified encapsulated nanoparticles showed higher in vitro cytotoxicity and more protective antitumor activity compared to the controls in her overexpressing cells and tumor-bearing mice, respectively. in addition, they reported well tolerability for maximum tolerated dose, as well as reduced systemic toxicity when modified nanoparticles were administered to mice model compared to control (chen et al., ; gao et al., ) . nanotechnology has promoted greater opportunities for higher specific drug delivery with minimum side effects. bio-conjugation strategies of therapeutic agents loaded nanoparticles with mabs have exhibited a targeted drug delivery approach both in vitro and in vivo. mabs can act as highly specific probes when they are attached to nanoparticles to aid in targeted delivery of various antitumor cytotoxic agents (sutradhar and amin, ) . the combination therapy of nanopartilces of mabs and other chemotherapeutic agents, such as doxorubicin, -fluorouracil, paclitaxel, docetaxel, campothecin, and topotecan, could bring about synergistic tumor growth inhibition. some nanoparticulate systems combining antibody with these chemotherapeutic agents are listed in table . . neovascularization is a major cause of visual loss in a number of ophthalmic diseases. it consists of new blood vessel growth from preexisting vascular structures. this process occurs in pathologies of the anterior and posterior segments of the eye, such as corneal neovascularization, age-related macular degeneration (amd), diabetic macular edema, viral retinitis, proliferative vitreoretinopathy, posterior uveitis, retinal vascular occlusions, choroid neovascularization (cnv), and diabetic retinopathy, to name a few. anti-vegf agents have demonstrated efficacy in reducing neovascularization in both animal models and clinical trials. specifically, anti-vegf antibodies have shown initial therapeutic success. bevacizumab is a full-length, humanized murine mab that recognizes all isoforms of vegf. bevacizumab was initially approved by the u.s. food and drug administration (fda) to treat metastatic colon cancer, but it has also shown efficacy in the treatment of various neovascular ocular diseases and is used off-label to treat neovascular age-related macular degeneration. topical delivery is a relatively easy and a less risky method of drug administration. however, delivery to the posterior segment via this route is considered inefficient and unsuccessful, since less than % of the topically applied dose enters the eye and an even smaller fraction of it ( . %) is expected to reach the posterior segment. intravitreal administration involves the direct administration of drug solution/suspension into vitreous humor via pars plana using a -g needle. in contrast to the topical roth et al. ( ) and systemic routes, intravitreal injection makes high concentrations of drug locally available to the internal eye tissue, including the choroid and the retina. similarly, the intravitreal administration of macugen (pegaptanib sodium; pfizer) and lucentis (ranibizumab; genentech/novartis), vascular endothelial growth factor (vegf) inhibitors, is highly successful for the control of amd. however, agents with molecular weight less than da when applied intravitreally tend to be drained off from the site of application with a half-life of less than days, indicating a need for repetitive injections. anyway, the period requiring a repeat dose may extend from a few days to several months for macromolecular antibodies. for example, three is the mean number of injections of bevacizumab (avastin; roche) required to be administered per year for the treatment of amd. on the other hand, the recommended dosing frequency of ranibizumab (lucentis) is once a month ( . mg; µl) for at least months (rosenfeld et al., ) , whereas pegaptanib sodium (macugen) needs to be injected intravitreally at -week intervals for year (kitagawa and yuzawa, ) . nevertheless, repetitive intravitreal injections, even if spaced widely, are invariably associated with complications, such as vitreous hemorrhage, retinal detachment, cataract, and endophthalmitis. the rate of endophthalmitis and retinal detachment being observed with intravitreal injection is . and . %, respectively (edelhauser et al., ) . moreover, patient compliance is lower with such regimens because of the painful and invasive procedures requiring hospitalization and specially trained physician for administration, in addition to the high cost of the medicine per se (kaur and kakkar, ) . biodegradable polymeric nanoparticles offer properties that make them suitable candidates to overcome these administration issues. in this regard, abrishami et al. ( ) studied encapsulated bevacizumab into liposomesand and found that the intravitreal injection of liposomes was well tolerated through days in rabbits. the clearance of this drug in vitreous from liposomal formulations was slower than the soluble form. the concentration level of bevacizumab after intravitreal injection suggested that intravitreal injection of drug by these carriers provide sufficient concentration of therapeutic drug for weeks for diabetic neovascularization and probably for other neovascular eye diseases (abrishami et al., ) . other authors developed nanoparticles of plga loaded with bevacizumab and stabilized whit albumin. they found that the vitreous concentration of bevacizumab was sustained for about weeks at values greater than ng ml − and that the nanoparticles presented a drug vitreous medium retention time (mrt) . times higher than the control. in addition, it was confirmed the nanoparticles persistence in ocular tissues for days (varshochian et al., (varshochian et al., , . recently lu et al. ( ) studied the effects of intravitreal injection of bevacizumab-chitosan nanoparticles on pathological morphology of retina and the expression of vascular endothelial growth factor (vegf) protein and vegf mrna in the retina of diabetic rats. the results have offered a new approach for inhibiting angiogenesis of diabetic retinopathy and indicated that the intravitreal injection of bevacizumab inhibited vegf expression in retina. in addition, bevacizumab-chitosan nanoparticles presented a longer period of action (zhou et al., ) . chronic respiratory diseases (crd) are chronic diseases of the airways and other structures of the lung. among the most common, asthma, chronic obstructive pulmonary disease (copd), and respiratory allergies can be mentioned. the airway inflammation in these diseases cannot always be controlled with conventional therapies. the critical role of the combination therapy of an inhaled corticosteroid (ics) and a long-acting β-adrenoceptor agonist (laba) in the treatment of patients also suffering from severe asthma and chronic obstructive pulmonary disease (copd) explains why there is a strong interest within the pharmaceutical industry in developing new pharmaceutical alternatives. biological therapies represented in particular by mabs against selective targets, could improve the outcome of these diseases. many current publications have shown the important role of nanomedicine in the treatment of respiratory diseases (card et al., ; mansour et al., ; smola et al., ) . systems releasing nanoparticles have several possibilities to improve treatment of upper respiratory tract since they can protect drugs from degradation by enzymes present in the epithelial lining fluid, the particle size may permit circumvent pass macrophages, and crossing the endothelium will allow systemic treatment of nanoparticles and pulmonary drug intravenously administration. identifying asthma phenotypes has given impetus to the search for biomarkers to help classify patients, pointing to new therapies and predicting different pathological mechanisms of disease progression with strong benefits for affected patients . an ideal biomarker is easy to detect and measure, noninvasive, and inexpensive, and it can be used to identify phenotypes either for clinical response or treatment, assessing changes in disease activity, or confirming a diagnosis. copd is currently the fourth leading cause of death in the world, and if current trends continue, it is likely to rank as the third leading cause of death by (world health organization, ) . both cytokines and chemokines play a fundamental role in the organization of copd, so the use of mabs in the treatment options is very valuable to provide more targeted therapies (li et al., ; yamagata and ichinose, ) . numerous antibodies directed to cytokines, chemokines, growth factors, and their receptors are considered for the treatment of copd and some have already been studied in clinical trials in patients with copd. the macrophage imaging using mri, together with the use of magnetic nanoparticles of iron oxide, has recently emerged as a promising noninvasive technique for preclinical and clinical studies of inflammatory diseases. however, limited research on inflammation and passage of macrophages in the lung, using imaging technology has been performed due to difficulties in imaging the body (i.e., the signal loss due to the pulsations and heart breathing, low proton density, and susceptibility artifacts). novel perspectives for imaging, diagnosis, and treatment of respiratory diseases, such as copd can appear due to technical improvements and the development of techniques of magnetic resonance pulse sequence detection. studies reported the possibility of noninvasive monitoring of subsets of macrophages in an inflammatory model using high resolution mri after intravenous administration. the possibility of noninvasive monitoring of subsets of macrophages was reported in an inflammatory model using high resolution mri after intravenous administration. furthermore, other studies have proven that polarization and proliferation was not affected using iron oxide nanoparticles with ex vivo labeled subpopulation of macrophages. the coupling of a specific antibody with the iron oxide nanoparticles directed to a particular subpopulation of macrophages may provide a promising strategy for early diagnosis and improved various inflammatory diseases noninvasively using mri. al faraj et al. ( ) evaluated the in vivo effect of intrapulmonary administration of spion on the profiles of alveolar macrophage polarization in a model of copd and developed a protocol mri noninvasive to specifically target and monitor a subpopulation of macrophages using specific antibodyconjugated spion. an interesting approach was performed to target a subpopulation of macrophages in the lung of a copd animal model using biocompatible antibody-conjugated iron oxide nanoparticles. this conjugation allowed noninvasive tracking using a free-breathing mri protocol. antibodies to il- abgenix improved dyspnea but not lung function. this limited clinical benefit is attributed to suboptimal dosing into the airways because of intravenous administration (mahler et al., ) . therefore, the use of nanoparticles may allow administration by inhalation for local concentrations and avoiding side effects. so far, clinical trials with anti-tnfalpha antibody infliximab have been carried out, but no effects in patients with copd were observed (dentener et al., ; rennard et al., ; van der vaart et al., ) . therefore, the release of cetuximab (currently undergoing clinical trials for cancer therapy) could be a valuable option for the treatment of copd. molecularly targeted nanoparticles were chosen as the best strategy for imaging inflammation to make progress in health care (mccarthy and weissleder, ; weissleder, ) . magnetic nanoparticle's versatility makes them well suited for applications to enable early detection and prevention, and so improving diagnosis, treatment, and follow-up of diseases (oghabian and farahbakhsh, ) . al faraj et al. ( ) developed an assay to quantify the antibodies cd or cd conjugated to spion, which involves the reduction of cu + to cu + by proteins and the appearance of a purple-blue copper-protein complex in alkaline medium. some significant trends that appear to be developing include bispecific antibodies, adcs, and companion diagnostics, which should help to identify the most appropriate patient populations for treatment. even though the health benefits of mab therapeutics are proven, controversies will continue about their economic viability because of their expensive price. high prices, however, are generally associated with early innovative treatments, and mabs are not the only drugs to have staggering prices. the cost of cancer treatments, for example, has more than doubled in the past two decades, leading to an outcry by many european and american cancer specialists. another reason why mabs are so expensive is the fact that many of them are still protected by patents. a drug's patent life is years from the date of filing, in order to help developers recover some of the research and development costs involved in getting a drug to market. some of the patent life is in fact reduced because some of that time, an average of years, is taken up by clinical trials and regulatory approval (siddiqui and rajkumar, ) . the slow progress of mab therapeutics for infectious diseases can be attributed in part to the large arsenal of other antiinfective drugs, such as vaccines and antibiotics. because they are specific to a single pathogen, mab drugs are also commercially less attractive than traditional drugs because they cover a narrower spectrum of patients. in addition, mabs need to be administered by intravenous or subcutaneous injection, unlike other antiinfectives that can be taken orally, so they are unsuitable for patients in developing countries who have limited access to healthcare. mabs are also more effective at preventing infection rather than treating established ones, and unlike vaccines provide only short-term prophylaxis. and finally, the high development and manufacturing costs associated with mabs and the poor record in winning approval for treating infectious diseases with mabs lessen their commercial appeal (reichert, ) . more promising are recent developments to enhance the potency and efficacy of mabs, so as to make it possible to prescribe lower doses and potentially reduce costs. a number of approaches have been adopted to augment the efficacy of mabs. one of the most encouraging is the use of genetic engineering to remove glycosylation sites from the variable domain of the antibody. this enhances the effector function of mabs, such as antibody-dependent cell-mediated cytotoxicity (adcc), which activates the patient's innate immune cells to kill a target cell like cancer. although the new generation of mabs may greatly improve the treatment of cancer and autoimmune disorders, which are well-established disease targets for mab therapeutics, it remains to be seen whether mab therapeutics will be effective in other areas. nowhere is this question more urgent than in the case of infectious diseases. the fact that mab therapeutics is pathogen-specific could hinder its use for treating mixed infections. one solution might lie in the use of a cocktail of mabs to target the diverse range of antigens that viruses carry. such a strategy would effectively mimic the natural immune response: once infected, the body tends to develop several antibodies in response to the antigens presented by a virus, each of which attaches to one of the different antigens. it is this diversity of antibodies that helps the immune system in fighting the invader. the use of a cocktail of mabs is already being investigated for the treatment for rabies recent advances in mab engineering have opened up new opportunities for serum therapy. importantly, mabs offer the means to prepare standardized agents, which, when combined in a cocktail, can yield a product that is more precise and more potent than traditional serum therapy. to date, the development of antiinfective mab products has attracted little commercial investment. in part this is because infectious diseases are short-lived and therefore have a limited market. this is in contrast to chronic conditions like cancer and autoimmune diseases, which require regular treatment and therefore have greater profit potential. nonetheless, the pharmaceutical climate is changing, fueled by concern over the rise in new pathogens (such as west nile and corona viruses); the reemergence of old pathogens (like tuberculosis), increasing antibiotic resistance among microorganisms; and the rise of superbugs like mrsa, as well as the growing epidemic of patients who are immunocompromised as a result of hiv infection, organ transplantation, chronic degenerative diseases, and improvements in cancer care. likewise, mabs have been used to investigate and treat cancer, providing powerful tools for identifying and targeting different antigens on tumors. although these applications did not quickly translate into successful mab therapeutics for cancer, in more recent years mabs therapeutics have offered alternatives to drugs with a broad spectrum and high toxicity. this has transformed the care of cancer patients, who no longer face the prospect of losing hair and other serious side effects associated with other cytotoxic drugs. the advantage of mabs is that they can be given as maintenance therapies. this is reshaping our perceptions of some cancers from what was once seen as inevitably fatal to a chronic condition. mabs have also enabled the prescription of specific therapeutics for particular tumor antigens in individual patients. this allows a greater degree of personalization in the management of cancer than in the past. indeed, mabs are expected to be an increasingly important component in personalized cancer therapies. in a world of antibiotic-resistant superbugs and an 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semiconductor quantum dots agents against cytokine synthesis or receptors theranostic nanoparticles carrying doxorubicin attenuate targeting ligand specific antibody responses following systemic delivery drug targeting with nano-sized carrier systems targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-her trastuzumab a rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles targeted co-delivery of docetaxel and siplk by herceptin-conjugated vitamin e tpgs based immuno-micelles quantitative control of targeting effect of anticancer drugs formulated by ligand-conjugated nanoparticles of biodegradable copolymer blend photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles beyond: a randomized, doubleblind, placebo-controlled, multicenter, phase iii study of first-line carboplatin/paclitaxel plus bevacizumab or placebo in chinese patients with advanced or recurrent nonsquamous non-small-cell lung cancer further reading formulation and delivery issues for monoclonal antibody therapeutics sterically stabilized anti-her immunoliposomes: design and targeting to human breast cancer cells in vitro anti-her immunoliposomes: enhanced efficacy attributable to targeted delivery key: cord- -wu k u authors: hofmann, tim; krah, simon; sellmann, carolin; zielonka, stefan; doerner, achim title: greatest hits—innovative technologies for high throughput identification of bispecific antibodies date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: wu k u recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. the plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. in this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the duobody concept, paired light chain single cell production approaches, sortase a and transglutaminase, the spytag/spycatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies. the human body is continuously exposed to potentially harmful and life-threatening opponents such as pathogens or malignant cells. in order to assert oneself, several layers of innate defense mechanisms such as pathogen recognition receptors enable the early detection of 'danger signals' and, consequently, the elimination of the respective invader [ , ] . moreover, the human immune system can also respond in an adaptive fashion to a foreign antigen via antibody-mediated processes, for instance antibody-dependent cellular cytotoxicity (adcc) or complement-dependent cytotoxicity (cdc) [ , ] . as such, antibodies are crucial players in host defense. inspired by the natural mechanisms of antibody-mediated pathogen elimination, monoclonal antibodies emerged as one of the most promising classes of therapeutic molecules, with about entities now being approved either in the usa or europe [ , ] . however, one of the main limitations of antibody-derived therapeutics relies in their monospecific nature. this restricts targeting to one antigen only. since most diseases are typically rather complex, involving multiple disease mediators or factors simultaneously [ , ] , much effort was made within the last decades to combat the multifaceted nature of oncology and immunology diseases more efficiently. in this respect, the advent of bispecific antibodies opened up new avenues for disease treatment such as effector cell redirection or the simultaneous blocking of two different disease mediators, with encouraging results in (mostly early stage) clinical trials [ ] [ ] [ ] . consequently, an unprecedented multitude of bi-and multi-specific formats employing flexible valences and overall architectures were engineered to facilitate a plethora of modes of action (moa), as elegantly described elsewhere [ , ] . for the generation of bispecific antibodies, one of the main tasks relies in the identification of paratope pairs that fulfill the desired moa in the best way possible when reformatted as bispecific. nowadays, antibody selection campaigns such as immunization combined with b-cell cloning, phage display, or yeast surface display of naïve, synthetic, as well as immune libraries typically enable the identification of up to hundreds of different initial hit candidates [ ] [ ] [ ] [ ] [ ] . hence, testing of every combination of hits for each binding arm in a bispecific antibody format would result in the expression of several thousands of different molecules, making the whole process rather cumbersome and tedious. in this regard, kitazawa and co-workers initially expressed and analyzed , bispecific antibody combinations in order to identify a molecule that mimics the cofactor function of coagulation factor viii sufficiently [ ] . in addition, other factors such as the order of paratopes in a given architecture or steric hindrance might impact biological activities, e.g., crosslinking abilities for effector cell redirection, and thus need to be taken into consideration [ ] [ ] [ ] . consequently, full coverage of this multidimensional screening space would require huge efforts with respect to molecular biology, i.e., cloning, antibody expression, and purification. in order to overcome this bottleneck, technologies such as controlled fab-arm exchange used for duobodies [ ] , paired light chain single cell production approaches [ ] , microbial transglutaminase [ ] or sortase a [ ] mediated bioconjugation, the spytag/spycatcher system [ , ] , or split inteins [ ] were adapted to enable broad bispecific antibody screening ( figure ). these methodologies harbor the benefit that bispecific entities can be generated from a pre-existing set of antibody fragments in a mix-and-match manner, significantly reducing hands-on time as well as overall efforts for bispecific screening. in this review, we aim at giving an overview of the most relevant protein bioconjugation technologies for the creation of bifunctional antibodies. we further discuss benefits as well as limitations of each specific methodology in the context of bispecific screening. [ , ] , much effort was made within the last decades to combat the multifaceted nature of oncology and immunology diseases more efficiently. in this respect, the advent of bispecific antibodies opened up new avenues for disease treatment such as effector cell redirection or the simultaneous blocking of two different disease mediators, with encouraging results in (mostly early stage) clinical trials [ ] [ ] [ ] . consequently, an unprecedented multitude of bi-and multi-specific formats employing flexible valences and overall architectures were engineered to facilitate a plethora of modes of action (moa), as elegantly described elsewhere [ , ] . for the generation of bispecific antibodies, one of the main tasks relies in the identification of paratope pairs that fulfill the desired moa in the best way possible when reformatted as bispecific. nowadays, antibody selection campaigns such as immunization combined with b-cell cloning, phage display, or yeast surface display of naïve, synthetic, as well as immune libraries typically enable the identification of up to hundreds of different initial hit candidates [ ] [ ] [ ] [ ] [ ] . hence, testing of every combination of hits for each binding arm in a bispecific antibody format would result in the expression of several thousands of different molecules, making the whole process rather cumbersome and tedious. in this regard, kitazawa and co-workers initially expressed and analyzed , bispecific antibody combinations in order to identify a molecule that mimics the cofactor function of coagulation factor viii sufficiently [ ] . in addition, other factors such as the order of paratopes in a given architecture or steric hindrance might impact biological activities, e.g., crosslinking abilities for effector cell redirection, and thus need to be taken into consideration [ ] [ ] [ ] . consequently, full coverage of this multidimensional screening space would require huge efforts with respect to molecular biology, i.e., cloning, antibody expression, and purification. in order to overcome this bottleneck, technologies such as controlled fab-arm exchange used for duobodies [ ] , paired light chain single cell production approaches [ ] , microbial transglutaminase [ ] or sortase a [ ] mediated bioconjugation, the spytag/spycatcher system [ , ] , or split inteins [ ] were adapted to enable broad bispecific antibody screening ( figure ). these methodologies harbor the benefit that bispecific entities can be generated from a pre-existing set of antibody fragments in a mix-and-match manner, significantly reducing hands-on time as well as overall efforts for bispecific screening. in this review, we aim at giving an overview of the most relevant protein bioconjugation technologies for the creation of bifunctional antibodies. we further discuss benefits as well as limitations of each specific methodology in the context of bispecific screening. is selected to generate a bispecific igg full-length format with correct chain pairing. for the sake of simplicity in this example, fab fragments are reconstituted with one-armed monovalent fragments. the depicted antibody fragments contain a heterodimerization technology indicated by striped ch regions. the bispecific antibody reconstitution is performed in a combinatorial setup to increase the number of variants. (c) high throughput antibody screening of reconstituted bispecific antibodies is performed in plate format, depending on the mode of action. (d) biological functional screening of several t-cell engager combinations is conducted in to weeks, depending on the combinatorial sample size. a very elegant and generic approach for the generation of bispecific antibodies was adapted from processes occurring within the human immune repertoire itself. igg antibodies are dynamic molecules that physiologically form bispecifics by a process called fab-arm exchange (fae). herein, half molecules (consisting of a paired heavy and light chain) from different igg s recombine and form a polyclonal mixture of bispecific antibodies in vivo. by losing their ability to crosslink antigens and generate immune complexes, it is believed that igg may dampen inflammatory reactions by interfering with immune complex formation of other antibody isotypes in chronic inflammatory diseases [ ] . the natural mechanism of fae also seems to be attractive to generate bispecific antibodies in vitro, because it may overcome limitations of other bispecific antibody platforms like knobs-into-holes [ ] , wherein additional methods like crossmab [ ] or paratope scfv grafting need to be applied to circumvent light chain mispairing [ , ] . in , genmab published an "efficient generation of stable bispecific igg by controlled fab-arm exchange" [ ] . this work was inspired by the observation that human/rhesus intraspecies igg enhance fae (due to igg specific ch residues) and that -mercaptoethylamine-hcl ( -mea), as a mild reducing agent, enables fae with antibody pairs harboring native igg -hinge regions. in controlled fab-arm exchange ((c)fae), two igg s with matched ch mutations are produced separately and are subsequently recombined under reducing conditions. ch mutations need to destabilize the ch homodimer interaction and promote heterodimer formation. for this, the igg specific r mutation was incorporated into one igg ch and combined with surrounding mutations on positions l , k , d , f and y (all amino acids except for c and p) on the opposite chain. in a dual-binding elisa, matched point mutations for all five antibody positions were obtained, enabling bispecific binding by fae. in depth analyses of (c)fae were conducted using igg -f l and igg -k r mutants, the variants that are also applied in genmab's proprietary duobody technology. the general process of bispecific antibody generation applying duobodies involves three basic steps: ( ) separate production of monospecific antibodies harboring respective mutations in mammalian cell cultures, ( ) purification according to standard processes (e.g., protein-a), ( ) (c)fae under tailored laboratory conditions followed by another purification step. this typically yields bispecific antibodies with more than % heterodimer content. the beauty of duobodies lies in the fact that the bispecifics fully retain igg structure and that fc-mediated mechanisms, such as fcrn mediated recycling and fcγ-receptor interactions, stay unaffected [ ] . today, several bispecifics based on (c)fae are under clinical development. one of these molecules is the met × egfr bispecific antibody jnj- , jointly developed by genmab and janssen, currently in phase i, for the treatment of advanced nsclc, that was screened from five anti-met × anti-egfr parental antibodies in the duobody format [ ] . similar to the bioconjugation methods described in later sections, (c)fae serves as an ideal platform for bispecific antibody screening. for the given example of , antibody variants individually produced and screened by kitazawa et al. [ ] , only + = parental monospecific would need to be produced using complementary duobody mutations, while × = , variants could be screened, drastically reducing lab work and timelines applied during classical screening efforts [ , ] . associated with that, astrazeneca recently published a method for high throughput bispecific antibody screening, applying matched f l and k r mutations [ ] . in addition, h r and y f (also known as rf-mutation) were incorporated into one of the ch mutants (k r). the rf-mutation completely abrogates the interaction of an antibody-fc with protein-a (however, it should be noted, that antibodies harboring a vh segment will bind to protein-a, regardless of the rf-mutation in the antibody-ch ). in a proof of concept study, bispecific antibodies were generated from four parental antibodies (anti-her ; anti-egfr; anti-nip ; anti-igf r). all six resulting bispecific antibodies could be produced with high heterodimer purity as demonstrated by liquid chromatography-mass spectrometry (lcms) and reverse phase liquid chromatography (rplc) and showed bispecific target engagement proven by bli. compared to the conventional duobody generation, this approach enables the formation of bispecifics directly from culture-supernatants without any pre-purification steps of monospecific antibodies, making it perfectly suited as a bispecific screening tool [ ] . finally, bispecific antibodies can be generated by the duohexabody platform, developed by genmab, combining the before mentioned duobodies with a hexameric formation of antibody complexes. the duohexabody technology offers a broad applicability for bispecific antibody generation with target-mediated enhanced potencies for cdc-mediated effector functions as elegantly demonstrated by oostindie et al. [ ] . when aiming at screening of heterodimeric fc-based bispecific antibodies not affected by light chain (lc) pairing issues, the combination of two binding moieties' panels can also be executed on the dna rather than the protein level. genentech developed a tethered-variable cl bispecific igg (tcbsigg) platform based on knobs-into-holes heterodimerization combined with linked light chains. this approach allows robust production of intact bispecific antibodies in a single cell line, concurrently ensuring cognate light chain pairing and preserving structural and functional properties of heterodimeric fc antibodies, hence rendering it applicable to high throughput screenings [ ] . another elegant example is a combination of a common lc with a dekk heterodimerization fc portion single cell production of bispecific antibodies for a successful unbiased her /her combinatorial screen at merus [ ] . in addition to methodologies for screening of classical igg and heterodimeric fc architectures, plentiful approved or clinically-evaluated non-fc formats represent a need for further high throughput screening technologies beyond fc heterodimerization strategies. enzyme-mediated bioconjugation, up to now mainly applied for adc generation, could be a solution. enzyme-mediated bioconjugation has evolved in the last decades to a robust and versatile tool for protein labeling and covalent attachment of two or more protein building blocks by site-specific ligation. it also obviates the need for harmful chemicals as employed for chemical ligation. finally, also enzymatic bioconjugation avoids cumbersome genetic engineering or subsequent expression of individual multivalent binders but offers options for one-pot reactions yielding bispecific antibodies amendable to high throughput or functional screening approaches. first clues for the presence of transglutaminases (tgases) date back to the work of clarke and colleagues in describing the transamidation activity by pig liver [ ] . microbial transglutaminases (mtgases) were first isolated from streptomyces mobaraensis [ ] and are now widely used in the food, textile, and cosmetic industry as "biological glue", e.g., for meat [ ] . belonging to the class of protein-glutamine γ-glutamyltransferases, tgases catalyze the transfer of primary amines (e.g., the ε-amino group of lysines as acyl-acceptor) to γ-carboxamides (e.g., γ-glutamyl group of glutamine as acyl-donor) under ammonia release, yielding a stable isopeptide bond resistant to proteolytic degradation [ ] . although mtgases accept a promiscuous acyl-acceptor substrate repertoire [ ] , surface-exposed glutamines of glycosylated native human igg antibodies are not accepted by mtgase [ ] . mtgase has been engineered by directed evolution and rational design for improved catalytic performance and alternative recognition sequences [ , ] , and has been exploited in particular for the generation of antibody-drug conjugates [ , , ] or labeling of antibody fragments [ , ] . multimerization of a single domain antibody fragment targeting tnf could be achieved by using mtgase and compatible peptidyl linkers [ ] -an approach that could be generically employed for high throughput screening of non-fc bispecific antibodies. the staphylococcus aureus transpeptidase sortase a (srta) was identified in to be essential for cell wall assembly of gram-positive bacteria, recognizing a conserved "lpxtg" motif within secreted cell surface proteins, cleaving between the threonine and the glycine residues, forming an thioester acyl-enzyme intermediate and catalyzing the covalent linkage of the carboxyl group of the threonine to a nucleophilic amino group of a n-terminal pentaglycine within the cell wall anchored peptidoglycan [ ] . the reaction is reversible, hence, dependent on an excess of donor and acceptor substrate to favor high yields, with the acylation step being the rate-limiting factor [ ] . removal of unwanted byproducts can limit the reversibility of the reaction and could provide the basis for an equimolar ratio of the substrates. sortase a engineering yielded variants with improved reaction rates and the reduction of by-products [ , [ ] [ ] [ ] [ ] with versatile applications also termed "sortagging" [ ] [ ] [ ] such as protein circularization [ ] and antibody drug conjugates [ ] . srta has been employed for the generation of bi-and multi-specific antibodies of several formats, e.g., conjugations of fab [ ] or scfv fragments [ ] as well as c-terminally linked antibody heterodimers in combination with click chemistry [ ] . for a high throughput screening approach, the group of plückthun recently established a one-pot srta-mediated coupling reaction for the alternative binding protein darpin (designed ankyrin repeat proteins) with compatibility to functional assays, due to minimal levels of monovalent side products. combinations of darpins, resulting in bispecific molecules targeting c-met and epcam, were analyzed for cell proliferation inhibition and yielded a novel bifunctional darpin with superior cellular activity [ ] . this illustrates the applicability of enzymatic bioconjugation for pharmaceutical high throughput biotherapeutics' functional screening approaches. another bacterial transpeptidase served as the basis for the engineering of a ligase, for the conjugation of two proteins. this system, also known as the spytag/spycatcher system, provides a fully intrinsic bioconjugation capability without the need to add enzymes and is described below. the spytag/spycatcher system originates from covalently-stabilized pilin polymers found in the pilus of gram-positive bacteria which usually undergo intramolecular amide bond formation to impart mechanical and proteolytic stability to pili [ ] , precisely streptococcus pyogenes, inspiring the name spy technology. when split and engineered, zakeri and co-workers generated first generation isopeptags binding to pilin-n or -c [ , ] , mediation of irreversible peptide-peptide interactions via spyligase [ ] , and finally, the spycatcher and spytag [ ] . this approach makes use of the collagen adhesin domain (cnab ) of fibronectin binding protein (fbab) that possesses an internal isopeptide bond between n-and c-domains' amino acids lys and asp . when engineered, the domain is split between lys and asp into two fragments resulting in an n-terminal fragment (spycatcher) of aa and a c-terminal fragment (spytag) of aa. putting both fragments again in close proximity, a spontaneous formation of a covalent peptide bond between lys and asp occurs, resulting in a double hydrogen bond between glu and asp , facilitating the peptide bond formation by nucleophilic attack and forming a zwitterionic intermediate. the bioconjugation of spytag and spycatcher is redox insensitive, efficient in a broad range of ph ( to ) and temperatures ( to • c) [ ] . the ligation reaction rate is fast and was further increased -fold by engineering an optimized spycatcher variant yielding complete bioconjugation after a few minutes [ ] . the technology is a promising tool for diverse biotechnological applications as reviewed by sutherland et al. [ ] , as discussed also above, ranging from protein ligation via peptide bond formation to protein stabilization by cyclization [ ] [ ] [ ] . specifically, the spytag/spycatcher fragments can either be fused c-or n-terminally or to internal positions within the protein, unlike, for example, split inteins, which are restricted to c-or n-terminal fusions. within the years, a plethora of applications has been published, ranging from the generation of bispecific antibodies to robo [ ] , a generic trivalent scfv platform [ ] , and the generation of site-specific conjugated adcs with high efficiency [ ] for therapeutic approaches. akiba et al. described an elegant technology for generating biparatopic antibodies through two-step targeting using a pair antibody-spytag or spycatcher-fusions targeting different epitopes of the same target that spontaneously react to form a covalent bond between fragments to generate a biparatopic antibody in situ [ ] . the system is applied in several modular "plug-and-display" approaches for the generation of enhanced immunostimulants against cancer or infectious diseases such as influenza or mers-cov [ ] [ ] [ ] [ ] [ ] . further applications include optimized protein purification procedures (in parts, in combination with inteins) [ , ] , specific immobilization of targets for enhanced phage display antibody discovery campaigns [ ] , site-specific fluorescence labeling of antibodies for in vivo optical imaging [ ] , up to in vivo assembling of proteins or enzymes [ , ] , and versatile further applications, in depth reviewed by hatlem et al. [ ] . in addition to a two-component spy system, veggiani and co-workers identified an orthogonally acting snooptag/snoopcatcher pair by engineering streptococcus pneumoniae adhesin rrga in a similar fashion, which also forms a spontaneous isopeptide bond with > % yield and no cross-reaction to spytag/spycatcher [ ] . similar to spy approaches, three-part splitting of rrga resulted in the respectively called snoopligase, catalyzing the formation of an isopeptide bond between the two peptide tags, snooptagjr and dogtag [ ] . many biotherapeutics currently in pre-clinical research and early development are complex molecules, mostly bispecific antibodies, and require both a specific spatial arrangement and flexibility of their binding moieties to not only specifically bind but also fully elicit their functionality of often several desired moas [ , ] . therefore, in-format screening of bispecific antibodies is desirable and can significantly impact the identification of the best binders' combination [ ] . spytag/spycatcher fusions leave a peptide imprint of aa to the protein of interest after bioconjugation. a aa shortened, truncated version of an n-terminal spycatcher fragment has been developed by li et al. [ ] to decrease an immune response in mice and to shorten the peptide footprint. in case of screening for effector cell engagers, the remaining kda spy domain could lead to altered flexibility and paratope distances, to impaired biological functionality, and finally, to potentially suboptimal choice of binders or epitopes. despite these potential limitations for screening of certain biotherapeutics classes, the spytag/spycatcher system has, only ten years after its initial publication, been applied to a tremendous extent and very versatile manner and will continue to drive basic science as well as pharmaceutical development. another option for in vitro bioconjugation and screening of bispecific antibodies are split inteins. found in every organism of life, n-and c-termini of split inteins are located in their natural origin on two different genes to elicit splicing in trans at the protein level with well-understood mechanisms [ ] . the process is called protein trans splicing (pts), and it yields-similar to enzymatic bioconjugation or spy technology-a stable peptide bond and is very specific for each split intein [ ] . pts is a single irreversible turnover reaction, not dependent on thermal or energetic co-factors such as temperature or atp. the most common and well investigated split inteins are ssp dnab from cyanobacterium synechocystis [ ] or npu dnae originated from nostoc punctiforme with a reported splicing rate of t / = min at • c [ ] [ ] [ ] . most investigated split inteins are dependent on a mild reducing environment by the addition of reducing agents like tris ( -carboxyethyl) phosphine (tcep) or dithiothreitol (dtt) to undergo pts. pts is dependent on intein and extein sequences. exteins flank both split intein parts holding specific recognition sequences to initiate the pts mechanism. the amino acid residue at position + consists either of a cysteine, serine, or threonine, depending on the split intein. pts takes place in four steps: ( ) a nucleophilic attack by the first amino acid residue of either a cysteine or serine located in the n-terminal intein part on the carbonyl carbon of the preceding amino acid residue (− position) located in the flanking n-extein leads to an n-s or n-o acyl shift; ( ) a linear (thio) ester intermediate being trans-esterificated by a nucleophilic attack of the first amino acid residue of the so-called c-extein (+ position) forms a branched intermediate [ ] ; ( ) the n-terminal intein part is now cleaved from its fusion protein and transferred to the n-extein. as a result, a succinimide ring is generated through cyclization of the conserved asparagine residue of the c-terminal intein part after the nucleophilic attack of the previously formed intein extein junction; ( ) finally, the cleavage reaction of the c-terminal intein part followed by a spontaneous s-n or o-n acyl shift ligates the esterified c-and n-exteins by a native stable peptide bond [ , ] . the process of pts is similar to native chemical ligation (ncl), which is well described elsewhere [ ] . similar to (c)fae, mild reducing conditions for the activation of certain inteins have been proven to minimize the potential risk of interchain disulfide bond breaks but retain native lc pairing and the desired functionalities of resulting bispecific antibodies. non-antibody cysteine residues within extein sequences are essential for certain inteins and could lead to disulfide scrambling or the generation of trisulfide bonds [ ] . of note, some split inteins rely on serine or threonine for catalytical activity, hence do not require cysteines and are engineered to undergo pts triggered by a shift in ph, salinity conditions, light or temperature, making them more applicable for a variety of biological applications [ , ] . recently, the engineered cysteine-free version of the split intein aes polb intein was developed by bhagawati and coworkers, with fast kinetics and not depending on a reducing environment, enabling the screening of antibodies exhibiting disulfide bonds sensitive to reduction [ ] . in addition, and similar to spytag/spycatcher, most inteins require the incorporation of a one to six extein amino acid footprint that may cause altered flexibility and geometry. to overcome this limitation, serine-or threonine-based inteins [ ] could be applied for seamless bioconjugation at respective, naturally occurring sequence positions or exteins could replace amino acids within the igg hinge region or linker sequences in further formats during screening. finally, the incorporation of split inteins into a nonnative host protein generally could alter splicing kinetics resulting in non-ligated products [ , ] , hence this needs to be individually assessed for each bispecific antibody format. han et al. first described in vitro bioconjugation of a full-length bispecific antibody via intein fusions to precursor antibody fragments within the hinge region and successfully demonstrated in vivo activity of a reconstituted cd xher t-cell engager mediated by npu dnae [ ] . a similar approach yielded a cd xprlr bispecific antibody for t-cell activation and cytokine release towards prlr expressing breast cancer cells [ ] . in addition to fc-based bispecifics, also non-fc, circularly connected vhh fragments (cyclobody) have been developed via siclopps (split intein circular ligation of peptides and proteins) reaction between both c-and n-termini, forming a cyclic conformation after pts [ ] . these cyclobodies are, as discussed before for other applications, protected from proteolysis due to their cyclic topology, yet they retain their dual specificity. an anti-egfrxcd cyclobody was successfully generated to show cytotoxicity against egfr-positive cancer cells, able to bind simultaneously egfr and cd on the cell surface [ ] . applicability of the aforementioned split intein aes polb intein was demonstrated by successful bioconjugation of several therapeutically relevant formats like full-length igg, fc, and vhh fusions [ ] . split inteins have been used to bioconjugate toxic components to antibodies [ ] , avoiding toxicity issues during antibody production, as exemplified by an anti-her immunotoxin conjugated via split intein derivative (m ) of the ssp dnab intein [ ] . next to several split inteins applied in bioconjugation of single binders in diverse formats, we recently described the application of split inteins for automated high throughput screenings in pharmaceutical research and development [ ] . similar to other presented methodologies, split intein screening could enable the comparison of complex formats with feasible low production needs and faster development times. in addition to such two component systems, and akin to the spy/snoop combinations discussed earlier, orthogonally-acting split intein pairs could enable three to multi component screens [ ] , e.g., for trispecific antibodies. the group of pinto et al. [ ] presented a mutually orthogonal split intein library for in vivo applications and further split intein pairs to be used for the in vitro seamless assembly of large repetitive proteins with biotechnological or pharmaceutical relevance. the past years have seen the unparalleled development of monospecific antibodies with natural moas as well as a broad range of engineered bispecifics to multi-functional biologics for therapeutic intervention in cancer or immunological diseases. the concept of bispecific antibodies for effector cell recruitment dates back to the s, with reports on production [ ] and effective ctl retargeting [ ] and the design of a universal t cell engager [ ] . however, it was only until a decade ago that catumaxomab (revomab ® ) for targeting epcam-positive tumors by fresenius biotech as well as micromet and amgen with cd -specific blinatumomab (blincyto ® ) were the first to bring bispecific t cell engaging antibodies to clinical approval [ ] . this resulted in a tremendous increase and diversification of bispecific antibody approaches for cancer and immunotherapy in research and clinical development [ , , ] . many investigational bispecific antibodies still aim at retargeting t cells to kill tumor cells [ , ] of both solid and hematological malignancies, exemplified by a bite platform review for cancer immunotherapy [ ] . a similar, flourishing strategy in cancer immunotherapy involves the recruitment and activation of nk cells, as applied in bispecific or trispecific killer cell engagers (bikes or trikes), recently reviewed for approaches towards solid [ ] and hematological diseases [ ] . other moas aim at intervening with two different disease mediators such as cell surface receptors, soluble ligands, and other proteins [ ] . diverse architectures allow various numbers and spatial relationship between different binding sites, valences, several secondary immune functions, and pharmacokinetic half-life. this plethora provides great opportunity to tailor the design of bispecific antibodies to match the desired moas and the intended clinical application to address the unmet need in several cancer indications [ ] . advances have been reported in production, often by site-specific bioconjugation and self-assembly technologies, and reviewed elsewhere [ ] . the sheer size of the combinatorial screening space spanning between diverse panels of targeting moieties, formats, and moas demands innovative methodologies for automated high throughput functional screenings of bispecific and even more complex formats in pharmaceutical development. as the plentiful formats and moas of the biology-driven discovery of bispecific antibodies was recently elegantly reviewed by nie et al. [ ] , we sought to cover methodologies technically enabling broad bispecific screenings. until today, the successful development of highly innovative bispecific antibodies was carried out by efficient automated screening with appropriate robotics, as exemplified by the work of kitazawa et al. [ ] or geuijen and co-workers [ ] . in addition to the technologies described herein, classical screening will continue to yield highly differentiated biotherapeutics such as bispecific antibodies with optimized moas. complementary to this, most screening methodologies described herein share the opportunity to greatly reduce production needs and development times for complex biotherapeutics by bioconjugation on the protein level or when produced in a small scale from one cell. classical drug candidate screening is often straight forward and allows for functional interrogation in the desired final format, whereas methodologies presented herein add a layer of complexity with inherent advantages and drawbacks, discussed in the following paragraph. the choice of an appropriate method will often be based on locally established automation, expertise, and the respective desired format and associated moa to be screened. for heterodimeric fc architectures, (c)fae, tethered variable-cl, or common lc bispecifics avoid light chain mispairing issues and represent viable options with minimal engineering but robust conversion, enabling high throughput screenings in early stages even in crude supernatant. similarly, pts can be performed both in vitro and in vivo, hence enabling bioconjugation without preceding purification within the production cell or in the supernatant [ , ] . this represents an advantage over enzymatic ligation for early bispecifics hts. enzyme bioconjugation, as exemplified by mtgase and srta, can be applied for non-fc formats, leaving minimal peptide footprints, but requires enzyme addition and, potentially, purification. unlike duobodies, the spy technology and split inteins are not restricted to the full-length igg antibody format and are applicable to pharmaceutical development high throughput screening of multiple formats by fusing binding moieties to the termini of a fc portion, or to each other and also overcome light chain pairing issues during screening. although the spytag/spycatcher system has been harnessed for versatile applications in the past years, the significant imprint of one additional protein domain in the resulting bispecific product might hinder its application for the screening of formats strongly relying on the respective paratope distance to elicit the desired moa, e.g., effector cell recruitment. split inteins generate a comparatively low scar of to amino acids remaining that, e.g., in linker sequences, can be engineered to mimic the envisioned format, representing a viable option for close-to-format or, under certain conditions, in-format screening. both the combination of spy/snoop technology and orthogonal split inteins offer the option to screen even larger combinatorial spaces of tri-to multivalent biologics, such as biparatopic effector cell engagers, bispecific, adcs, or trispecific antibodies. in addition to antibody-derived bispecifics, the plethora of alternative scaffolds fused to relevant antibody portions might further enlarge the combinatorial space that could be covered by the discussed screening methodologies, as recently exemplified for pts-generated, lectin-based antibody fusions termed "lectibodies" [ ] . limitations and advantages apply similarly to the above discussed two component approaches. table gives an overview of the properties, advantages, and shortcomings of the respective methodologies. the future will tell whether a broader coverage of these large screening spaces by application of reviewed or similar technologies will enable the identification of best-in-class innovative biotherapeutics. although this review discussed mainly the screening of igg-based therapeutic bispecific antibodies, as well, isotype-switched iga, igd, ige, and igm antibodies play important roles in the healthy and diseased immune system, with distinct differences in the architecture, tissue distribution, receptor binding, and effector function properties. respective isotypes and their application for bispecific antibodies were recently comprehensively reviewed [ ] and might bear great potential for future bispecific antibody therapies. in summary, the reviewed methodologies offer great tools for broad functional screening campaigns of diverse bi-to multi-specific antibodies and formats, with versatile moas in early drug discovery. these methods could greatly shorten development times and enhance the probability of identifying optimal combinations, ultimately leading to the generation of better biotherapeutics. table . comparison between different bioconjugation technologies for posttranslational antibody modification. the technologies presented here are able to reconstitute antibody fragments in vitro on the protein level. tethered variable cl as well as common lc bsabs are generated on a molecular biology basis and not recombined on the protein level. pathogen recognition and innate immunity role of toll-like receptors in pathogen recognition boosting therapeutic potency of antibodies by taming fc domain functions the ligands for human igg and their effector functions. antibodies , , antibodies to watch in development of therapeutic antibodies for the treatment of diseases engineering igg-like bispecific antibodies-an overview simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin bispecific antibodies: a mechanistic review of the pipeline bispecific antibodies: design, therapy, perspectives. drug des a review of bispecific antibodies and antibody constructs in oncology and clinical challenges the making of bispecific antibodies therapeutic antibody engineering by high efficiency cell screening phage display-derived human antibodies in clinical development and therapy designing human antibodies by phage display yeast surface display for screening combinatorial polypeptide libraries single b cell cloning and production of rabbit monoclonal antibodies a bispecific antibody to factors ixa and x restores factor viii hemostatic activity in a hemophilia a model ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design trifabs-trivalent igg-shaped bispecific antibody derivatives: design, generation, characterization and application for targeted payload delivery the effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody efficient generation of stable bispecific igg by controlled fab-arm exchange unbiased combinatorial screening identifies a bispecific igg that potently inhibits her signaling via her -guided ligand blockade transglutaminasecatalyzed covalent multimerization of camelidae anti-human tnf single domain antibodies improves neutralizing activity high-throughput generation of bispecific binding proteins by sortase a-mediated coupling for direct functional screening in cell culture post-translational assembly of protein parts into complex devices by using spytag/spycatcher protein ligase catching a spy: using the spycatcher-spytag and related systems for labeling and localizing bacterial proteins efficient generation of bispecific igg antibodies by split intein mediated protein trans-splicing system anti-inflammatory activity of human igg antibodies by dynamic fab arm exchange knobs-into-holes' engineering of antibody ch domains for heavy chain heterodimerization immunoglobulin domain crossover as a generic approach for the production of bispecific igg antibodies engineering bispecific antibodies with defined chain pairing how the bispecific antibody targeting egfr and cmet has superior preclinical activity and potentially better safety profile fab-arm exchange combined with selective protein a purification results in a platform for rapid preparation of monovalent bispecific antibodies directly from culture media duohexabody-cd ® , a novel biparatopic cd antibody with enhanced fc-mediated hexamerization as a potential therapy for b-cell malignancies tethered-variable cl bispecific igg: an antibody platform for rapid bispecific antibody screening the incorporation of amines into proteins purification and characteristics of a novel transglutaminase derived from microorganisms transglutaminases: nature's biological glues comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors site-specific and stoichiometric modification of antibodies by bacterial transglutaminase microbial transglutaminase for biotechnological and biomedical engineering location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates locked by design: a conformationally constrained transglutaminase tag enables efficient site-specific conjugation enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase site-specific cross-linking of functional proteins by transglutamination staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall kinetic mechanism of staphylococcus aureaus sortase srt a a general strategy for the evolution of bond-forming enzymes using yeast display ca +-independent sortase-a exhibits high selective protein ligation activity in the cytoplasm of escherichia coli recent advances in sortase-catalyzed ligation methodology sortase-mediated ligations for the site-specific modification of proteins sortase-mediated protein ligation: a new method for protein engineering sortagging: a versatile method for protein labeling protein-protein fusion catalyzed by sortase a sortase-catalyzed transformations that improve the properties of cytokines engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase a-mediated protein ligation preparation of bispecific antibody-protein adducts by site-specific chemoenzymatic conjugation bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity pili in gram-positive pathogens spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin superglue from bacteria: unbreakable bridges for protein nanotechnology synthetic biology: a new tool for the trade evolving accelerated amidation by spytag/spycatcher to analyze membrane dynamics spytag/spycatcher cyclization enhances the thermostability of firefly luciferase enhanced stability of a rumen-derived xylanase using spytag/spycatcher cyclization use of spytag/spycatcher to construct bispecific antibodies that target two epitopes of a single antigen a novel synthetic trivalent single chain variable fragment (tri-scfv) construction platform based on the spytag/spycatcher protein ligase system enzyme-based labeling strategies for antibody-drug conjugates and antibody mimetics generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using spytag and spycatcher improving the malaria transmission-blocking activity of a plasmodium falciparum / based vaccine antigen by spytag/spycatcher mediated virus-like display surface display of classical swine fever virus e glycoprotein on gram-positive enhancer matrix (gem) particles via the spytag/spycatcher system particulate multivalent presentation of the receptor binding domain induces protective immune responses against mers-cov a novel rapid modularized hepatitis b core virus-like particle-based platform for personalized cancer vaccine preparation via fixed-point coupling a self-adjuvanted, modular, antigenic vlp for rapid response to influenza virus variability spy chemistry-enabled protein directional immobilization and protein purification spy & go purification of spytag-proteins using pseudo-spycatcher to access an oligomerization toolbox integrating spycatcher/spytag covalent fusion technology into phage display workflows for rapid antibody discovery site-specific fluorescent labeling of antibodies and diabodies using spytag/spycatcher system for in vivo optical imaging genetically encoded spy peptide fusion system to detect plasma membrane-localized proteins in vivo crispr/cas-directed programmable assembly of multi-enzyme complexes programmable polyproteams built using twin peptide superglues snoopligase peptide-peptide conjugation enables modular vaccine assembly epitope distance to the target cell membrane and antigen size determine the potency of t cell-mediated lysis by bite antibodies specific for a large melanoma surface antigen format and geometries matter: structure-based design defines the functionality of bispecific antibodies in-format' screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules structural analysis and optimization of the covalent association between spycatcher and a peptide tag inteins: structure, function, and evolution protein trans-splicing as a protein ligation tool to study protein structure and function protein trans-splicing by a split intein encoded in a split dnae gene of synechocystis sp unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources highly efficient protein trans-splicing by a naturally split dnae intein from nostoc punctiforme the naturally split npu dnae intein exhibits an extraordinarily high rate in the protein trans-splicing reaction inteins-a focus on the biotechnological applications of splicing-promoting proteins protein splicing mechanisms and applications chemical science a functional interplay between intein and extein sequences in protein splicing compensates for the essential block b histidine native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations disulfide bond structures of igg molecules: structural variations, chemical modifications and possible impacts to stability and biological function salt-inducible protein splicing in cis and trans by inteins from extremely halophilic archaea as a novel protein-engineering tool mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions recent advances in in vivo applications of intein-mediated protein splicing intermolecular disulfide bonds between unpaired cysteines retard the c-terminal trans-cleavage of npu dnae a novel bispecific antibody targeting cd and prolactin receptor (prlr) against prlr-expression breast cancer construction of a circularly connected vhh bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites site-specific modification of ed-b-targeting antibody using intein-fusion technology generation of potent anti-her / immunotoxins by protein ligation using split inteins intein mediated high throughput screening for bispecific antibodies segmental isotopic labeling of a central domain in a multidomain protein by protein trans-splicing using only one robust dnae intein an expanded library of orthogonal split inteins enables modular multi-peptide assemblies bispecific monoclonal antibodies from hybrid hybridomas human ovarian carcinoma lysis by cytotoxic t cells targeted by bispecific monoclonal antibodies: analysis of the antibody components universal bispecific antibody for targeting tumor cells for destruction by cytotoxic t cells bispecific antibodies for cancer therapy: a review t cell-redirecting bispecific antibodies in cancer immunotherapy: recent advances alternative molecular formats and therapeutic applications for bispecific antibodies utilizing the bite (bispecific t-cell engager) platform for immunotherapy of cancer monoclonal antibody therapy of solid tumors: clinical limitations and novel strategies to enhance treatment efficacy bispecific antibodies in the treatment of hematologic malignancies site-specific bioconjugation and self-assembly technologies for multi-functional biologics: on the road to the clinic biology drives the discovery of bispecific antibodies as innovative therapeutics intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for nmr studies an off-the-shelf approach for the production of fc fusion proteins by protein trans-splicing towards generating a lectibody in vitro sagacity in antibody humanization for therapeutics, diagnostics and research purposes: considerations of antibody elements and their roles key: cord- -eifh zm authors: owji, hajar; negahdaripour, manica; hajighahramani, nasim title: immunotherapeutic approaches to curtail covid- date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: eifh zm covid- , the disease induced by the recently emerged severe acute respiratory syndrome coronavirus (sars-cov- ), has imposed an unpredictable burden on the world. drug repurposing has been employed to rapidly find a cure; but despite great efforts, no drug or vaccine is presently available for treating or prevention of covid- . apart from antivirals, immunotherapeutic strategies are suggested considering the role of the immune response as the host defense again the virus, and the fact that sars-cov- suppresses interferon induction as an immune evasion strategy. active immunization through vaccines, interferon administration, passive immunotherapy by convalescent plasma or synthesized monoclonal and polyclonal antibodies, as well as immunomodulatory drugs, are different immunotherapeutic approaches that will be mentioned in this review. the focus would be on passive immunotherapeutic interventions. interferons might be helpful in some stages. vaccine development has been followed with unprecedented speed. some of these vaccines have been advanced to human clinical trials. convalescent plasma therapy is already practiced in many countries to help save the lives of severely ill patients. different antibodies that target various steps of sars-cov- pathogenesis or the associated immune responses are also proposed. for treating the cytokine storm induced at a late stage of the disease in some patients, immune modulation through jak inhibitors, corticosteroids, and some other cognate classes are evaluated. given the changing pattern of cytokine induction and immune responses throughout the covid- disease course, different adapted approaches are needed to help patients. gaining more knowledge about the detailed pathogenesis of sars-cov- , its interplay with the immune system, and viral-mediated responses are crucial to identify efficient preventive and therapeutic approaches. a systemic approach seems essential in this regard. severe acute respiratory syndrome coronavirus (sars-cov- ), a newly emerged betacoronavirus, is responsible for coronavirus disease (covid- ) , which was first reported in wuhan, china in december . sars-cov- , the third fatal virus of its group, is an enveloped positive-sense, singlestranded rna virus [ ] . while the other viruses of this family induce only mild cold symptoms, severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov), the two other virulent betacoronaviruses, have been presented with higher fatality rates than sars-cov- . on the other hand, sars-cov- has shown a higher transmission rate and therefore a wider spread around the globe [ ] . the burden of coronavirus disease (covid- ) pandemic, caused by severe acute respiratory syndrome coronavirus (sars-cov- ), has been very huge on health, economy, and many other aspects of life. it has already claimed more than , lives (as of julyaugust according to who covid- dashboard at https://covid .who.int/). the urgency of this threat has prompted scientists in many countries to seek solutions through drug repurposing and repositioning of the previously approved drugs, while the fast-tracking of vaccine and drug development are seriously followed. however, some of the repurposed candidate drugs have already failed in some clinical trials [ ] . some antiviral drugs, developed for other similar viruses, are suggested, which may inhibit the cell entry or replication of the virus [ ] . on the other hand, supporting the immune system's potential to function properly and fight with the virus is another viable strategy. normalization of the dysregulated immune responses or even their suppression at the final stages of the disease may also be required [ ] . generally, following the entry of an invading pathogen into the body and its recognition by the first-line immune cells, the innate immune system would be stimulated, which may be later supported by adaptive immune responses as well. the interaction of each pathogen with the immune cells could define the defense the body against it, thus understanding such interplays is fundamental. although the role of the the immune system function in the control or overcoming covid- is indisputable, and many therapeutic and preventive approaches implicate modulation of the immune system activity, there are still many questions to be answered in this regard. for instance, the pattern of cytokine secretion during the disease course has been the subject of vast investigations. the complexity of the immune system responses as well as the variations in the level of cytokines that happens during the disease period and may lead to cytokine storm, highlights the need for gaining a deep overview on such events as well as the interventional possibilities to be able to cure or prevent the disease. in this review, the pathogenesis of sars-cov- and its interplay with the immune system , which are discussed in detail in other publications [ ] [ ] [ ] , are briefly stated. a classification of possible immune-based approaches to combat covid- is presented with a focus on convalescent plasma therapy, manufactured antibodies (monoclonal and polyclonal antibodies), and immunomodulators that could potentially modify the immune system activity. vaccines, as a potential immune-based approach to curtail covid- , were discussed previously [ ] [ ] [ ], so will not be discussed in detail here. despite the large volume of ongoing studies on sars-cov- , parts of our current knowledge on its pathogenesis is based on the studies of sars-cov and mers-cov, since the clinical manifestations of sars-cov- highly resemble the ones observed in the two latter viral infections [ ] . sars-cov- may enters the lungs from through the nasopharyngeal mucosal membrane and infect. alveolar alveolar macrophages and type i and ii epithelial cells in the lungs are shown to be infected by sars-cov- [ ] . the most prominent way of the viral entry was shown to be through the attachment of s protein and angiotensin-converting enzyme (ace ) receptors, which. some studies clarified that viral entry may be enhanced through some proteases such as a serine protease called tmprss (transmembrane protease, serine ) or cathepsin l/b (ctsl/b) [ ] . tmprss may, in fact, proteolytically activates sars-cov- by cleaving the s protein and promote replication and syncytium formation in the virus. hence, tmprss -expressing cells were shown to be highly susceptible to sars-cov- infection [ ] . cathepsin, which is expressed in endosomes, was shown to facilitate the fusion of the virus and endosome membrane [ ] . recent studies revealed that a combinational effect between tmprss and cathepsin is needed for an effective viral entry [ ] . another serine exopeptidase receptor, a serine exopeptidase, called dipeptidyl peptidase (dpp ) or cluster of differentiation (cd ), also was also shown to provide additional interactions with sars-cov- spike beside the ace receptor [ ] . despite the noteworthy pieces of evidence supporting the role of ace and the associated proteases in the viral entry, iit was shown revealed that the virus can also enter the cell through clathrin-dependent and -independent endocytosis pathways. for instance, sars-cov- may attack lymphocytes through the jak-stat pathway [ ] . covid- patients manifest mild to severe symptoms, including fever, non-productive cough, dyspnea, malaise, fatigue, lymphopenia, and pneumonia - days after the viral attack. moreover, laboratory results including leucopenia, elevated c-reactive protein (crp), and higher erythrocyte sedimentation rate (esr) are detected. a wide range of other clinical manifestations has been observed in covid- involving different organs, namely heart, eyes, nose, brain, pancreas, kidney, and bladder. as reported, - days upon the manifestation of the initial symptoms of the disease, the virus may cause a second attack and an aggravation of the disease symptoms in which severe pneumonia, ground-glass opacity, acute cardiac injury, and rnaaemia are observed [ ] . in this phase of the disease, the level of lymphocytes drops dramatically, and cytokine storm occurs. cytokine storm is an uncontrolled release of inflammatory cytokines, including ifn-α, ifn-γ, gm-csf, g-csf, il- ß, il- , il- , il- , il- , tnf-α, tgf-ß and chemokines, including ccl , ccl , ccl , cxcl , cxcl , cxcl . the cytokines level reduces as the patient recovers. cytokine storm has been recognized as the main cause of acute respiratory distress syndrome (ards), which causes lung injury, and multiple organ failure. moreover, patients who succumbed to covid- represented a higher level of neutrophils, d-dimer, blood urea nitrogen (bun), and creatinine than the survivors [ , ] . as the virus enters the cell, the viral rna is released and proceeds to the translation step. the viral proteins and rnas, produced by the host cell, are inserted into the endoplasmic reticulum and golgi, where the viral nucleocapsid is formed, and the viral particles are prepared to be released out of the cell membrane. the virus may enter the peripheral blood from the lungs and attack other organs that express the ace receptor, including heart, kidneys, and gastrointestinal tract. the viral antigen is presented to t cells and b cells via major histocompatibility complex (mhc) on antigen-presenting cells (apcs), thus innate and humoral adaptive immunities are activated. innate immunity is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [ ] . sars-cov- is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [ , ] thereby evading from the innate immune system defense [ ] . macrophage and dendritic cells were shown to play important roles in viral destruction and mucosal immunity. the level of cd + and cd + cells also increases, which leads to long-term immunity. it was shown that cd + and cd + cells are responsible for promoting neutralizing antibody proliferation and destruction of viral-infected cells, respectively [ ] . different immunoglobulins against viral s (spike), m (membrane), n (nucleoprotein) proteins, nsp (nonstructural proteins), and orf (open reading frame) are produced. however, the prevalent types of immunoglobulins are against s protein and among them igm and igg induce short-term and long-term immunities, respectively. moreover, it was shown that the immune system decision between th response (cellular response) or th response (humoral response), which is affected by the cytokine pattern, determines the viral infection control. in fact, it was reported that some infections are well controlled by th response and this response was observed in recovered covid- patients [ ] . sars-cov- was found to suppress antigen presentation through downregulation of mhc class i and ii molecules, which may lead to the impediment of t cell-mediated immune defense [ ] . what mentioned above are known as the most prevalent process of sars-cov- pathogenesis and clinical manifestations; however, a closer scrutiny recently revealed a wider range of clinical manifestation for covid- . in fact, a direct association has been observed between the expression of viral receptors, including ace and tmprss and the affected organ. herein, organs including heart, eyes, nose, brain, pancreas, kidney, and bladderfor instance, the abundance of ace receptors on endothelial cells explain why cardiovascular complications are considered as the second threat caused by sars-cov- after respiratory symptoms [ ] . moreover, sars-cov- may target the central nervous system and infect neurons in the nasal passage, which can be the cause of smell and taste disruption in some covid - patients. however, the loss of taste and smell is of the most initial inflammatory responses to sars-cov- and can be reversible as the body defeats the virus [ ] . were also shown to be affected by sars-cov- [ ] . in addition to the role of nasal cells in sars-cov- infection, the nasal secretory cells may be exploited by the virus for transmission to the other persons. viral antigens are presented to t cells and b cells via major histocompatibility complex (mhc) on antigenpresenting cells (apcs), thus innate and adaptive immunities are activated. innate immunity response is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [ ] . sars-cov- is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [ , ] thereby evading the innate immune system defense [ ] . moreover, it was shown that the immune system decision between th response (cellular response) or th response (humoral response), which is affected by the cytokine pattern, determines the viral infection control. in fact, it was reported that some infections were well controlled by a th response, and this response was observed in recovered covid- patients [ ] . sars-cov- was found to suppress antigen presentation through the downregulation of mhc class i and ii molecules, which may lead to the impediment of t cell-mediated immune defense [ ] . in the second attack phase of the disease (usually one or two weeks after the presentation of the first symptoms), the level of lymphocytes drops dramatically, and the cytokine storm occurs. cytokine storm is an uncontrolled release of inflammatory cytokines, including ifn-α, ifn-γ, gm-csf, g-csf, il- ß, il- , il- , il- , il- , tnf-α, tgf-ß, and chemokines, particularly ccl , ccl , ccl , cxcl , cxcl , cxcl . the cytokine level reduces as the patient recovers. cytokine storm has been recognized as the main cause of acute respiratory distress syndrome (ards), which causes lung injury, and multiple organ failure. taken together, the immune system is highly impaired in critically ill covid- patients. given the role of the immune system in host defense, immunomodulation could be regarded as an important strategy to curtail covid- considering the patient's immune system condition at various phases of the disease. such immunomodulatory interventions can be achieved using vaccines, interferons, convalescent plasma, anti-inflammatory agents, antibodies, and other classes of immunomodulators, which are described in the following. innate immunity, as the first immediate and general defense response, plays a key role in the protection against invading pathogens, which is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [ ] . sars-cov- is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [ , ] thereby evading from the innate immune system defense, as a mechanism of its pathogenesis [ ] . besides the yet questions in the initial responses to sars-cov- (e.g. types of receptors that are recruited for viral entry or cells that are affected), secondary responses (e.g. immune system responses) have been also under debate. for example, data concerning the beneficial or detrimental role of inflammatory mediators are controversial [ ] . cytokine storm, which is the result of increase in the levels of some cytokines as explained before, may also happen as a pathological situation of the innate immune system as well. another disruptive activity of the virus involves the functional exhaustion of lymphocytes, which would affect the adaptive immunity. the immunopathological conditions aroused in covid- , are mentioned in brief here, as they were discussed in detail elsewhere . there are still unknowns regarding the exact interactions of the immune and adaptive immunities with the virus as well as the methods of virus for confounding the host immune system. the in-depth understanding of viral-mediated responses and interplay of the virus and the host immune system might not be feasible without a systemic approach . nevertheless, the similarity of severe respiratory failure induced by sars-cov- to acute respiratory distress syndrome (ards) and the deterioration of patients' conditions in around a week following the first symptoms implicate the role of immunity dysregulation in covid- profile [ ] . therefore, immunotherapy and modulation of the immune system, which could modify these disorders, are potentially effective strategies to fight with sars-cov- . such interventions may be done through vaccines, which actively stimulates the immune responses, or administration of interferons i, which are discussed in previous publications. passive immunotherapy and the modulation of the immune system through anti-inflammatory or immunostimulatory agents are other alternatives, which will be examined in detail in the following. of note, these immunotherapeutic approaches should be carefully selected based on the stage of the disease and the state of the patient's immune system. the suppression of interferon i-mediated immune responses by sars-cov- is already confirmed [ , ] thereby evading from the innate immune system defense, as a mechanism of its pathogenesis [ ] . although interferon has beenwas shown to fight against the virus and are is suggested for to treatment of the disease [ ] , some contradictory data demonstrated that interferon may enhance ace expression and thus viral entry [ ] . on the other hand, positive results were found by using interferons type i, including interferon-beta- a in several clinical trials [ ] . the difference in the route of administration, either subcutaneous (s.c.) and intravenous (i.v.), was proposed as a reason for the diverse effects reported about interferon beta- a in some studies [ ] . interferon-beta is already being examined in a combination protocol in the international clinical trial launched by who in the partner countries, called as "solidarity" [ ] . the outcomes of the investigations on interferon therapy in covid- were presented in some other publications [ , [ ] [ ] and as a systematic review [ ] . interferon-beta is already being examined in a combination protocol in the international clinical trial launched by who, called the "solidarity" trial, in the partner countries [ ] . vaccines are believed as the ultimate protection for saving the public from the novel virus. the lack of previous exposure of the human immune system to sars-cov- [ ] is regarded as the major contributor to its high risk. hence, active immunization through vaccines could prepare the body to resist against this infection. very soon after finding the virus genetic sequence, vaccine development was started and followed with unprecedented speed by several research groups and pharmaceutical companies. huge investments are dedicated by several public and private bodies to advance the this project [ ] . various platforms are employed in these investigational vaccines, including inactivated, killed or weakened pathogen, non-replicating viral vector, rna, and dna, vlp (virus-like particle), and protein subunit structures virus. there also some inactivated as well as replicating viral vector-based vaccines under development at preclinical stage. each of these platforms has its own advantage and limitations. while dna and rna vaccines are intensively studied mainly because of their rapid development, easy production, and safety, there are novel types not commercially available previously. thus, their largescale production might take need more time to be set up, due to novelty and lack of previous experience in their commercial production. additionally, more than one dose of these vaccine types is required for the immunization because of their short half-life [ ] . as tthis topic is reviewed in details elsewhere [ ] [ ] [ ] . all in all, vaccine development is a time-consuming process. moreover, the induction of memory in the immune system is still under question. therefore, despite preliminary promising results, the efficiency of the investigational vaccines should be confirmed in large clinical trials. considering the lack of an approved drug or vaccine against sars-cov- , taking advantage of a helpful alternative intervention is an urgent need [ ] [ ] [ ] . passive immunotherapy via antibodies has been considered as a possible strategy for defeating covid- apart from anti-viral therapeutics and vaccines ( fig. ). antibodies can be either isolated from a convalescent patient or produced in a lab [ ] . these two approaches would be discussed in more detail in the following. active immunization is provided through vaccines, which are still under development for covid- . passive immunization can be performed via natural antibodies using convalescent plasma therapy (cpt) or antibodies that are manufactured. in cpt, natural neutralizing antibodies derived from a hyperimmune patient would be administered to a covid- patient through plasma transfusion. this approach is already being used and investigated in many countries with acceptable levels of success. on the other hands, different polyclonal or monoclonal antibodies could be produced via using hybridoma cell-lines, animals, or cell-free protein synthesis. neutralizing antibodies derived from a hyperimmune patient through plasma transfusion is the most prevalent and accessible empirical approach used to treat several viral infections previously. this method, so-called convalescent plasma therapy (cpt), is also considered as a viable therapy for covid- [ ] [ ] [ ] . it has been used to reduce the hospital stay and the mortality rate of critically ill patients with severe acute respiratory syndromes [ , ] and shown was found beneficial in some previous epidemics of infectious diseases [ ] . the use of this approach had been suggested for the first time during the outbreak of spanish influenza (pandemic of - ) [ ] . subsequently, plasma transfusion was recommended as a safe and effective way for the prevention or treatment of the ebola virus in and also several other severe viral infections, including mers, sars-cov, and avian influenza a [ , ] . in fact, neutralizing antibodies in the convalescent plasma (cp) could suppress the viremia through binding to the external antigens of the viruses and blocking their entry into the host cells [ , ] . the effectiveness of cpt could vary according to the type of microorganism, it's pathogenesis, and treatment protocols, such as timing, dosing, and volume of administration [ ] . according to the previous evidence for plasma therapy of other coronaviruses, such as sars-cov and mers, the early transfusion of cp can probably be more effective and improves the survival rate of critical covd- patients at the early disease stage. it could be explained by the fact that in most viral infections, viremia rises in the first week of the disease [ , ] . it should be noted that cpt may not be useful for mild or end-stage patients. indeed, cpt is not able to significantly reduce the mortality rate well among end-stage patients because of their disease severity. on the other hand, mild patients can be self-recovered and cpt would not be required [ ] . the titer of sars-cov- neutralizing antibodies in the cp could be another important factor to increase treatment efficacy. although, the antibodies level in the donor plasma before transfusion is not determined, some studies indicated that the specific igg increases about three weeks after symptom onset and peaks at week . therefore, the cp from donors who are at week after the initiation of the symptoms is predicted to be more efficient [ , ] . generally, patients with primary and secondary antibody deficiencies, need intravenous immunoglobulin (ivig) treatment as the standard replacement therapy [ ] ; and historically, administration of ivig has been one of the important treatments in immunodeficient patients [ ] . these patients are considered as a high-risk group, which can encounter several severe complications if infected with sars-cov- virus [ ] . therefore, an effective treatment is required to help such patients survive. cp extracted from the sars-cov- survivors may be a promising approach for the protection of covid- patients with antibody deficiency before the development of an effective vaccine [ ] . however, the data about the potency of cpt in covid- patients with primary and secondary humoral immunodeficiency is limited and has not been fully established, there are some case studies that have reported its proper effectiveness. for example, clark et al. reported a -year-old covid- case with lymphoma who was treated with a combination of bendamustine and rituximab that could lead to the impairment of humoral and cellular responses. after the failure of different treatment protocols against sars-cov- , the administration of hyperimmune plasma resulted in a rapid recovery in this patient [ ] . in another report, an immunosuppressed covid- patient with myeloid malignancy, disseminated tuberculosis, and kidney disease, was successfully treated after transfusion of cp and tocilizumab [ ] . male patient with covid- , whose health condition was improved following cp administration [ ] . presently, several clinical trials investigating the usage of cpt in covid- are ongoing (as recorded in https://clinicaltrials.gov/), a number of which are summarized in table . the trials are selected according to the recruitment status of the study (recruitment or completed state), age ( years and older), and severity of symptoms in participants (admitted to the hospital or icu with severe symptoms). several other publications have discussed the usage of cpt in covid- [ , , ] , thus no more details would not be addressed here. all in all, cpt has demonstrated acceptable safety and in the current situation and seems a promising choice for treating severe covid- patients besides other therapeutic strategies [ ] . as mentioned before,the previous experiences and efficacy in other similar diseases, as well as its feasibility, are the important advantages of cpt. moreover, according to different reports, cpt is welltolerated by receivers [ ] , but as all treatment approaches, this method may have some minor adverse effects as well. generally, the most common adverse effects of cpt is related to transfusion events, such as chills, fever, rash, allergic reactions, circulatory overload, and hemolysis [ , ] . additionally, several other problems are attributed to the mentioned approach, including lack of plasma donors, risk of cross-contamination, inconsistency from batch to batch, non-scalability, and the possibility of host reaction [ ] . these pitfalls behoove pharmaceutical companies to manufacture polyclonal or monoclonal antibodies. the first and most common targets are spike (s) proteins, which are located on the virus surface, generating its specific 'crown' shape [ ] . sars-cov- starts its pathogenesis through the attachment of receptorbinding domain (rbd), located in the s subunit of the s protein, with angiotensin-converting enzyme (ace ). thus, s proteins are considered as the most antigenic part of the virus with the main responsibility for the host immune responses [ ] . it has been widely suggested that the previously-experimented sars-cov rbd neutralizing antibodies can be repurposed for sars-cov- [ , , ] . the cross-neutralization capacity of antibodies relies on the conservation of the particular residues that are essential for the formation of specific bonds between rbd and the antibody, between the two types of viruses. for instance, an analysis was performed to determine which of the previously-proposed monoclonal antibodies against sars-cov can also cross-neutralize sars-cov- . in the mentioned study, the residues for the formation of a salt bridge and electrostatic interaction between the m antibody and rbd were conserved between sars-cov and sars-cov- . by contrast, antibodies, including r and f g failed to interact with sars-cov- rbd in a similar way they did with sars-cov rbd due to the differences in their residues [ ] . surprisingly, it was shown that f g neutralized sars-cov- more potently than sars-cov through other interactions [ ] . studies including cryoelectron microscopy of sars-cov- spike and analysis of the protein-protein interactions of sars-cov- and ace receptor with energy-based methods revealed the amino acids - of sars-cov- rbd as important residues; the latter study also introduced the linear and conformational epitopes within this region as antibody targets [ , ] . though the rbd has been considered as the main target of interest, some neutralizing or blocking antibodies have been shown to recognize other epitopes, including domains in s subunit, s-ectodomain, hr and hr domains in the s subunit, nucleoprotein (np), or envelope (e) protein [ ] [ ] [ ] . for example, cr cross-neutralized sars-cov- more strongly than other neutralizing antibodies against sars-cov, while it did not compete with ace for binding to sars-cov- . this observation indicates that cr neutralizes sars-cov- through binding epitopes other than rbd [ ] . another study showed that the hr domain with an identity of % is highly conserved between sars-cov and sars-cov- , and thus neutralizing antibodies that target the hr domain, including b , a , b , and g potently cross-neutralized sars-cov- [ ] . similarly, the s monoclonal antibody, retrieved from convalescent sars-cov patients, potently neutralized sars-cov- through a highly conserved domain distinct from rbd and did not interfere with the binding of s protein with the ace receptor [ ] . moreover, in contrast to the above-mentioned importance of rbd in designing monoclonal antibodies, some studies have revealed the antibody-dependent enhancement of viral entry when the monoclonal antibody targets the rbd. it was shown that the binding of monoclonal antibody to the rbd triggers conformational changes that are similar to the alterations made following the binding of viral receptors to the viral rbd; hence, the binding mediates the virus entry to the cell via viral receptor-dependent pathways. however, as stated in the mentioned study, this mechanism depends on the monoclonal antibody dosage, expression of particular viral receptors (e.g. fc receptors), and particular features of the monoclonal antibody [ ] . although ace has been introduced as the main receptor of sars-cov, it may not be sufficient for the interaction between the cell and the virus. other cellular factors, such as vimentin, a cytoskeleton protein, were revealed to be important in the formation of the ace -sars-cov complex [ ] . therefore, surface vimentin was recognized as a potential target for sars-cov. ds-sign/cd is also a transmembrane adhesion molecule, which is mainly expressed on interstitial dendritic cells and lung alveolar macrophages. it was shown that ds-sign also mediates the entry of sars-cov. a humanized monoclonal antibody was produced to interfere with the interaction of ds-sign and intercellular adhesion molecule (icam- ), and thus inhibit sars-cov entry to the cell [ ] . similar proteins may be identified in regards to sars-cov- . in addition to inhibiting the virus entry, antibodies can intrude into the biological activities of the virus thereby preventing its replication. fully human antibodies, which are capable of traversing across the cell membrane of infected cells and preventing virus replication, were made against several kinds of viruses, including influenza, hepatitis c virus, and ebola [ ] . papain-like proteases (plpro), cysteine-like protease ( clpro), and other non-structural proteins (nsps) can be suggested as targets of interests that hinder sars-cov- replication [ , ] . besides structural parts of the virus, various steps associated with the innate and the adaptive immune responses have been proposed as the most important targets of interest. for instance, the significant rise in the level of chemokines and cytokines, including il- β, ifn-γ, ip- , and mcp- , which is called the cytokine storm, can be inhibited by antibodies [ ] . the preliminary studies of critically-ill covid- patients have shown that il- may cause severe inflammatory responses that lead to acute respiratory distress syndrome [ ] . herein, tocilizumab, an il- inhibitor monoclonal antibody, has gained significant attention. in a -patient clinical study recruited in china, tocilizumab resulted in the reduction of oxygen need in % of patients, lung lesion opacity absorption in . % of patients, and correction of lymphocyte and c-reactive protein levels [ ] . the significance of tocilizumab can be better explained since a noticeable number of clinical trials regarding its use against sars-cov- related pneumonia and respiratory tract infections were recorded in nih until may , . an increase in the level of some of these proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (gm-csf) results in a positive feedback in the number of other inflammatory mediators, including il , il- , and tnf. gm-csf along with il and il also induce th /th differentiation and the polarization of macrophages to m phenotypes, which in turn boost the inflammatory responses [ ] . th immune response also can aggravate the cytokine storm by raising the level of il , gm-csf, il , and il [ ] . high levels of gm-csf and th cells were observed in the plasma of severe-to critically-ill covid- patients [ , ] . herein, harnessing the upregulation of gm-csf can prevent a cascade of inflammatory responses, which result in acute respiratory syndrome. since monoclonal antibodies targeting one inflammatory mediator, may fail to control the whole cytokine storm and prevent the lung injury induced by acute respiratory distress syndrome, a newer approach to prevent lung injury was proposed. it was shown that physical stress such as excessive mechanical stress caused by ventilators upregulates the expression of a gene, called nicotinamide phosphoribosyltransferase (nampt). as the bioavailability of nampt increases, toll-like receptor (tlr ), which is responsible for lung inflammation, gets activated [ , ] . herein, neutralization of circulating nampt by monoclonal antibodies can be another viable approach in preventing the lung injury caused by sars-cov- . in addition to the role of inflammatory mediators, understanding the adaptive immune responses helps us to repurpose or invent immunomodulatory antibodies to defeat sars-cov- . for example, cd + and cd + t-cells are expected to promote the proliferation of neutralizing antibodies and the destruction of infected cells, respectively. however, lymphocytopenia is identified to play a role in the pathogenesis of severely-ill covid- patients [ ] , which can be prevented or restored by regulating lymphocyte proliferation and apoptosis [ ] . herein, some studies have shown that sepsis may occur secondary to inflammatory responses. the immune imbalance, which occurs in sepsis, maybe as a result of t-cell depletion. pd- and ctla- receptors are immune checkpoints that are expressed on the surface of t-cells and play a role as the negative regulator of t-cell function. therefore, inhibiting the immune checkpoints by monoclonal antibodies is also an intriguing approach in defeating sars-cov- [ ] . furthermore, cd + cells express a receptor called c-chemokine receptor (ccr ), which was established as a way of hiv entry to the cell [ ] . this receptor could also be a potential target for sars-cov- . the level of different immunoglobulins is raised in response to sars-cov- . even though an increase in the level of immunoglobulins is attributed to pose neutralizing effect on sars-cov- , a rise in anti-s igg is associated with lung failure [ , ] . complement systems also play a role in conferring both humoral and natural immunity; however, they have been also attributed to the refractory inflammatory diseases. herein, c a and c a receptor, the members of the complement system, have been successfully targeted in different clinical trials of inflammatory diseases and thus can be recognized as possible targets in new diseases such as covid- [ ] . additionally, tlr , cd , immunoreceptor tyrosin-based activation motif (itam), g-csf, monocyte chemoattractant protein (mcp ), tnfα, il , and il are the other members of the immune system for which anti-sars-cov monoclonal antibodies were patented. these monoclonal antibodies could be reevaluated for sars-cov- [ ] . besides the main strategies described above for designing monoclonal antibodies against sars-cov- , some more novel approaches with a completely different mechanism were suggested. one of these strategies is using dewetting monoclonal antibodies. dewetting transition is a process in which hydrophobic pores of the ion channels inhibit water transmission and thus impair the cellular performance. this phenomenon can be deployed to block viruses, bacteria, and autoimmune activities. dewetting monoclonal antibodies are antibodies with a lipophilic fragment that target the transmembrane receptors and hinder the physiological water flow inside the channel. such antibodies were produced against the influenza virus [ , ] . recently, viroporins were identified in sars-cov- , the ion channel proteins that are generated by the virus e protein and are responsible for different parts of the virus life cycle, including virus entry, assembly, release, and the whole pathogenesis cycle [ ] . as suggested, dewetting monoclonal antibodies could be developed against sars-cov- viroporins and administered through the nose. these antibodies can deactivate the virus by targeting viroporins even before the virus will be able to bind to the host cells [ ] . generally speaking, tthe antibodies used for treatment could be usually produced at large-scale, either as monoclonal or polyclonal antibodies, using hybridoma cell-lines, animals, or cell-free protein synthesis either, [ ] . monoclonal antibodies can be produced via several technologies, including the production of high-affinity human antibodies in immunized transgenic mice (e.g. xenomouse® or humab® mice), various phagedisplay systems such as generating antibodies from immunoglobulin cdna libraries in bacteria or mammalian cells, and obtaining memory b cells of convalescent patients that are immortalized by ebv transformation. all of these techniques were previously recruited to produce monoclonal antibodies against sars-cov [ ] . production the production of monoclonal antibodies against sars-cov- is in its incipient phase. currently, our data about sars-cov- antibodies greatly comes from the studies in which the antibodies derived from the plasma of convalescent patients were analyzed. a recent study of the convalescent patients' antibodies demonstrated that anti-sars-cov- antibodies were versatile among the convalescent patients and each patient represented a unique pattern of antibody biodistribution. these results explain why it may be difficult to design specific anti-sars-cov- antibodies [ ] . therefore, the introduced antibodies in this study were mainly tested against sars-cov or approved for other immune inflammatory diseases such as rheumatoid arthritis, cancers, and other viral infections. an extra method for designing antibodies against sars-cov- can be based on the antibody-antigen computational simulation [ ] . for instance, an online docking server using the codockpp engine is constructed to predict the docking modes between antibodies or other peptides. this server is freely available at http://ncov.schanglab.org.cn. identified structural parts of sars-cov- and the homologous parts of other coronaviruses were gathered to produce the mentioned docking server [ ] . table consists of tthe antibodies retrieved from the previous studies on sars-cov or computational studies concerning sars-cov- , which mainly target the virus structure or host receptors are shown in table . despite a great homology between sars-cov and sars-cov- , the cross-reactivity of sars-cov antibodies against sars-cov- is still under debate. for instance, some highly-conserved regions are found in sars-cov- , which are absent in sars-cov. the c-terminal of sars-cov- rbd highly differs from that of sars-cov. moreover, an extra furin cleavage site was found between s and s subunits in sars-cov- , which is absent in sars-cov. these differences may not affect the ability of both viruses in interacting with the ace receptor but explain the different levels of affinity among neutralizing antibodies with the two viruses [ , , , ] . moreover, a recent study revealed that the antibodies targeting rbd of the coronavirus family are virus species-specific, while those that target viral parts outside rbd are capable of cross recognition [ ] . in an antibody epitope computational analysis, it was revealed that . % of antibody epitopes in the sars-cov- spike were novel in comparison with those in sars-cov [ ] . therefore, antibodies suggested in table ought to be reevaluated to be used against sars-cov- . taken together, among the introduced monoclonal antibodies in these study f g , cr , and d were shown to cross-neutralize sars-cov and sars-cov- . antibodies, including s . , s . , s . , s . , s . , s . , s . , s . , s . , neutralize spike by binding to residues - analysis of immune sars-cov patients' serum and in vivo study in mice [ ] s . , s . , s . , s . , s . , s . [ ] further information in this study concerning monoclonal antibody data against sars-cov- was retrieved from clinical trial data recorded in clinicaltrials.gov or biotechnology and pharmaceutical companies' websites, which are summarized in tables and and harbour biomed, are designed to neutralize sars-cov- structure. however, the mechanisms and specificities of the latter antibodies have not been elaborated to date and thus should be followed in companies' websites. not mentioned binds to highlyconserved epitopes within sars-cov and sars-cov- vir biotechnology with wuxi biologics and biogen enters clinical trial within - months aimed to confer short-and long-term immunity and use as prophylaxis [ ] not for effective passive immunotherapy, several epitopes ought to be targeted rather than one epitope. moreover, the design of monoclonal antibodies and their testing at a clinical stage is a long pathway followed by the fact that the massive production of monoclonal antibodies might be costly, time-consuming, (table ). polyclonal antibodies produced by immunized animals or a particular cell line technology are expected to mimic convalescent plasma therapy with a higher potency than their plasma-derived equivalents as well as better clinical outcomes. furthermore, the risk of contamination and host reactions will be reduced compared with their plasma equivalents; dosing and kinetics also would be more predictable and scalable. moreover, targeting more than one epitope can cause synergistic effects in neutralization and would limit the formation of escape-mutants. for instance, a recent study revealed that a cocktail of antibody noticeably enhanced sars-cov- neutralization compared with the use of one monoclonal antibody [ ] . no doubt the immune system is highly impaired in critically ill covid- patients, which denotes the possibility of using immunomodulators in this disease. monoclonal or polyclonal antibodies, interferons, and hydroxychloroquine are immunomodulators, which were widely discussed in previous sections. in this section, other several classes of immunomodulators including tyrosine kinase inhibitors, mtor inhibitors, calcineurin inhibitors, antimetabolites, tnf blockers, metal-based agents, and other anti-inflammatory agents are might be of value in the treatment of covid- .discussed janus-associated kinase (jak) inhibitors are of high interest among the mentioned immunomodulators. although ace receptors have been recognized as the main receptor for the entry of sars-cov- , it was shown that sars-cov- also attacks the cells that do not have ace receptors, including lymphocytes. it inhibitors have a marginal effect on il , which is responsible for b cell function, and do not disrupt innate immune response, since the inhibition caused by jak inhibitors is transient and reversible [ ] . another member of this family, baricitinib, is of special interest due to its advantages over other jak inhibitors and has been highly studied. baricitinib inhibits another regulator of endocytosis, called cyclin g associated kinase, through which it can defeat the viral infection [ ] . baricitinib has been also suggested as the best choice among other jak inhibitors due to its acceptable profile of side effects, the possibility of once-daily dosing, higher potency, and advantageous pharmacokinetics. the inhibitory doses of baricitinib were welltolerated by patients with inflammatory diseases in comparison with other kinase inhibitors [ , ] . moreover, baricitinib represents low protein binding and minimal interaction with drug transporters or metabolic enzymes; thus, it is preferred over other jak inhibitors for administration along with an antiviral regimen [ ] . in contrast to the above-mentioned benefits of baricitinib, some clinical studies suggested that baricitinib may not be an ideal option for the treatment of covid- due to the possibility of causing lymphocytopenia, neutropenia, viral reactivation, and enhancement of coinfection [ ] . other jak inhibitors, including upadacitinib and filgotinib, were also shown to impair interferon-mediated antiviral responses and perpetuate sars-cov- infection. herein, the incidence of secondary viral infections, including herpes zoster was observed, which was shown to be more prevalent in immunocompromised patients. hence, jak inhibitors, particularly baricitinib, should be administered with meticulous consideration, especially in susceptible and immunocompromised patients [ ] . next, ruxolitinib was listed as one of the top hit compounds in an advanced bioinformatics analysis of available medications for sars-cov- . the mentioned study aimed to identify compounds that counteract the expression of sars-cov- -related genes [ ] . to date, only three jak inhibitors have entered clinical studies on covid- patients, including baricitinib, ruxolitinib, and tofacitinib, among which many of the studies are related to baricitinib and ruxolitinib (table ) . bruton tyrosine kinase (btk) inhibitors are another group of tyrosine kinase inhibitors, which has been repurposed to modulate the cytokine storm ensuing covid- infection. btk signaling leads to b cell proliferation and activation of cytokine pathway. btk inhibitors are mainly approved for the treatment of an aggressive form of b cell lymphoma, called mantle cell lymphoma. this group includes acalabrutinib, zanubrutinib, tirabrutinib, and ibrutinib, among which ibrutinib was shown to have less efficacy and more toxicity [ ] . astrazeneca ® incorporation has designed a clinical trial to assess the efficacy of one of these btk inhibitors, called acalabrutinib, to alleviate the cytokine storm of sars-cov- infection (table ). in addition to btk inhibitors, other kinase inhibitors, including erlotinib and sunitinib were also shown to interfere in viral entry through inhibiting jak and aak . however, they are not classified as jak inhibitors and are not preferred over jak inhibitors due to less efficacy and higher toxicity [ , ] . sorafenib was also hypothesized to be repurposed in covid- based on a drug-gene interaction analysis [ ] . another mechanism of immunosuppression, which has been proposed, is related to the use of mtor inhibitors. the cytokine storm in covid- patients is attributed to a mechanism, called antibodydependent enhancement, in some systematic reviews [ ] . this phenomenon happens when the virus triggers the production of cross-reactive antibodies by memory b cells. these cross-reactive antibodies enhance virus delivery to the macrophages and thus contribute to the massive replication of the virus without being captured by the immune system. mtor inhibitors were found to inhibit the activation of memory b cell and prevent the antibody-dependent enhancement mechanism. mtor inhibitors also were shown to inhibit the replication of mers-cov in the in vitro studies [ ] . some mtor inhibitors, including rapamycin or sirolimus, were hypothesized to be repurposed in covid- clinical studies. sirolimus was shown to inhibit viral replication and release in patients with severe pneumonia and acute respiratory failure [ ] . the computational analysis of protein-protein interactions and gene-enrichment network also suggested that sirolimus can be repurposed for sasr-cov- [ ] . moreover, a clinical study of sirolimus on covid- patients has been recently started (table ). papain-like protease (plpro) is a viral protease responsible for coronavirus genome replication with deubiquitinating activity [ ] . plpro has been mainly the target of viral inhibitor class of medications. however, antimetabolites were also shown to be effective on plpro due to their pharmacological action. for instance, -mercaptopurine ( mp) and -thioguanine ( tg) were shown to be the specific inhibitors of sars-cov plpro [ ] . mycophenolate mofetil is another immunosuppressant that was shown to target plpro in sars-cov and mers-cov in both in vitro and in vivo studies. however, further clinical studies are needed to assess its efficacy against sars-cov- [ ] . there is no definitive evidence about the efficacy of other antimetabolites including methotrexate in covid- patients [ ] . calcineurin inhibitors such as tacrolimus might be effective against sars-cov- , since they inhibit calcineurin thereby blocking t cell activation. tacrolimus, which is mainly used in organ transplant, was shown to be effective against mers-cov in a renal transplant patient compared with a similar patient who did not receive tacrolimus as a part of the transplant regimen [ ] . tacrolimus was also found to be effective against sars-cov in a study on cell lines. however, further studies undoubtedly are required to assess its efficacy against sars-cov- [ ] . there is no definitive evidence about the efficacy of other calcineurin inhibitors including cyclosporine [ ] . metal-based agents with different metal centers, including gold, ruthenium, and bismuth were suggested to be used in covid- patients [ ] . the gold compound, called auranofin (ridaura®) is an fda approved compound, which was initially proposed for rheumatoid arthritis. the exact mechanism of auranofin is still unclear; however, it is classified as an immunomodulatory and anti-inflammatory agent. in recent years, auranofin has gained attention in viral infections, including hiv. in the case of hiv, it was revealed to be more effective than hydroxychloroquine in control of viral production, latency, and viral reactivation [ ] . it was also hypothesized that auranofin can interfere with il- signaling by inhibiting jak and stat pathways [ ] . it was shown that a low micromolar concentration of auranofin strongly inhibited sars-cov- viral replication and reduced the viral-induced cytokine expression in human cells [ ] . tnf-α was shown to be associated with sars-cov pulmonary injury; therefore, tnf-α blockers, which are mainly used in the treatment of autoimmune and inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, and psoriasis, can be suggested as a potential target for sars-cov- [ ] . besides the monoclonal antibodies that modulate the tnf-α responses, etanercept was suggested as another immunomodulator for covid- patients [ ] . lenalidomide and thalidomide, which are not specifically classified as tnf-α blockers, were hypothesized to be repurposed based on a drug-gene interaction analysis [ ] . thalidomide has antiinflammatory, anti-fibrotic, and immunoregulatory effects, which proved to be safe and effective in the treatment of lung injuries with different etiologies, including h ni-induced lung injury [ ] . it has also entered a clinical trial of covid- , as shown in table . cd fc, which is a fusion protein constituted of human cd attached to the human igg fc region, is another biological immunomodulator. cd fc was demonstrated to successfully ameliorate cytokine responses in viral infections and reduce the graft versus host disease [ , ] . hence, cd fc could be effective against the sars-cov- cytokine storm and the associated pneumonia. cd fc has entered a clinical trial by oncimmune ® incorporation (table ). the rationale for usinge of corticosteroids in covid- patients relies on their inhibitory effects on the inflammatory factors. corticosteroids inhibit a massive proportion of cytokines, chemokines, inflammatory enzymes, and receptors that are overexpressed in response to the viral infection [ ] . results regarding the beneficial effects of corticosteroids against coronavirus are controversial. a metaanalysis including patients from studies revealed that the use of corticosteroids resulted in higher mortality rate, the longer length of stay, a higher rate of bacterial infection, hypokalemia, and hypercalcemia [ ] . furthermore, russell and colleagues in a comment published in the lancet did not advocate the use of corticosteroids in covid- -induced lung injury or shock, except in the clinical trial settings [ ] . by contrast, a retrospective analysis of patients with severe sars revealed that corticosteroids led to reduced mortality rate and shortened hospital stay [ ] . taken together, corticosteroids are indicated for critically-ill critically ill covid- patients and its use in mild to moderate patients is highly disregarded. for instance, the use of corticosteroids is recommended in mechanically-ventilated covid- patients with respiratory failure (with ards), while is inappropriate for covid- patients without ards [ ] . in fact, administering corticosteroids in the onset of disease was shown to deter the immune system and increase the viral load; thereby inducing additional complications, such as diabetes and vascular necrosis [ , ] . the timing and dosage of corticosteroids are of special importance in the outcomes of treatment in critically-ill critically ill patients [ ] . for instance, in patients with high inflammatory responses including severe deterioration of oxygenation indicators and rapid imaging progress, a short term ( - days) of corticosteroids doses equivalent to - mg/kg/day methylprednisolone is recommended [ ] . moreover, a recent study revealed that low doses of corticosteroids (e.g. - mg/day methylprednisolone) did not delay viral clearance and did not increase the mortality rate compared with the control group [ ] . recently dexamethasone was introduced as the first medicine presented with a survival benefit in treating critically-ill critically ill covid- patients, and who welcomes preliminary results regarding its use [ , ] . according to a large, multi-center and randomized clinical trial called recovery (randomized evaluation of covid- therapy) trial, the use of mg/day dexamethasone in covid- patients on mechanical ventilation or oxygen supplementation was associated with reduced mortality rate compared with those who received standard treatment. in the patients who did not need respiratory support, no improvement was observed [ , ] . this well-known steroid is believed to support the suppression of hyperactive inflammatory responses, so-called cytokine storm [ ] . interestingly, an extra mechanism in defeating sars-cov- by dexamethasone was suggested. a computational study revealed that dexamethasone tightly binds to c-like protease (the main protease) in sars-cov- as remdesivir does; however, dexamethasone surprisingly forms a better contact with the enzyme active site than remdesivir [ ] . in addition to the importance of dose and timing, other considerations concerning the use of corticosteroids ought to be made according to a recent correspondence consensually made by experts in the lancet, including: ( ) the pros and cons of corticosteroids should be carefully evaluated prior to administration; ( ) prudent consideration should be made for the further use of corticosteroids in patients who are on the regular use of corticosteroids for chronic diseases [ ] . modulation of the immune system is obviously a major strategy in the control and treatment of covid- , which should be adjusted according to different stages of the disease. it can be done through vaccines, interferons, antibodies (either as convalescent plasma therapy or monoclonal and polyclonal antibodies), or potential therapeutic immunomodulators. at the pre-disease or disease prevention stage, active immunization by vaccines could be an optimal approach. however, vaccine development is a time-consuming and complicated process. despite enormous efforts on finding vaccines against sars-cov- , there is still a fairly long way to the market availability of a proper vaccine. following covid- disease onset, regulating the activities of the aberrant immune system may be a valuable treatment goal. at the early stages of the disease, stimulation of the immune system through passive immunotherapy is regarded as a therapeutic objective to support the patient's immune system and despite the similarities of sars-cov- with sars and mers, some differences such 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convalescence. zhonghua yi xue za zhi the pathogenesis and treatment of thecytokine storm'in covid- effectiveness of glucocorticoid therapy in patients with severe coronavirus disease : protocol of a randomized controlled trial low-dose corticosteroid therapy does not delay viral clearance in patients with covid- : . who welcomes preliminary results about dexamethasone use in treating critically ill covid- patients covid- ) treatment guidelines. national institutes of health effect of dexamethasone in hospitalized patients with covid- : preliminary report. medrxiv coronavirus breakthrough: dexamethasone is first drug shown to save lives deciphering the binding mechanism of dexamethasone against sars-cov- main protease: computational molecular modelling approach on the use of corticosteroids for -ncov pneumonia key: cord- - nxk e authors: sanders, john w.; ponzio, todd a. title: vectored immunoprophylaxis: an emerging adjunct to traditional vaccination date: - - journal: trop dis travel med vaccines doi: . /s - - - sha: doc_id: cord_uid: nxk e the successful development of effective vaccines has been elusive for many of the world’s most important infectious diseases. additionally, much of the population, such as the aged or immunocompromised, are unable to mount an effective immunologic response for existing vaccines. vectored immunoprophylaxis (vip) is a novel approach designed to address these challenges. rather than utilizing an antigen to trigger a response from the host’s immune system as is normally done with traditional vaccines, vip genetically engineers the production of tailored antibodies from non-hematopoietic cells, bypassing the humoral immune system. direct administration of genes encoding for neutralizing antibodies has proven to be effective in both preventing and treating several infectious diseases in animal models. while, a significant amount of work has focused on hiv, including an ongoing clinical trial, the approach has also been shown to be effective for malaria, dengue, hepatitis c, influenza, and more. in addition to presenting itself as a potentially efficient approach to solving long-standing vaccine challenges, the approach may be the best, if not only, method to vaccinate immunocompromised individuals. many issues still need to be addressed, including which tissue(s) makes the most suitable platform, which vector(s) are most efficient at transducing the platform tissue used to secrete the antibodies, and what are the long-term effects of such a treatment. here we provide a brief overview of this approach, and its potential application in treating some of the world’s most intractable infectious diseases. from the early practice of scarification to prevent smallpox through the creation of targeted, recombinant vaccines, the development of effective vaccines has been one of the great achievements in public health and medicine, resulting in millions of lives saved. modern vaccines typically protect by eliciting immunity following exposure to an inactivated or attenuated whole pathogen or recombinant components of a pathogen [ ] . this approach works well for diseases in which natural infection leads to immunity and protection against re-infection and has resulted in the eradication of smallpox and dramatic declines in such diseases as diphtheria, measles, and polio [ ] . however, it has been more challenging to develop effective vaccines against diseases for which prior infection does not offer full future protection, such as hiv, malaria, hepatitis c virus, and influenza a [ ] . although cellular immunity is certainly important, humoral immunity appears to play the most significant role in the protection associated with most vaccines [ ] . passive immunization achieved through the infusion of serum has played a significant historical role in the treatment and prevention of infection [ , ] . the recent development of hybridoma technology and humanized monoclonal antibodies have resulted in a new class of antibody-based drugs with demonstrated and potential efficacy in cancer, inflammatory diseases, addiction, and infectious diseases [ ] . within this context, there has been an increased interest in passive immunization utilizing monoclonal antibodies produced in plants or transgenic animals for infections such as ebola virus and mers-cov [ , ] . however, logistical requirements including the need for high antibody concentrations requiring repeated injections due to the short half-life of antibodies, a cold-chain for delivery, and trained medical personnel for delivery create potential limitations to the use of this therapy, especially in low resource areas [ , ] . the development of passive immunization by gene therapy could be a solution to some of those logistical issues and holds potential promise as either an adjunct to standard vaccination in populations who do not generate a sufficient immune response or for pathogens able to evade current vaccination strategies due to antigenic variability. originally proposed as a concept in [ ] , passive immunization by vector-mediated delivery of genes encoding broadly neutralizing antibodies for in vivo expression has been referred to as immunoprophylaxis by gene transfer (igt) [ ] , vector-mediated antibody gene transfer [ ] , or vectored immunoprophylaxis (vip) [ , ] ; and for sake of consistency, 'vip' is used here. rather than passively transfering pre-formed antibodies, vip is a process in which genes encoding previously characterized neutralizing antibodies are vectored into non-hematopoietic cells which then secrete the monoclonal antibodes encoded by those genes [ ] (see fig. .) this vectored delivery and production of specified antibodies allows for protection without generating a standard immune response and results in endogenous antibody production that has the potential to be sustained [ ] . the approach has several benefits, including: ) it does not require the host have the ability to respond immunologically, ) the antibody can naturally be selected for a specific pathogen targets, as well as specific epitopes, ) the antibody can be genetically modified to further enhance its activity, and, ) vectors can be selected or engineered to have tropic characteristics targeting specific tissues and cells, potentially allowing either systemic or enhanced localized antibody production [ ] . vip has been demonstrated to be effective in a host of animal models for the prevention of infection with several pathogens, especially those commonly afflicting travelers (see table ), including influenza a virus [ , ] , malaria (plasmodium falciparum) [ ] , hepatitis c virus [ ] , respiratory syncitial virus [ ] , bacillus anthracis [ ] , dengue virus [ ] , and chickungunya virus [ ] . in addition to the protection conferred by systemic neutralizing antibodies, protection against infection with influenza a virus has also been demonstrated following intranasal administration of vectored local antibody production [ ] . by far, the most extensive and promising exploration of vip for an infectious disease has been against hiv. in the initial study demonstrating the potential of vip, a recombinant adeno-associated virus (raav) vector using a dual-promoter system generated both light and heavy chains of igg b , one of the early broadly neutralizing antibodies described for hiv. the raav was injected into the quadricep muscles of immunodeficient mice and biologically active antibody was found in sera for over months [ ] . this study provided the first evidence that raav vectors could transfer antibody genes to muscle, and muscle tissue was a suitable platform to produce and distribute the antibodies throughout the circulation [ ] . follow-on studies used a native macaque siv gp -specific fab molecule as an immunoadhesin, a chimeric, antibody-like molecules that combine the functional domain of a binding protein with immunoglobulin constant domains, which were considered to be superior to single chain (scfv) or whole antibody (igg) molecules with respect to achievable steady-state serum concentrations [ ] . six of nine rhesus macaques were completely protected against intravenous challenge with virulent siv and still had stable immunoadhesin levels years after injection [ ] . the three subjects not protected were found to have developed an immune response to the immunoadhesin by weeks after injection [ ] . another group used an raav vector injected into the quadriceps muscle of a humanized mouse to express an array of broadly neutralizing antibodies: g , igg b , f , e and vrc . though vrc serum levels as low as . μg/ml provided the genetic sequences of the antibody variable regions are determined. c the genetic sequence for the bna can then be placed downstream from an appropriate promoter (prom) within a suitable vector. d the vector can then be administered to the subject in an appropriate tissue platform, such as muscle. the bna produced by the vector and secreted by the tissue confers the host subject with broad and lasting protection from the targeted pathogen protection from an intravenous challenge with hiv, they achieved concentrations as high as μg/ml for at least months [ ] . they followed-up that study by optimizing the broadly neutralizing antibody, and although muscle was chosen as a platform for expression and secretion of the igg isotype, antibodies were found to effectively reach the vaginal mucosa. animals receiving vip that expressed a modified vrc antibody (concentration of nearly μg/ml in the serum and μg/ml in vaginal wash fluid) were completely resistant to repetitive intravaginal challenge by a heterosexually transmitted founder hiv strain [ ] . saunders, et al., used an raav serotype vector to produce a full length igg of a simianized form of the broadly neutralizing antibody vrc in macaques which was protective against simian-human immunodeficiency virus (shiv) infection . weeks after treatment [ ] . shivs are chimeric viruses constructed to express the hiv envelope glycoprotein to be used in vaccine experiments to evaluate neutralizing antibodies. the antibody reached levels up to μg/ml for weeks, but immune suppression with cyclosporine was needed to sustain expression due to the development of anti-idiotypic antibodies [ ] . the approach to preventing hiv was enhanced further by fusing the immunoadhesin form of cd -ig with a small ccr -mimetic sulfopeptide at the carboxy-terminus (ecd -ig). ecd -ig is more potent than the best broadly neutralizing antiody and binds avidly to the hiv- envelope glycoprotein. rhesus macaques expressed - μg/ml of fully functional rhesus ecd -ig for more than weeks after injection with a self complimentary serotype aav (scaav ) vector and were completely protected from multiple challenges with a simian/human immunodeficiency virus, shiv-ad [ ] . of note, the rhesus ecd -ig was also markedly less immunogenic than rhesus forms of four well-characterized broadly neutralizing antibodies [ ] . in addition to disease prevention as noted above, studies have also demonstrated an application for vip in the effective treatment of previously-infected animals. using hiv- -infected humanized mice, horwitz, et al., demonstrated that following initial treatment with anti-retroviral therapy (art), a single injection of adeno-associated virus directing expression of broadly neutralizing antibody - , produced durable viremic control after the art was stopped [ ] . the first human trial using the vip approach started in january and is a phase , randomized, blinded, doseescalation study of an raav vector coding for pg , a potent broadly neutralizing antibody, in high risk, healthy adult males (clinicaltrials.gov number, nct ). another study evaluating using vip in hiv-positive subjects is scheduled to get underway soon [ ] . many options exist for vectoring the transgene into the host tissue, each with distinct advantages and limitations. naked plasmid dna is relatively easy to use, does not elicit significant immunogenicity, and has the potential for inexpensive large-scale production [ , ] . recent advances in both the mechanism of delivery [ ] and optimization of plasmid and electroporation conditions [ ] have improved the concentration and duration of antibody production, but it has yet to prove as potent as viral vectoring. viral vectors offer the advantage of efficient, rapid delivery of the transgene into host cells and the potential for integration into the host genome, allowing for sustained expression [ ] . the life cycle of a virus consists of attachment, penetration, uncoating, replication, gene expression, assembly and budding. replication and gene expression typically take place in the nucleus where viral genomes persist episomally or integrate into the host genome (i.e., a provirus). vectors that persist episomally can provide sustained transgene expression in post-mitotic tissue, but since they do not alter the host genome, they may be lost if and when the cells divide. vectors that integrate into to the host genome may provide life-long transgene expression in dividing cells but could also lead to insertional mutagenesis resulting in apoptosis or malignant transformation [ ] . adenoviral vectors produce rapid, but transient, gene expression that could be ideal for responding to a disease outbreak, but would have limitations for long term protection [ ] . adenovirus serotype (ad ) has successfully transduced protective antibodies for respiratory syncytial virus (rsv), influenza a virus (iav), and bacillus anthracis [ , , ] . the ad genome is easy to engineer and remains episomal, but there is significant pre-existing immunity to ad , estimated at % of the adult population worldwide and even higher in sub-saharan arica, which decreases the ability to transfer the transgene. additionally, it can result in systemic cytokine release creating a sepsis presentation and there is significant tissue tropism for the liver when delivered intravenously. alternative adenoviral vectors are being researched [ ] . lentivral vectors are better suited for long term expression since they typically integrate into the genome and can transduce dividing and non-dividing cells. they have successfully been utilized to transduce hematopoietic stem cells to produce broadly neutralizing antibodies against hiv in mouse models [ , ] . however, because they can integrate into the host genome, there is concern for mutagenesis. newer generation lentiviral vectors contain deletions in their long-terminal repeat (ltr) and a self-inactivating (sin) ltr, leaving them replication incompetent, which should make them much safer, but this question is not fully answered [ ] . although other viral vectors are being explored, raav vectors are currently the favored vehicle for delivering the antiody genes into the host tissue due to their efficiency in gene transfer [ ] . in contrast to other viral vectors, such as adenovirus, raav's have not been associated with any human diseases and do not stimulate signficant immunologic reaction, and are therefore able to induce long-term expression of non-self-proteins [ ] . they are engineered to consist of the antibody gene expression cassette flanked by the aav itrs (inverted terminal repeats), which are the only part of the aav genome present in the raav vector and are required for raav vector genome replication and packaging. despite a relativley small packaging capacity of kb, both heavy-and light-chain antibody genes can be incorporated into a single vector, either using a promoter for each gene cassette or a single promoter for expression with the heavy and light chain separated by a foot-and-mouth disease virus a peptide [ ] . all studies to date have targeted skeletal muscle as the platform for transfection and antibody production. muscle offers some significant advantages. it is easily accessible for localized vector administration, and some muscle groups can be removed in the event of mutagenesis or auto-immunity without functional consequence. however, muscle has certain disadvantages as well. it is a tissue that does not normally produce circulating proteins and therefore may not do it efficiently. it also contains antigenpresenting dendritic cells that could induce immune responses which might eliminate transduced cells or induce auto-immunity. additionally, the removal of muscle tissue would likely have a significant effect on a subject's lifestyle in the event of a potential unexpected vip-induced pathology. other platforms have been considered. for example, some authors have suggested the liver as an alternative site [ ] . unlike muscle, it is designed to secrete circulating proteins. it is also thought to be less immunogenic. however, transduction would require systemic administration of the vector, and there would be no simple means of eliminating expression in the event of a complication. another potential site could be the salivary glands. while it is well-know that the salivary glands secrete proteins into the oral cavity, it may be less well appreciated that they have also been used as a platform to deliver therapeutic proteins, including the igg fc fragment and a host of other proteins, into the systemic circulation [ , ] . transgenes delivered to the salivary gland tend to favor being sorted either into the saliva or the blood, though it is currently a challenge to predict which direction a particular protein will sort [ ] . the major paired salivary glands are also easily accessible, and the parotid glands are encapsulated, which minimizes vector spillage into the general circulation. futhermore, in the event of complications, the transfected glands could be removed without creating major disability. safety concerns associated with vip include genotoxic events typically associated with any viral vector mediated gene therapy, such as inflammation, a random insertion disrupting normal genes, activation of proto-oncogenes, and insertional mutagenesis [ ] . there are many factors which affect the likelihood of developing a genotoxic event including the vector, the targeted insertion site, the transgene, the targeted cell type, and host factors including age and underlying disease [ ] . the risk of genotoxicity or carcinogenicity can potentially be decreased by selection of the promoter and the integration site, using novel techniques such as clustered regularly interspaced short palindromic repeat (crispr)-cas (an rna-guided gene-editing platform that allows for cutting of dna in a specified gene), but much more work needs to be done to better characterize safety and efficacy of these methods [ ] . as the purpose of vip is to produce a monoclonal antibody, the possibility of producing a paraproteinemia similar to that caused by multiple myeloma, other hematologic malignancies, primary amyloidosis, or a monoclonal gammopathy of undertermined significance (mgus) is a concern. the most benign of these is mgus, but it has been increasingly recognized to have pathologic associations including is nephropathy secondary to monoclonal gammopathy of renal significance (mgrs), neuropathy, oculopathy, and dermopathy as well as possible associations with autoimmunity and coagulopathy and an epidemiologic association with early mortality from a variety of apparently unrelated causes [ ] [ ] [ ] . any of these conditions could result from a monoclonal gammopathy produced by vip. however, it should be noted that mgus is very common, occurring in % of the population older than years old, and most of these associations remain either unclear or uncommon [ ] . however, the potential for autoimmunity should be of particular concern. it is possible that the monoclonal antibody could interact with self-antigen and either stimulate an autoimmune antibody that interacts with self-antigen [ ] or neutralizes the intended effect of the monoclonal antibody. vectored immunoprophylaxis has demonstrated great promise in a variety of pre-clinical studies as a potential adjunct to vaccination in patients not able to respond effectively to immunization or as an alternative to vaccination for infectious diseases not effectively covered by current vaccines. the rapid identification of specific neutralizing antibodies is likely to increase the potential for this method. one could imagine uses for vip such as an adjunct to vaccination for influenza in the elderly and immunocompromised, for hiv protection in high risk populations, or as part of a ring vaccination strategy in an outbreak of a disease such as ebola. many important questions remain, including the ability to produce equally effective clinical results in human trials, the duration of response, and the potential for side-effects. mutagenesis at the site of transfection is a common concern, but the development of an immune response to the transgene product or the off-target binding of the antibodies are more likely scenarios, either of which could result in decreased efficacy of the procedure or a significant auto-immune reaction. questions also remain concerning the best vector and the optimal tissue site for transfection. despite these questions and concerns, the advantages offered in settings ranging from chronic protection of the aged or immunocompromised to rapid protection for early responders in the event of a bioterror or emerging infection event are significant and intriguing. further preclinical and clinical studies are certainly warranted. engineering humoral immunity as prophylaxis or therapy vaccine-preventable disease table working g. historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states contributions of humoral and cellular immunity to vaccine-induced protection in humans hark back: passive immunotherapy for influenza and other serious infections meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h n treatment? passive immunization against hiv/aids by antibody gene transfer human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo plant-based vaccines against viruses emerging vaccine technologies. vaccines (basel) generation of neutralizing activity against human immunodeficiency virus type in serum by antibody gene transfer vector-mediated in vivo antibody expression antibody-based protection against hiv infection by vectored immunoprophylaxis broad protection against influenza infection by vectored immunoprophylaxis in mice passive immunization with a recombinant adenovirus expressing an ha (h )-specific single-domain antibody protects mice from lethal influenza infection vectored antibody gene delivery protects against plasmodium falciparum sporozoite challenge in mice broadly neutralizing antibodies abrogate established hepatitis c virus infection genetic delivery of an anti-rsv antibody to protect against pulmonary infection with rsv rapid/sustained anti-anthrax passive immunity mediated by co-administration of ad/aav protection against dengue disease by synthetic nucleic acid antibody prophylaxis rapid and long-term immunity elicited by dna-encoded antibody prophylaxis and dna vaccination against chikungunya virus intranasal antibody gene transfer in mice and ferrets elicits broad protection against pandemic influenza vector-mediated gene transfer engenders long-lived neutralizing activity and protection against siv infection in monkeys vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission broadly neutralizing human immunodeficiency virus type antibody gene transfer protects nonhuman primates from mucosal simian-human immunodeficiency virus infection aavexpressed ecd -ig provides durable protection from multiple shiv challenges hiv- suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice monoclonal antibodies produced by muscle after plasmid injection and electroporation a novel electroporation device for gene delivery in large animals and humans optimized and enhanced dna plasmid vector based in vivo construction of a neutralizing anti-hiv- envelope glycoprotein fab interaction of vectors and parental viruses with the host genome development of novel adenoviral vectors to overcome challenges observed with hadv- -based constructs inhibition of in vivo hiv infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-hiv antibody engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-hiv antibody after in vitro maturation to human b lymphocytes aav vectors vaccines against infectious diseases viral vectors take on hiv infection advances in salivary gland gene therapy -oral and systemic implications in vivo secretion of the mouse immunoglobulin g fc fragment from rat submandibular glands general considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination viral vectors: the road to reducing genotoxicity monoclonal gammopathy: the good, the bad and the ugly criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the international myeloma working group monoclonal gammopathy of renal significance: when mgus is no longer undetermined or insignificant none. none. availability of data and materials not applicable.authors' contributions jws and tap contributed equally to the development of this manuscript. both authors read and approved the final manuscript. the authors declare that they have no competing interests. ethics approval and consent to participate not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -nrodyagi authors: schutzer, steven e. title: the use of host factors in microbial forensics date: - - journal: microbial forensics doi: . /b - - - - . - sha: doc_id: cord_uid: nrodyagi advances have been made in the forensic analysis of microbes and toxins. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. principal methods that are sufficiently developed are characterization of the humoral immune response to microbial antigens including vaccine-induced immunity and detection of antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a representative example, which, in the appropriate context, has a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim. this chapter also presents information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. considerable advances have been made in the forensic analysis of microbes and toxins. these advances include sequencing, genomics, and microscopy. an underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. for forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. two principal methods that are sufficiently developed are characterization of the humoral immune response and identification of vaccine-induced immunity or antibiotics that may be present in a possible perpetrator. this chapter presents central elements of the host response in a simplified fashion and describes a few representative examples, which, in the appropriate context, have a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim who has been exposed to a particular microbe or by-product, such as a toxin. this chapter also presents nonmicrobial forensicedirected information about the immune system so that the interested reader can have a fuller understanding of the immune response in general. the primary aims of a microbial forensics are to identify the biological agent, its source, and the individuals responsible for the event (budowle et al., ) . analytic approaches differ when the suspected biothreat agent is encountered in a container or the environment, as opposed to in vivo in a human, animal, or plant. analyses of trace elements, pollens, growth media, latent fingerprints, and microbial and nonmicrobial nucleic acids are all applicable to the container and environmental sample (states et al., ) . however, once the microorganism or its toxin is in the living host, it is no longer possible to analyze the preceding items except the microbial nucleic acid. however, the host's response to the biological agent may be available for analysis for clues. this is akin to other forensic studies where physical traces of bite marks, scratches, wound trajectories, and sizes of wounds are often surrogate evidence of the teeth, fingernails, and bullets (averill and odontology, ) . while the forensic pathologist is familiar with evidence related to determining the manner of death including the host response, those involved with healthcare alone are more familiar with the host response. in the context of microbial forensics, it is important to integrate all of these with intelligence information so that they may be included in the analytical data and attribution picture. the physician and other healthcare providers may be among the first to realize that a patient is a victim of a biocrime. in the case of a covert attack, it may be the physician or medical examiner who first recognizes the index case. these healthcare workers are in key positions to preserve critical evidence and, thereby, contribute to the investigation (schutzer et al., ) . there are a number of steps that should be followed when the possibility of a biological attack arises, either with the consent of the patient or because individuals are compelled by law to interact with public health and law enforcement. a joint statement by the fbi, the cdc, and the dhs advises calling the fbi and public health authorities if a suspicious situation arises (investigation et al.) . some guidelines on the procedure(s) to report of suspicions of biocrimes are provided by the centers for disease control and prevention (cdc; http://www.cdc.gov), the federal bureau of investigation (fbi; http://www.fbi.gov), and the department of homeland security (dhs; http://www.dhs. gov) and detailed in previous article (schutzer et al., ) . the host response to a microorganism or other foreign substance is often a wellorchestrated series of events, which may protect the individual from harm (zabriskie, ). at the same time, these host responses may provide clues as to identity of the offending microorganism or toxin as well as a rough chronology of when it occurred and for how long it has been persisting. emerging technologies such as transcriptional arrays and bioinformatic analysis will eventually be refined and methods validated to provide even greater help in delineating more of the pathways and components of the host response to an infectious agent (sala et al., ; popper et al., ; ko et al., ) . other technologies are sufficiently mature to be of use today. the immune system and its components are a mainstay of our protection against infections and malignancies (zabriskie, ; paul, ; murphy and weaver, ) . inflammation is often a side effect as the immune system contains and eradicates a microorganism or eliminates foreign tissue. specific arms of the immune system can be used as markers in support of or against the presence of an infection. the humoral or antibody response to an invading microorganism is one example of a specific arm that can have forensic value. some of the antibodies produced may have a protective role together with other parts of the immune system by eradicating the pathogen or neutralizing a toxin. other antibodies may not be as effective in this role. however, in their ability to recognize unique and specific microbial antigens, they can serve as indicators that a specific microorganism was recently present or was present in the past. in the case of a vaccine, specific antibodies may recognize highly specific epitopes of one microbe versus those of a related microbe (e.g., influenza virus). this is especially so with different recombinant vaccines and could have forensic importance. substances such as antibiotics, which can rapidly kill a pathogen, may modify the immune response by removing or reducing the infectious driving force for a full-scale response. as noted above, in clinical and veterinary medicine, measurement of the immune response helps the diagnostician decide what infection was present and how recently. in these situations, the intent is to provide treatment. the forensic scientist may exploit parts of the immune response to discover who is likely a victim of an attack and who might be responsible. this chapter will discuss the basics of the host immune response in a simplified manner that can have utility in a forensic sense. examples will provide a sense of what information is achievable and what is . the use of host factors in microbial forensics not likely to provide clues with a high degree of certainty. in response to a new exposure to a microbe, the innate immune system may be the first line of defense. then the immune system starts to activate the antibody system. typically, a cell known as a macrophage ingests and degrades some of the invading pathogens. it then presents part (antigens) of the microorganism to a helper t cell (a lymphocyte), which then directs other lymphocytes known as b cells to produce antibodies to those antigens of that particular microbe that were presented. it usually takes at least days before any microbe-specific antibody can be detected (parslow, ) . antibodies are a specific form of the proteins known as immunoglobulins (igs). igm, igg, iga, and secretory iga are the principal classes of immunoglobulins with prime relevance to this chapter and will be discussed in more detail. in an infection, immunoglobulins usually appear in the order of igm, igg, and iga. b cells first begin to produce igm, and then some b cells undergo an irreversible switch to those that produce igg. later, some of these b cells undergo a switch to become iga-secreting b cells. immunoglobulins persist for varying times; for example, the half-life of particular igm antibodies is approximately days, while that of igg can be as long as e days (table . ) (paul, ; murphy and weaver, ) . in certain circumstances of ruling in or ruling out a suspect, the specific ige may be of value. those individuals unfortunate to have allergies have problems due to ige against allergens (such as ragweed, peanut, or cat dander). in this case, the ige molecules sit on the surface of mast cells and basophils. these cells can release histamine and other allergic mediators when the offending allergen bridges two ige molecules. similar to the effect from an infection with a live microbe, vaccines are often designed to provoke an antibody response. the vaccine can be composed of a live or attenuated microbe, a whole nonproliferating microbe, or an antigenic (recombinant) part of the microbe or a toxoid. regardless, the intent of immunization with a vaccine is to engender protection, often by the generation of protective neutralizing antibodies. although the half-life of an individual igg molecule is less than a month, a population of antibodies of the igg isotype form may persist for life. memory b cells can sustain these antibodies and retain the ability to quickly generate the appropriate antibodies when challenged. when the immune system encounters another infection or is subjected to a revaccination (booster), the result is an accelerated production of the particular antibody and increase in the levels of antibodies that circulate in the blood ( fig. . ) . perhaps, the pattern of antibody response which has the most forensic value, by providing a timeframe, is the appearance of igm first, followed by a b-cell switch to the longer-lasting igg. during the early phase of exposure, igm predominates, as time goes on, igg may wax and wane and igm is no longer found ( fig. . ) . the antibody response to a particular agent may be directed to different antigens at different times, that is, early or later after the initial exposure. that response often involves igm at the early stage and igg later. late in the course of (bennett et al., ) , a virus known to cause mononucleosis. during acute early disease, it is common to find high levels of antibody of the igm isotype to the viral early antigen (ea) and viral capsid antigen (vca). it is rare to find igg antibody to the vca or to epsteinebarr nuclear acid (ebna) in anything but low titers (levels). as the patient recovers from their first infection with ebv, it is rare to find anything but low levels of igm to ea or vca, but igg to vca in higher or increasing levels is common. antibodies to ebna are often very low during this stage. several months after clinical recovery, igm to ea and vca remain at low levels, whereas igg to vca and ebna are present at high levels, often for years. a controlled experiment or normal clinical event illustrates what happens when the immune system responds to the infectious agent or a vaccine again. the controlled experiment may be in a laboratory animal or a patient illustrative concepts receiving a booster vaccine. the uncontrolled but normal clinical event occurs when the patient is reexposed to the infectious agent. consider a generic antigen exposure. the first time the immune system encounters antigen x (agx), it responds as shown in figs. . and . . initially, antibodies to agx are barely discernible; then levels rise and later fall to a plateau. if a simultaneous exposure were to occur with agx and a new agy from another microorganism, the immune system would quickly mount a brisk response with high levels of ab to agx, while the course of ab to agy would be slow and delayed, just as it was in the response to the first exposure to agx. this phenomenon is termed immunological memory or an amnestic response. this can be useful when the symptoms and signs of exposure to either x or y are similar. this is the case with the early flu-like symptoms of pulmonary anthrax (raymond et al., ; waterer and robertson, ; bush et al., ) and with the influenza virus itself (meltzer et al., ; cao et al., ; lessler et al., ) . another example common to all of us is repetitive exposure to different strains of flu viruses (meltzer et al., ; janeway, ) . as illustrated in table . , a person infected for the first time with one strain of the influenza virus has a response to most of its antigens (as a theoretical example, ag , , , , , ). three years later, the same individual exposed to a partially similar influenza virus responds preferentially to those antigens that were also present on the original influenza virus. the person also makes a smaller initial antibody response to new antigens, that is, those not shared with the first virus. ten or years later, during a new flu season and exposure to a third strain of influenza, the most brisk responses would be to antigens previously recognized by the immune system. this is the scientific basis for giving the flu vaccine, which contains a variety of possible antigens common to multiple strains of the flu virus so that a rapid and protective antibody response will occur. utility of serologic analysis of people exposed to anthrax: strengths and limitations our knowledge of the humoral response to infection with biothreat microbes is limited compared with our knowledge of the kinetic response to common human infections. nevertheless, in the appropriate context and with sufficient background information, detection of antibodies to a particular microbe and its antigens can have important value for a microbial forensic investigation. this information may have critical probative value or it can guide investigative leads. the absence of a specific antibody response may also have value in a particular investigation. certainly, its importance is increased in the context of information of what organism could be involved, when the exposure was likely to have occurred, the route of exposure, what symptoms and signs are manifesting in the host, and other data such as presence of antigens and microbial nucleic acids (jackson et al., ) . other information such as how many hosts (people or animals) have had this infection in the geographic region, what is the incidence, and background prevalence of antibody titer to the organism in question or a related organism, in the population being studied, is also important. vaccination responses can have forensic value. the current protective antigen (pa) vaccine has small amounts of ef and lethal factor (lf), which are responsible for some of the side effects, so one might expect to see antibodies against these antigens as well as to pa. the recombinant pa is just pa so anti-lf and anti-ef would be absent in an immunized individual. the anthrax-letter attacks raised multiple questions for every person infected, possibly exposed, vaccinated, or treated. some of these questions included how these persons were infected by spores, if at all; that is, through breaks in the skin (cutaneous anthrax); by inhalation of spores (pulmonary anthrax (bennett et al., ) ), or by ingestion (gastrointestinal anthrax (bravata et al., ; tutrone et al., ) ). or, were they among the "worried well"? consider the situation where a close associate comes down with symptoms compatible with inhalational anthrax after receiving a letter containing powder and that material is no longer available. until this is shown not to be anthrax, great worry will ensue. in several cases of documented exposure, there was not enough time for the patient to develop antibody to a specific anthrax antigen, at least as probed for igg. serial serum samples obtained on november , , , and of were tested for igg antibody to the pa component of the anthrax toxins by enzymelinked immunosorbent assay (elisa); all samples were nonreactive. serial tests for serum igg antibody to the pa toxin of anthrax were performed on workplace-exposed persons. all but one test was negative. most of the specimens were collected on october and (traeger et al., ) . it is instructive to look at the positive antibody case in the context of the nature and utility of serologic analysis of people exposed to anthrax: strengths and limitations duration of that individual's symptoms when he developed a positive test. ernesto blanco, a -year-old mailroom clerk (case ), experienced fatigue on september . he worked in the mailroom of the ami building and delivered mail to the index case. on september , he developed a nonproductive cough, intermittent fever, runny nose, and conjunctivitis. these signs worsened through october when he was hospitalized. in addition, he had shortness of breath with exertion, sweats, mild abdominal pain and vomiting, and episodes of confusion. his temperature was elevated to . c ( . f), heart rate was rapid at /min, respiratory rate was slightly fast at /min, and blood pressure was / mm hg. he had bilateral conjunctival injection and bilateral pulmonary rhonchi. at the time of admission, his neurologic exam was normal. no skin lesions were observed. the only laboratory abnormalities were low albumin, elevated liver transaminases, borderline low serum sodium, increased creatinine, and low oxygen content in the blood. blood cultures were negative on hospital day , after antibiotics had been started. the chest x-ray showed a leftsided pneumonia and a small left pleural effusion but no "classical" mediastinal widening (dewan et al., ) . the patient was initially given intravenous azithromycin; cefotaxime and ciprofloxacin were subsequently added. a nasal swab obtained on october grew bacillus anthracis on culture. computed tomography (ct) of the chest showed bilateral effusions and multilobar pulmonary consolidation but still no significant mediastinal lymphadenopathy. pleural fluid aspiration was positive for b. anthracis dna by pcr. bacterial cultures of bronchial washings and pleural fluid were negative. immunohistochemical staining of a transbronchial biopsy demonstrated the presence of b. anthracis capsule and cell wall antigens. during hospitalization, his white blood count rose to , /mm , and fluid from a second thoracentesis was positive for b. anthracis dna by pcr. immunohistochemical staining of both pleural fluid cells and pleural biopsy tissue demonstrated the presence of b. anthracis capsule and cell wall antigens. serial serum samples demonstrated > fourfold rise in serum igg antibody to the pa component of the anthrax toxins by an elisa assay. the patient was able to leave the hospital on october on oral ciprofloxacin. table . illustrates both the clinical and microbial forensic approach and context in which to analyze such a patient. it is likely to be common to most situations where a biocrime ( ). these questions include was the infection acquired naturally or was it an intentional action that led to the infection; how did the particular individual acquire it if it was not a natural infectiondwas he the target or a bystander. an alternative possibility in the right circumstances is a laboratory-acquired infection. this case also demonstrates that cultures may be negative at different times from different fluids and tissues because of early administration of antibiotics. however, the remnants of the infection, even dead organisms, can be found by probing for antigens and dna. this patient's response demonstrated a classic principle of infectious disease, a rising antibody titer over time. in this case, it was igg to a particular antigenic component of the anthrax toxins (friedlander and little, ; cunningham et al., ) . the subject's antibody response may have been detected earlier if igm to this component or to other antigens of anthrax had been sought. the case also points out the utility of integrating the presence of antibody with other indications of an anthrax infection such as culture, pcr, and antigen detection. these take on their greatest significance during clinical illness in someone who was possibly exposed. early administration of antibiotics can prevent or interfere with the isolation of a pathogen by culture (kaeberlein et al., ) . of the first pulmonary anthrax cases associated with the letter attacks, three patients had no isolate of b. anthracis from any clinical samples; however, culture was attempted after initiation of antibiotic therapy. history of exposure in conjunction with compatible symptoms and signs of disease and objective laboratory findings were the basis for the diagnosis. b. anthracis was identified in pleural fluid, pleural biopsy, or transbronchial biopsy specimens by reactivity with b. anthracis-specific cell wall and capsular antibodies or by the detection of dna in pleural fluid or blood by pcr (jernigan et al., ) . it is important to understand the limitations of any assay used in medicine or forensics (budowle et al., ; schutzer et al., ). an igg-based elisa for anti-pa illustrates the importance of understanding the limitations of an assay. the elisa was developed at the us army medical research institute of infectious disease (usamriid) and put into operation after optimization and internal validation at the cdc for functional sensitivity and specificity in detecting an antibody response to b. anthracis infection. its major limitation was that only one antigen was used and only igg was measured. therefore, a negative result shortly after exposure may, in effect, be a false-negative result. a gap such as this may be filled by development of an assay for antigenspecific igm, and by probing for other b. anthracis antigens or epitopes yet to be characterized. the assay may be very useful in its present form to screen asymptomatic people with possible exposure. the study by dewan et al. (dewan et al., ) provide a contemporary background database on a group of postal workers who may have been exposed to b. anthracis. beginning on october , , postal employees and others who had been to the washington d.c. postal facility went to the d.c. general hospital for antibiotics in addition to those people whose treatment began on october , . serum samples were also obtained from the individuals who had been to the washington d.c. postal facility during the precious weeks. all were negative for specific anti-pa igg, including three individuals who reported a remote history of anthrax vaccination. the consistent negative findings may be explained by the fact that antibiotic therapy was initiated before serum testing and that there were no baseline serum samples available for testing. in addition, the time period from exposure to sampling was very short. among individuals in the capitol region with culture-positive nasal swabs who received prophylactic antibiotics immediately, none had a positive culture from a nasal swab repeated days later, and none developed igg to pa antigen days after exposure. this again emphasizes the limitation and interpretation of a test in someone who had early antibiotic treatment. it does raise forensic utility considerations. even with these easily disseminated spores, an antibody response may be aborted or modified with antibiotics by early eradication. furthermore, antibiotics taken before exposure would likely be effective in preventing laboratory and clinical signs of an infection. detection of microbial dna, antigen, or the organism itself on a person's body, clothing, or possessions should raise a red flag for exposure. the route of infection is important in interpreting results and the limitations of the assay used. the example of cutaneous anthrax in paraguay illustrates this notion, as well as the need to search for other antigens as markers of exposure (harrison et al., ) . in an analysis of an outbreak of cases of cutaneous anthrax that followed contact with raw meat from a sick cow, sera from cases and colony and noncolony controls were examined by western blot for antibodies to pa and lf weeks after the outbreak. an elisa was used to probe for antibodies to the poly-d-glutamic acid capsule. of the cases, had antibody to pa, for a sensitivity of . %; none of the controls was positive. only of cases had antibody to lf; all controls were negative. anticapsule antibodies were positive in of but were also positive in of controls. the results of this study demonstrate the need to consider other antigens. some of the principles discussed above are highlighted by a report on severe acute respiratory syndrome (sars). the appearance alone of this coronavirus responsible for this disease evoked concern of a possible terrorist origin at the onset. a report in the morbidity and mortality weekly report (mmwr (cfdcap, ) ) on the "prevalence of igg antibody to sars-associated coronavirus in animal traders" discussed the need to validate and interpret tests in appropriate populations. also discussed was the inability to date the time of infection by the igg assay, and the possibility of assay crossreactivity to a near neighbor that might be unknown. in a promed bulletin, dr. steve berger looked at the same data from a different perspective and reported "this week's study in mmwr indicates that animal contact may indeed promote infection; however, the most striking finding seems to have eluded the authors: . percent to . percent of individuals in a healthy control group of adults were also found to be seropositive! the population of guangdong province is . million ( ), of whom . million are adults over age . if we assume that the seropositivity rates among controls is representative of the province as a whole, , to , , adults in guangdong have at some time been infected by the sars virus. these figures are -to -fold the total number ( ) of sars patients reported worldwide to date!" this comparison is a good illustration of the advantage of open dissemination and discussion of information as well as the need to question the methodology of acquisition of data before accepting their application in formulas or for analyses for forensics and epidemiology. it is also of value to remember that many infections include many with sars coronavirus have been asymptomatic or mildly symptomatic. plague, is a zoonotic infection caused by yersinia pestis, which occurs in the western united states with regularity and has an animal reservoir (bennett et al., ) . the situation with the naturally occurring yersinia is in contrast to the appearance of a case of smallpox which would raise an immediate red flag for a bioterrorist event. cases need to be approached from an epidemiologic standpoint first to determine whether it is a naturally acquired case or whether the facts point to a deliberate introduction of the organism. analytic techniques could include genomic analysis of an isolated organism and immunological response of the host. in the new era of rapid and deep sequencing, our capacity to investigate the genomics is growing (mardis, ; stavnsbjerg et al., ) . in consideration of animal reservoirs, elisa assays were compared with other tests for detection of plague antibody and antigen in multimammate mice (mastomys coucha and. mastomys natalensis) (shepherd et al., ) , which were experimentally infected and then sacrificed at daily intervals. igg elisa was equivalent in sensitivity to passive hemagglutination and more sensitive than the igm elisa and complement fixation. antibody was detectable by day after infection using all four tests. igm elisa titers fell to undetectable levels after weeks. plague fraction antigen was detected in of bacteremic sera from m. coucha and m. natalensis. this antibody pattern comparison shows that the principle of igm versus igg to this pathogen works to temporally situate the infection as an early versus late or past event. it also shows that when the information is combined with antigen detection, it engenders more confidence in the results. it should be noted that conclusions from this older reference has been substantiated with more defined antigens and assay technologies. melioidosis is caused by burkholderia pseudomallei (ashdown, ) . key clinical signs and laboratory results may raise the possibility of an infection with this pathogen. whether it is an acute, persistent, or past infection can be determined by assessing several host responses. often a simple indicator such as erythrocyte sedimentation rate or c-reactive protein (crp) can raise a clinical suspicion of an infection. in a study of patients with clinical melioidosis, ( culture-positive and culturenegative) had relatively uneventful disease courses. initially, they had elevated serum crp that decreased with antibiotic therapy and returned to normal as the disease resolved. in another series of patients, igm and igg were measured by elisa in sera from septicemic cases and sera from cases with localized melioidosis (chenthamarakshan et al., ) . sixty-five sera from culture-negative cases seronegative for other endemic infections but suspected of melioidosis were also examined. other controls included serum from nonmelioidosis cases, high-exposure risk cases, and healthy individuals. the igg-elisa was % sensitive and % specific. all sera from cases with septicemic and localized infections and of sera from clinically suspected melioidosis cases were positive for igg antibody. the sensitivity and specificity of the igm elisa were % and %, respectively. a geometric antibody index for igm antibody in the sera of the melioidosis cases was significantly higher in cases compared with that of the noncase controls. in another study by some of the same authors, a rapid test for igg and igm was shown to have clinical utility (cuzzubbo et al., ) . a study with the intent of evaluating the utility of an igg assay compared with other assays illustrates how the clinical and temporal context must be integrated for interpretation (dharakul et al., ) . it also illustrates how there is room for technical improvement in tests but the best setting is often the endemic area itself or at least using samples from that area in which the infections are occurring. these tests were evaluated in the actual clinical setting in an area endemic for melioidosis. specificity of igg ( . %) and igm ( . %) assays was significantly better than that of an indirect hemagglutination test (iha) ( . %). the sensitivity of the igg assay ( . %) was higher than that of the iha test ( . %) and the igm test ( . %). specific igg was found in septicemic cases ( . %) and localized infections ( . %). the igg test was also better than the igm test and the iha test in identifying acute melioidosis cases in the first days after admission. igg antibody to a b. pseudomallei antigen remained high for longer than years in recovered, disease-free patients. because this is a disease that may have an incubation of days to years, an acute case may very well be detected by a rise in specific igm if it were a matter of days from infection. although endemic for southeast asia, if b. pseudomallei was used as a biothreat agent in a different environment, its course and manifestations may not be recognized due to unfamiliarity with the disease. the above example also points out how the context in which a test is used determine is valuable. the concept of predictive value is instructive in determining how useful a test may be. in terms of disease detection, a high positive predictive value indicates the test is useful in determining that the disease is present. a high negativity predictive value would indicate that the test is useful in excluding the presence of the disease. another zoonotic agent is rift valley fever virus (rvfv), which can be transmitted via aerosols (clark et al., ) . one study with the intent at looking for improved tests showed the utility of igm to determine an early exposure to rvfv (niklasson et al., ) . two elisa igm tests detected specific igm antibodies to rvfv during the first weeks after vaccination. three inactivated vaccine doses were given on days , to , and to . elisa serum igm on days e were negative or in the lower range of detection; on days e the serum antibody values were strongly positive; on days e , they were waning and in later collected samples were negative. the plaque reduction neutralization test was negative on days e and became positive in later samples. similar to the examples shown above, these data suggest that three doses of rvfv vaccine induced a prolonged primary antibody response. the authors of that study concluded that the elisa igm may be useful for early diagnosis of acute human infection. good correlation of a neutralization test and elisa igg would indicate a later infection. taken together, these examples illustrate that an ideal test or analysis for both clinical and forensic use would incorporate endemic and incident area controls, historical contextual information, knowledge of the route of exposure, background incidence, and kinetics of transmission. each of these scenarios must take into account multiple factors and the limitations of any analytic process to be applied. on one extreme is the situation that occurred with the onset of acquired immunodeficiency syndrome (aids) from the human immune deficiency virus (hiv) in the united states. initially, there were no cases, and therefore a precise highly sensitive and specific test with excellent positive and negative predictive values (such as exists now when a combination of tests are used) would not likely yield a positive result in an area where there was little hiv infection and disease at the onset such as kansas. a positive test by today's methodologies from a serum sample from kansas would be considered a probable falsepositive and warrant further investigation. today, several viral and nucleic acid assays are available that would provide a definitive diagnosis in a short period of time (bennett et al., ) . however the same sample tested at the beginning of hiv testing could have been positive if the person had adult t-cell leukemia, which is caused by human t-cell leukemia virus- (htlv- ) because the original tests for what became known as aids involved whole viral lysates in which up to % of the htlv- sera cross-reacted. questions regarding the interpretation of the test results could be raised by knowledge of different presentations of the infection. for example, htlv- can actually be used in the laboratory to immortalize cells. in the patient, it actually increases the t-cell count, as is the nature with leukemia, instead of decreasing them, as with hiv infections. other laboratory indicators such as hypercalcemia would now raise the leukemia as a consideration. interpretation of a positive laboratory test must also take into account the health status of the person being tested. this is important for the practice of medicine and can have relevance when extended to forensic analysis (schutzer et al., ) . the following examples illustrate this concept. individuals who have syphilis, a treponemal bacterial infection, can typically have a positive fluorescent treponemal antibody test result for years, even after successful treatment. however, while infected they would have a positive venereal disease research laboratory (vdrl) test, which reverts to negative following successful antibiotic therapy. the vdrl test detects nonspecific anticardiolipid antibodies and can produce false-positive results with other conditions (e.g., pregnancy). there are some notable exceptions related to crossreactive epitopes or autoimmune diseases. these are readily distinguishable by history and clinical information. similarly, individuals infected with active tuberculosis will likely have a positive skin test (mantoux) or a positive interferon-gamma release assay (dewan et al., ; ota and kato, ) , whereas the uninfected healthy person will be negative. in certain instances, a sick person with a cell-mediated immune deficiency will be anergic, that is, he/she will be negative to multiple skin tests including common antigens such as candida. the key difference here is that there is a great difference between the healthy person being tested and an ill or immunocompromised individual being subjected to the same test. tests may also discriminate between the length of the infection (i.e., acute or chronic); limitations of these tests may lead to different interpretations unless one is familiar with those limitations. an example of this occurred with the bacterial infection of borrelia burgdorferi, which causes lyme disease. antibiotics can abrogate the antibody response because elisa results were negative in % of patients with known disease who were treated early (dattwyler et al., ) . in early cases, reactivity to a unique antigen, ospa, was also negative in serological assays despite a demonstrable t-cell possible scenarios of bioterrorism attacks: distinguishing victims from perpetrators response (krause et al., ) . analysis of these same sera found that there was antibody to b. burgdorferi, but it was below the threshold of detection by conventional assays. it was detectable in its bound form, in immune complexes (schutzer et al., ; schutzer and coyle, ) . anthrax can be used as an example where investigatory leads can be generated by considering a scenario in toto. the elderly woman who died in connecticut from inhalation of anthrax clearly had no occupational exposure nor was she known to have had contact with anyone who had anthrax. it was possible that she had contact with cross-contaminated mail. however, if this case had occurred as the index case or out of context of the mail attacks, it would have been reasonable to question her travel history, what her work if any was, or if she received or used spore-contaminated products from an anthrax endemic area. similarly, the vietnamese woman who died of inhalation anthrax in new york city would also have had these questions investigated. it would have been useful to search for direct or indirect evidence of anthrax by physical examinations of her contacts or close neighbors. inspection and cultures from her workplace, apartment, and apartment complex (especially contiguous neighbors) are important for detecting the presence of b. anthracis. coworkers, friends, neighbors, and other contacts could have had their serum analyzed for antibody to antigens of b. anthracis. these samples could have been frozen so that if one were positive it would be available for a comparison study in the future. at a minimum, these types of studies could serve as future control data for the geographic region. with molecular methods, even trace amounts might be detectable (lasken and egholm, ) although parallel investigation as to background control would be necessary. although hypothetical, several results could have occurred, and each will be considered separately. first example, a close contact is positive for igm to one of the b. anthracis antigens, such as pa. this finding would suggest that this person had recent exposure and, if nothing else, should be treated. this individual could conceivably be the one who knowingly or unknowingly passed the spores to the patient. given the october onset of illness, which is late in the mailing sequence, it would be less likely that this individual was a perpetrator but rather a recent victim. however, if this person were igg-positive, then there are several other possibilities. perhaps, this person had past exposure in an endemic region and was treated (e.g., haiti, where anthrax is known as "charcoal disease"). or this person could have been vaccinated for bona fide reasons such as a researcher who received it for occupational exposure. or this person could have obtained the vaccine originally for legitimate or illegal purposes but was nevertheless vaccinated. the vaccine usage may have been for a clinical trial or animal experimentation. animal vaccines may be more obtainable without strict record keeping. this person could have loaded the mail with relative impunity if there was protective antibody generated from the vaccination. situations similar to this one will require intelligence information regarding access, ability, and motive. in an area where recombinant vaccines are being developed or used antibody response would be different between someone using one type of recombinant vaccine as compared with someone using another type of vaccine. nevertheless, finding igg to one or more antigens of b. anthracis could point investigators toward such a seropositive individual, whereas an igm finding could justify critical therapy. where information points to a particular individual, investigation could be extended to search for ingestion or injection of antibiotics as illustrated below in the ciprofloxacin example. questions would be raised regarding access to antibiotics, recent ingestion/injection of them, half-life of the antibiotic, half-life of the metabolites of the antibiotics, and in which body fluids or tissues can the residual be found. as illustrated from . the use of host factors in microbial forensics the data in the earlier sections, someone with antibiotics in their system may be protected following exposure to a potential pathogen. this person would be antibody-negative and likely antigen-and microbial dna/rnanegative, because the infection would have been eradicated before the organism can proliferate in any significant quantity. the widespread prophylactic use of ciprofloxacin during the period following the anthrax mailing attacks is illustrative of an understudied area. ciprofloxacin has been increasingly associated with tendonitis and ruptured achilles tendons (akali and niranjan, ; palin and gough, ; godoy-santos et al., ) . in the future, better methodology to follow the pharmacokinetics of an antiinfective compound may have forensic implications. in the last example, someone who takes an antibiotic prophylactically while manipulating a lethal microbe may exhibit side effects that in the proper context of an investigation may add to the picture of possible culpability. this area is far from established at this point in time. strategies can be employed to examine suspicious but possible accidental transmission of infections. this approach is illustrated by a recent study of avian influenza using a multitude of assays. tools to determine person-to-person spread as the mode of transmission included viral culture, serologic analysis, immunohistochemical assay, reverse transcriptasee polymerase chain reaction (rt-pcr) analysis, and genetic sequencing (ungchusak et al., ; meinel et al., ) . it is likely that future understanding of the immune system and evolving technologies will bring new analytic power to the field, but in the interim we can make good use of proven principles for forensic purposes. serial serum c-reactive protein levels as an aid to the management of melioidosis management of bilateral achilles tendon rupture associated with ciprofloxacin: a review and case presentation manual of forensic odontology mandell, douglas, and bennett's principles and practice of infectious diseases inhalational, gastrointestinal, and cutaneous anthrax in children: a systematic review of cases index case of fatal inhalational anthrax due to bioterrorism in the united states clinical features of the initial cases of pandemic influenza a (h n ) virus infection in china detection of immunoglobulins m and g using culture filtrate antigen of burkholderia pseudomallei systematic literature review of rift valley fever virus seroprevalence in livestock, wildlife and humans in africa from mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen evaluation of a new commercially available immunoglobulin m and immunoglobulin g immunochromatographic test for diagnosis of melioidosis infection seronegative lyme disease. dissociation of specific t-and b-lymphocyte responses to borrelia burgdorferi inhalational anthrax outbreak among postal workers diagnostic value of an antibody enzyme-linked immunosorbent assay using affinitypurified antigen in an area endemic for melioidosis advances in the development of next-generation anthrax vaccines fluoroquinolones and the risk of achilles tendon disorders: update on a neglected complication evaluation of serologic tests for diagnosis of anthrax after an outbreak of cutaneous anthrax in paraguay pcr analysis of tissue samples from the sverdlovsk anthrax victims: the presence of multiple bacillus anthracis strains in different victims immunobiology the immune system in health and disease bioterrorism-related inhalational anthrax: the first cases reported in the united states isolating "uncultivable" microorganisms in pure culture in a simulated natural environment what was old is new again: using the host response to diagnose infectious disease cellular immune reactivity to recombinant ospa and flagellin from borrelia burgdorferi in patients with lyme borreliosis. complexity of humoral and cellular immune responses whole genome amplification: abundant supplies of dna from precious samples or clinical specimens outbreak of pandemic influenza a (h n ) at a new york city school next-generation dna sequencing methods whole genome sequencing identifies influenza a h n transmission and offers superior resolution to classical typing methods laboratory surge capacity and pandemic influenza janeway's immunobiology detection of human immunoglobulins g and m antibodies to rift valley fever virus by enzymelinked immunosorbent assay risk of tuberculosis among air passengers estimated by interferon gamma release assay: survey of contact investigations rupture of the achilles tendon associated with ciprofloxacin medical immunology, tenth ed. lange medical books/mcgraw-hill medical pub. division fundamental immunology, sixth ed. wolters kluwer health gene transcript abundance profiles distinguish kawasaki disease from adenovirus infection specific, sensitive, and quantitative enzyme-linked immunosorbent assay for anthrax lethal toxin impairs il- expression in epithelial cells through inhibition of histone h modification dissecting regulatory networks in host-pathogen interaction using chip-onchip technology sequestration of antibody to borrelia burgdorferi in immune complexes in seronegative lyme disease biocrimes, microbial forensics, and the physician use of forensic methods under exigent circumstances without full validation comparative tests for detection of plague antigen and antibody in experimentally infected wild rodents handbook of forensic services comparison of two commercial broad-range pcr and sequencing assays for identification of bacteria in culture-negative clinical samples first case of bioterrorism-related inhalational anthrax in the united states cutaneous anthrax: a concise review probable person-to-person transmission of avian influenza a (h n ) bioterrorism for the respiratory physician essential clinical immunology key: cord- -aemjbkfj authors: kennedy, melissa title: methodology in diagnostic virology date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: aemjbkfj selection of proper assays and appropriate interpretation of results can be a challenge to the veterinary clinician. assays vary in methodology, sensitivity, and specificity. these assays can be invaluable in attaining the correct diagnosis, but a clear understanding of the assay and the results is essential. to this end, communication with the laboratory personnel is crucial. optimal sample selection, shipping recommendations, assay selection, and interpretation should be discussed with the laboratory staff. was submitted. for antibody assays, this is straightforward, because only serum is required. it may be advantageous to ship these samples cooled in warm climes or during hotter time periods to prevent bacterial growth and antibody destruction. proper sampling and shipment for virus detection may be more involved. for many assays (eg, antigen detection assays), the virus need not remain viable. in these situations, rapid shipment and cooling during transport are not required. for some assays, however, degradation of the virus during transport will necessitate cooling during shipment. this often is true for genetic detection that uses polymerase chain reaction. many viral genomes, especially those of rna viruses, are labile. for virus propagation, the virus must remain infectious from the moment it is collected until it reaches the laboratory. this requires rapid, cooled transport [ , ] . some viruses are inactivated by freezing; therefore, as long as shipment is speedy, refrigeration/cold packs generally are sufficient [ ] . communication with the laboratory regarding shipping requirements will assist in the proper transport. detection of the virus that causes an infection uses one of several methods: the virus may be propagated in the laboratory and characterized; the virus may be visualized by electron microscopy; the viral proteins may be detected using specific antibody; the viral nucleic acid may be detected; or an activity of the virus, such as red blood cell agglutination, may be assayed. identification of the virus that is associated with the disease is good evidence for identification of the etiologic agent. one must remember that the relationship between the agent that is detected and the disease is not always causal. some of the more common assays are discussed below. for many viruses, virus isolation remains the gold standard for identification [ , ] . this assay requires collection of a sample that contains viable virus and propagation of the virus in a cell culture system. for some avian viruses, propagation in embryonated eggs is required [ ] . after growth in the in vitro or in vivo system, the virus is characterized and identified. sample collection and transport are critical steps in successful virus isolation [ , , ] . usually, samples are collected from the disease lesions, using swabs, fluids, and tissue samples. various secretions/excretions can be collected, depending on the mode of virus shedding. viruses in which a viremic phase occurs following exposure may be isolated from blood; however, this most often is possible only in the early stages of infection. collection of virus for virus isolation optimally is done in the acute phase. later in infection or during chronic infection, the presence of host antibody may inhibit virus growth in the laboratory. the speed of shipment that is required depends on the lability of the virus; nonenveloped viruses tend to be hardier and less susceptible to inactivation than enveloped viruses [ ] . because the identity of the virus usually is not known, overnight shipment on cold pack is ideal. certain information should be provided to the laboratory to facilitate the chances of successful propagation (eg, species, history, clinical presentation). cell lines that are used vary with the suspected virus; any information that can assist the diagnostician in the appropriate selection of propagation medium is helpful. after viral growth has been detected, identification of the virus is done. this can be accomplished in several ways. some viruses produce characteristic cytopathic changes (eg, syncytia) in cell culture (fig. ) . for viruses whose identity is completely unknown, electron microscopy allows visualization and classification of the virus into a family based on morphology. specific identification usually is done through the use of virus-specific antibody in an assay, such as immunofluorescence. alternatively, a virus activity may be exploited to identify the virus. for example, the virus of newcastle disease, avian paramyxovirus- , agglutinates chicken red blood cells (rbcs). its presence in cell culture can be detected by agglutination of rbcs using the cell culture supernatant. virus isolation offers several advantages. it remains the gold standard for many viruses. it is more sensitive than the antigen detection assays (eg, immunofluorescence) because the virus is amplified effectively by propagation in the laboratory. virus isolation is a nonspecific assay (ie, any virus present in the sample) may be propagated. this is in contrast to antigen identification, where the assay is specific. for example, submission of cells from a tracheal wash for the virus of infectious laryngotracheitis (herpesvirus) of birds will be negative if the agent is that of infectious bronchitis (coronavirus); however, if the sample is submitted for virus isolation, either virus may be isolated. virus isolation also has certain disadvantages. generally, it is more timeconsuming and expensive than most routine antigen detection assays [ ] . it may take to weeks for the virus to be detectable and the increased material and labor increases the cost. obviously, the virus must remain viable during shipment which can be a problem with certain labile viruses (eg, herpesviruses). rapid shipment in refrigeration is required which may add to the cost. the presence of other components (eg, bacteria, toxins) can interfere with the ability of the virus to grow in the laboratory. for example, hepatic viruses can be difficult to isolate because of the presence of cell culture toxins in the liver tissue [ ] . this also is true if neutralizing antibodies are present. additionally, some viruses simply do not grow well in an in vitro system. this is true for some enteric viruses, for example. electron microscopy provides a means for visualizing virus from samples or laboratory propagation. samples are homogenized, pelleted by centrifugation, and a small amount is mixed with a stain (eg, phosphotungstic acid) [ , ] . this is aerosolized onto a small grid and viewed by transmission electron microscopy. the virus, if present, is shadowed by the stain which gives an image that is similar to a negative photograph. the identification is based on morphology of the virion which allows classification into the virus family (fig. ) . a more specific identification is provided by the addition of virus-specific antibodies to the mixture. this results in clumping of the virus [ , ] . like virus isolation, electron microscopy is a nonspecific assay (ie, it looks for any virus that is present in the sample). for example, in diarrheic samples, any virus could be seen if present, whether parvovirus, coronavirus, or rotavirus, whereas an assay that is specific for one will miss the others. where available, this assay is rapid and generally takes less than day. in addition, the virus need not remain viable for detection and samples may be stored before shipment. because feces is the most common sample that is submitted, storing and shipping under cooled conditions to keep down bacterial growth is recommended. the equipment that is required for electron microscopy is extremely expensive as is the maintenance. therefore, this service may not be widely available; it is primarily found at academic and research institutions. also, expertise is required to identify the virus which also may impact availability. the fee for this service varies with the laboratory, but usually is reasonable, despite the expensive equipment. a drawback to electron microscopy is its relative lack of sensitivity. generally speaking, approximately particles per gram of sample are required for visualization [ , , ] . more may be required for small viruses, such as parvovirus. because of this, electron microscopy usually is restricted to testing of feces in cases of enteritis where virus concentration is significant in the acute phase of disease. for example, in diarrhea that is caused by parvovirus, the animal may be shedding more than particles per gram. it also is used to identify viruses in cell culture, where the virus has been amplified through growth in the laboratory. it is too insensitive to detect virus in most tissue, fluid, or swab samples. these require propagation before detection. one exception to this may be poxviruses. because of their large size, they may be seen in samples from lesions. antigen detection assays involve the use of virus-specific antibody to detect or bind to the viral protein or antigen in the sample. the assays vary in the sample substrate, how the binding of antibody to antigen is visualized, sensitivity, and specificity. availability of these assays for the various viruses depends largely on the availability of antibody to the pathogen. this can be a challenge for many viruses of exotic species because antibody to them often are not commercially feasible to develop. in these cases, one may find testing available at laboratories that are involved in research on the particular pathogen. we will consider several of the more common assays that are available. immunofluorescence assays (ifas) are used in many diagnostic laboratories. they are fast and economical and can be done on a wide variety of samples. these include tissue impression smears or scrapings, cells pelleted from fluids, and cell cultures. generally, slides are made by the practitioner and submitted to the laboratory. neither fixation nor special storage is required before submission. the quality of the sample does affect the results. because the assay involves detection of infected cells, the more cells that are on the slide, the more likely it is that infected cells will be identified. most ifa assays use fluorochrome-labeled antibody, such as fluorescein ( fig. ; direct) [ , ] . therefore, it is important that before collection of ocular samples, fluorescein staining of the eye must be avoided. also, purulent discharge will result in background fluorescence because of the autofluorescence of granules in white blood cells and should be removed before collection of the sample. bacteria also may lead to background fluorescence. these assays may be direct or indirect (see fig. ). in direct assays, the pathogen-specific antibody is labeled. in indirect assays, the virus-specific antibody is followed with an antibody to the first antibody (antiglobulin) that is labeled. this extra layer of antibody may increase the sensitivity of the antigen detection assays as a result of increased fluorescence relative to direct assays [ ] . immunofluorescence assays are intermediate in sensitivity. this sensitivity is even lower in chronic infections where the presence of host antibody can block the binding of the detecting antibody [ , ] . in general, a positive result ''rules in'' but a negative result does not ''rule out.'' also, because it is a specific assay, it does not detect the presence of other pathogens (eg, an ifa assay for felid herpesvirus on a conjunctival scraping will be negative if the pathogen involved is calicivirus). finally, technical expertise is required to interpret ifa assays. the reader must be familiar with the pattern of fluorescence and have the ability to distinguish viral fluorescence from background or nonspecific fluorescence. experience is thus required for proper interpretation of ifa assays. elisas are available for some viral pathogens. these assays normally are provided as commercial kits and availability is dependent on marketability. some available assays include feline leukemia virus, canine parvovirus, rotavirus, equine influenza virus, and bovine viral diarrhea virus. most use pathogen-specific antibody for capture and detection as for ifa assays (fig. b) . the antibody is anchored in a solid substrate, such as a membrane or plastic well. the sample, usually in fluid form, is added. the initial sample collected may be fluid (eg, serum for feline leukemia virus) or the sample may be solubilized in a diluent using a swab (eg, feces for parvovirus). if the virus is present, it will be bound by the anchored antibody. this is followed by soluble pathogen-specific antibody with enzyme attached, which will bind the bound virus. addition of the substrate for the enzyme leads to a color change. washing is a critical step in many elisa assays to ensure unbound antibody is removed. variations on this protocol may be found with different assays, but the underlying technique of pathogen-specific antibody binding the virus in the sample is similar in all (fig. ) [ , , ] . elisas offer several advantages. generally, they are rapid assays that require only minutes to to hours, at most, for completion and most are economical. they are simple and often can be done patient-side. usually, elisas are more sensitive than ifas. because protein antigen is detected, the agent need not remain viable for identification. elisas, like ifas, are specific assays. thus, a negative result will not provide information on the presence of other pathogens. sensitivity of elisa is usually good; however, negative results may occur early or late in infection when concentrations are low. also, if the animal has large amounts of antibody present to the pathogen, these may bind and mask the agent and prevent binding by the assay antibody. false positive results may occur for a variety of reasons. the most common may be insufficient washing. other factors can lead to false positive results. for example, some feline leukemia virus elisas use antibody to feline leukemia virus that is prepared in mice as the anchored and soluble antibody. some cats have antibody to mouse antibody from hunting behavior; this can be bound by the anchored and soluble assay antibodies and lead to false positive results. tests that use rabbit antibody can lead to the same result in felids with antirabbit antibody [ ] . immunohistochemistry (ihc) is similar to elisa in that an enzymebound antibody is used to detect the virus in the sample (fig. ). the major difference is that the sample used is fixed tissue [ ] . sampling thus requires biopsy or necropsy. this allows identification of the virus associated with the microscopic lesion, if present. usually, the sensitivity and specificity of ihc are good and it is the gold standard for some diseases, such as chronic wasting disease of cervids and feline infectious peritonitis [ , ] . availability of this assay depends upon availability of the pathogen-specific antibody. also, expertise in sample preparation and processing is required which also may impact availability. some pathogens have the ability to agglutinate rbcs. this assay usually is used to detect virus in cell culture [ , ] . newcastle disease virus can be detected in the laboratory after propagation by detection of rbc agglutination. this assay is not done routinely on clinical samples for virus detection. agglutination of viruses also can be accomplished by the presence of pathogen-specific antibody in the assay; the immune complex formation can be visualized under certain conditions. one method is to attach the antibody to a solid substrate, such as a latex bead; addition of the sample with the antigen leads to clumping of the substrate which can be seen. the most common mechanism is through agar gel immunodiffusion (fig. ) . in this instance, the immune complexes precipitate out in the agarose and form fig. . example of agar gel immunodiffusion for antibody to equine infectious anemia virus. center well contains viral antigen; wells , , and contain positive control sera; wells , , and contain patient sera. well is positive, well is weak positive, and well is negative. a precipitin band. this assay is low in sensitivity and is not used widely for detection of viral pathogens [ ] . the assays that were described above involve detection of whole virus or protein components of the virion. an alternative method is detection of the genetic material of the virus. a variety of techniques is available to accomplish this and are described below. in situ hybridization (ish) uses genetic probes to detect nucleic acid within the cell. samples are as for ihc and usually involve fixed tissues, but pretreatment protocols differ from ihc. dna or rna probes are designed to hybridize to a portion of the pathogen genome or mrna; their binding is detected through the labeling of the probe. the labels that are used can vary and include radioactive isotopes, fluorescent dyes, or chemiluminescent materials. the presence of the agent within the cell of the tissue sample can be visualized [ ] . this technique allows visualization of the agent that is associated with the lesion and provides good evidence for identification of the etiologic agent of disease. there are several factors to consider with ish to ensure appropriate interpretation of results. the sensitivity of the assay depends primarily upon two factors-the probe design and the abundance of the target. the probe design includes such parameters as the probe composition and length. the abundance of the target influences directly whether binding can be detected. generally, for targets of low abundance, probes of increased length are required to increase the amount of label. the specificity also is impacted by the probe design. is the genetic region that is targeted highly conserved or heterologous among isolates? problems with nonspecific cross-hybridization also can be encountered. the availability of this assay for particular pathogens depends on the presence of the required expertise and on the availability of sequence data for the virus of interest that is necessary for probe design. this technique may not be widely available for some pathogens of nondomestic species. additionally, biopsy or postmortem tissue collection is required [ ] . polymerase chain reaction (pcr) has become more common and accessible for detection of many pathogens. this technique involves the extraction of nucleic acid from the sample and amplification of a portion of the genetic material of the agent of interest. this amplification is accomplished by repeated cycles of dna synthesis primed by small pieces of nucleic acid primers that are designed to target a specific genomic region (fig. a) . the resultant product is identified through gel electrophoresis (fig. b) , restriction enzyme digestion, probes, or nucleotide sequencing [ , ] . there are several variations to this assay. if the starting material targeted is rna rather than dna-such as with rna viruses or assays that target mrna of the agent-a reverse transcription reaction to convert the rna to dna is required before the actual amplification. nested pcr involves two rounds of multiple amplification steps using two primer sets, one internal to the other. this allows significantly more amplification of the starting material. quantitative pcr, also known as real time or taqman, allows quantification-either relative or absolute-of the starting material. multi-plex pcr uses multiple primer pairs to allow detection of more than one pathogen in a single reaction [ ] . one of the hallmarks of pcr is its exquisite sensitivity, especially with nested pcr, which can be a double-edged sword. it allows the detection of small amounts of the agent in the sample, which can be advantageous if low amounts are present [ ] [ ] [ ] . contamination or carry-over can lead to false positive results. in addition, subclinical or even latent infections (eg, herpesviruses) may be detected [ , ] . real time or quantitative pcr provides information on the amount of virus that is present in the sample. this can assist in distinguishing clinical from subclinical amounts of the virus in the sample. false negative results also can occur and may be due to a variety of reasons. these may include degradation of the sample during transport, presence of pcr inhibitors in the sample, or insufficient genetic homology between the primers that are used and the target. this last reason can be a problem with viruses that undergo significant strain variation. in this case, primers should target highly conserved regions ideally [ ] . for example, this has been an impediment to development of pcr assays that consistently work with all strains of fiv. rna templates require reverse transcription before amplification; this step can be inefficient and may lead to false negative results [ ] . specificity of pcr also depends upon primer design, but generally, specificity of pcr is high. depending upon the target, the assay can be family-, genus-, species-, or strain-specific. stringent controls and a significant level of expertise are required for accurate results using pcr. contamination of samples can lead to false positive results. nested pcr assays, because of their enhanced sensitivity and increased chance of sample contamination, are more likely to give false positive results than single rounds of pcr. inquiries to the laboratory personnel regarding these parameters can provide useful information that can aid in interpretation of results. often times, certain laboratories have more experience and expertise with certain pathogens. one laboratory that can test for all relevant pathogens is probably not feasible. a variety of sample types can be used for pcr; it depends, in large part, on the pathogenesis of the agent. for example, viruses that are shed in feces can be tested for using fecal material; viruses that produce significant viremia may be tested for using serum or whole blood; viruses that are shed in feather dander (eg, psittacine beak and feather disease virus) may be detected in feathers [ ] . again, communication with the diagnostic laboratory will provide helpful information. shipment of the sample usually requires refrigeration to prevent degradation of the genetic material of the agent. this is of particular concern with rna targets because rna is much more labile than dna. evaluation of the host immunologic response involves detection of antibody to a specific pathogen. this provides, at the least, a historical perspective of the pathogens to which the animal has been exposed. by doing paired antibody assays-testing in the acute and convalescent phases of disease-the current infection status can be determined. a fourfold increase between acute and convalescent samples indicates active infection. serology on herds or flocks can be used to establish the prevalence of the agent in the population [ ] . the assays that are used to detect antibody are similar to ones for antigen detection-the antigen that is detected in serology is a pathogen-specific antibody. unlike some antigen detection assays (eg, as virus isolation, electron microscopy), all serologic assays are specific (ie, they only detect antibody to a certain infectious agent). the assays for antibody detection can be divided into three basic categories [ ] : ( ) direct detection of the antibody, ( ) detection of antibody-facilitated activity, and ( ) detection of antibody-facilitated inhibition. the first category involves binding of the animal's antibody to antigen anchored to a solid substrate (eg, membrane, slide). detection of the bound antibody is done through anti-immunoglobulin (eg, antifeline igg) that is labeled with a dye or enzyme [ , ] . these assays include immunofluorescence, elisa, and western blot. this can be an obstacle in testing exotic species because there is limited availability of antiglobulin for most species of animals. some that are available include antibody to immunoglobulin of ferrets, raccoons, llamas, psittacines, deer, bears, rabbits, monkeys, and several species of rodents. testing for antibodies in other species may be available in some laboratories that are involved in research on the species [ ] . if no antiglobulin is available, then testing is restricted to one of the other two categories. the second category exploits the facilitation of an activity by the formation of the immune complex (patient's antibody; capture antigen, or virus); an activity occurs in response to formation of the immune complex formation. these may include complement fixation, agglutination, and precipitation. these assays detect igg and igm. because igm is an efficient activator of complement and enhances agglutination because of its pentameric structure, these assays are useful for detection of igm [ , ] . the third category exploits the inhibition of an activity by the formation of an immune complex (ie, an activity that the virus normally is capable of effecting does not occur because of binding of the patient's antibody to the assay virus). these include hemagglutination inhibition (hi) and virus neutralization. these two assays measure antibody to specific surface epitopes of the pathogen, usually viral attachment proteins [ , ] . serologic assays may be qualitative or quantitative [ ] . the former determines only the presence of antibody, not the level. this can be useful for agents that cause life-long infection, such as the retroviruses; the presence of antibody would indicate infection. quantitative assays determine the relative concentration of antibody by testing serial dilutions of the patient's serum. in this case, the titer is the highest dilution at which the presence of antibody is detected. detection of seroconversion or a fourfold increase in titer between paired samples indicates active infection [ ] . paired titers can be circumvented in populations by screening a proportion of the animals. for example, testing exposed animals, clinically ill animals, and recovered animals can mimic acute and convalescent sampling. basing a diagnosis on the magnitude of a single titer is unreliable; however, the laboratory may be able to provide some interpretation of a single titer. the usefulness of serology depends, in part, on the virus and disease. if onset of clinical signs coincides with antibody production, active disease may be diagnosed. for some pathogens, disease occurs before antibody production; serology would be retrospective. some pathogens cause disease months or years later, such as fiv; detection of antibodies is sufficient to diagnose infection [ ] . most of these assays use antibody to the patient's immunoglobulin for detection. they vary in the capture antigen matrix (eg, slides, membrane, plastic wells) and label (eg, fluorescein, enzyme, chemiluminescent). because these assays use species-specific antiglobulin, availability for exotic species is limited. antiglobulin to antibody of domestic species may be used in some situations (eg, anticanine igg in nondomestic canids like wolves). for those that are available, detection across species line is not always known. for example, in snakes, antiglobulin to igy of boids does not detect igy of elapids [ ] . these assays vary in sensitivity and specificity. ifa assays are intermediate in sensitivity, whereas elisas and western blots are higher in sensitivity [ ] . generally, specificity depends upon the capture antigen that is used. for example, assays that use antigen that is shared with other agents that are nonpathogenic may detect antibody to these pathogens. such an assay is antigen slides that contain whole rickettsia rickettsii (rocky mountain spotted fever) organisms for detection of antibodies [ ] . because this agent shares antigenic epitopes with nonpathogenic rickettsial organisms, the presence of antibody does not confirm infection with the pathogenic agent [ ] . western blots have the highest specificity because antibody to the individual proteins of the agent, including antigens that are specific for the pathogenic species, is detected. for example, the western blot for lyme disease can distinguish antibody to the vaccine from natural infection because of the difference in antibody profiles (vaccine: antibody to osp a; natural infection: antibody to outer surface protein (osp) c) [ ] . the basis for immunofluorescence for serology is the same as for antigen detection. in this case, the antigen, usually virus-infected cells, is fixed to a glass slide. the anchored antigen ''captures'' the antibody that is present in the patient's serum (see fig. ; indirect). the bound antibody is detected with fluorescein-labeled antiglobulin. ifa assays are quantitative assays. the titer is reported as the highest dilution at which fluorescence is detected. the antigen in elisas is anchored to a membrane or plastic well. as with ifa assay the patient's antibody is captured and detected with antiglobulin, to which an enzyme is attached. addition of the enzyme substrate leads to a color change (see fig. ). most elisas are qualitative, rather than quantitative, assays with results reported as positive or negative. most elisas are provided as kits by commercial suppliers and may be limited for diseases of exotic species. some may be adaptable to exotic species, such as the fiv antibody elisa, but one must recognize that these kits are not approved for use on nondomestic species and may not work consistently in all situations. western blot is similar to elisa. with western blot, however, the agent's proteins are separated on a gel before attachment to a membrane. in this way, the antibody response to specific proteins can be evaluated (fig. ) [ , ] . this can be helpful to confirm results of other assays. for example, fiv elisa positive results are confirmed through western blot [ ] . because western blot detects antibody to individual pathogen proteins, it is more specific than other assays. false positive results that occur as a result of detection of antibody to nonspecific proteins are avoided. as with elisas, western blots do not quantitate antibody levels. by this assay. as a result, antibody that recognizes these specific epitopes on one virus strain may not recognize a different strain in this antigenic region. for example, hi is used often to measure antibodies in reptiles to opv. we found that by using different isolates of ophidian paramyxovirus (opv) in the assay, different antibody titers will result with the same serum because of variability of the epitopes among virus strains (unpublished data). this assay is considered to be the most definitive serologic assay and may correlate with protection [ ] . binding of antibody to certain virus proteins inhibits the ability of the virus to infect a cell or neutralizes the virus. in this assay, the patient's serum is incubated with the virus, after which susceptible cells are added. if antibody is present, no infection will occur as evidenced by lack of cytopathic effects or detection of the virus in the cells by ifa assay. the absence of antibody allows the virus to infect the cells and this infection can be detected. the highest dilution at which no evidence of cellular infection (or infection of % of the cells) occurs is reported as the titer. as with hi, this assay commonly measures antibody to epitopes of surface proteins, and as a result, virus strain variation in these epitopes impacts the results. interpretation of serologic results can be a challenge. for quantitative assays, assigning an infection status to an animal based solely on the magnitude of a single titer is unreliable. antibody may be absent or decreased in the acute stage of disease. conversely, past infections with some agents lead to persistently elevated titers for significant periods of time [ ] . in addition, titers vary depending upon the type of assay and capture antigen that are used. finally, because laboratories vary in the methodology that is used, titers vary among them. paired titers offer the most information, but are not always feasible. communication with laboratory personnel can assist interpretation. for qualitative assays, a positive result does not correlate necessarily with active infection. exceptions to this are those that cause lifelong infections (eg, fiv). selection of proper assays and appropriate interpretation of results can be a challenge to the veterinary clinician. assays vary in methodology, sensitivity, and specificity. these assays can be invaluable in attaining the correct diagnosis, but a clear understanding of the assay and the results is essential. to this end, communication with the laboratory personnel is crucial. optimal sample selection, shipping recommendations, assay selection, and interpretation should be discussed with the laboratory staff. laboratory diagnosis of viral infections mosby year book virology: a laboratory manual veterinary virology same-day diagnosis of human virus infections evaluation of serologic and viral detection methods for diagnosing feline herpesvirus- infection in cats with acute respiratory tract or chronic ocular disease comparison of nested polymerase chain reaction, virus isolation, and fluorescent antibody testing for identifying feline herpesvirus in cats with conjunctivitis interpretation and misinterpretation of serological test results boenisch, editor. immunochemical staining methods recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium chronic wasting disease in deer and elk: scientific facts and findings diagnosis of polyomavirus-induced hepatic necrosis in psittacine birds using dna probes methods of detection of chlamydia psittaci in domesticated and wild birds comparison of an antigen capture enzyme-linked assay with reverse transcription-polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections laboratory diagnostic investigations for bovine viral diarrhea virus infections in cattle comparison of pcr, virus isolation, and indirect fluorescent antibody staining in the detection of naturally occurring feline herpesvirus infections high sensitivity polymerase chain reaction assay for active and latent feline herpesvirus- infections in domestic cats update on molecular techniques for diagnostic testing of infectious disease a universal polymerase chain reaction for the detection of psittacine beak and feather disease virus philadelphia: lippincott, williams and wilkins development of an enzyme linked immunosorbent assay (elisa) for the diagnosis of ophidian paramyxovirus (opv) immunology veterinary immunology: an introduction infectious diseases of the dog and cat canine borreliosis elementary body agglutination for rapidly demonstrating chlamydial agglutinins in avian serum with emphasis of testing cockatiels comparison of serologic tests used in canine brucellosis diagnosis laboratory diagnostic tests for retrovirus infections of small ruminants comparison of complement fixation and hemagglutination inhibition assays for detecting antibody responses following influenza virus vaccination this assay is confined to detection of virus-specific antibodies. although it has been invaluable in the past, it is not used commonly in most diagnostic laboratories. formation of an immune complex by the binding of patient antibody to the virus leads to activation of the complement cascade. in the assay system, the incubation of virus with patient's serum is done with the addition of guinea pig complement. if antibody is present, the complement is activated and effectively exhausted. after the incubation, sheep rbcs, to which antibody has been attached, are added. these are, in effect, immune complexes. if the complement has been exhausted, the rbcs are not lysed by complement. if no antibody was present in the patient's serum, addition of the sensitized rbcs leads to complement activation and rbc lysis [ , ] . most complement fixation assays quantitate the amount of antibody. because igm and igg can activate complement, both will be detected. generally, complement fixation is not used as widely as other antibody detection assays. formation of immune complexes can be visualized through agglutination or precipitation of the complexes. in the former, the viral antigen may be attached to a substrate that can be visualized (eg, latex bead) or clumping of the organism itself may be seen [ , ] . in the latter, the formation of immune complexes leads to precipitation in an agarose medium at optimal concentrations, which can be visualized [ ] . this is used commonly for detection of antibodies to caprine arthritis encephalitis virus and equine infectious anemia virus (see fig. ). this assay is used with certain viruses that have the ability to agglutinate rbcs. this assay is mediated by the surface proteins of the virus [ , ] . the patient's serum is incubated with the virus in a well. if present, binding of the patient's antibody to the virus will prevent the rbc agglutination by the virus. therefore, the rbcs will pellet in the well. the highest dilution at which agglutination of the rbcs is inhibited is reported as the titer (fig. ). this assay measures antibody to epitopes on the surface protein of the virus. these proteins are under immunologic pressure for variability; thus, virus strains may vary in the antigenicity of these proteins [ ] . antibodies to highly conserved proteins (eg, polymerase or core proteins) are not detected key: cord- -fxs d authors: ubah, obinna; palliyil, soumya title: monoclonal antibodies and antibody like fragments derived from immunised phage display libraries date: - - journal: recombinant antibodies for infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: fxs d morbidity and mortality associated with infectious diseases are always on the rise, especially in poorer countries and in the aging population. the inevitable, but unpredictable emergence of new infectious diseases has become a global threat. hiv/aids, severe acute respiratory syndrome (sars), and the more recent h n influenza are only a few of the numerous examples of emerging infectious diseases in the modern era. however despite advances in diagnostics, therapeutics and vaccines, there is need for more specific, efficacious, cost-effective and less toxic treatment and preventive drugs. in this chapter, we discuss a powerful combinatorial technology in association with animal immunisation that is capable of generating biologic drugs with high affinity, efficacy and limited off-site toxicity, and diagnostic tools with great precision. although time consuming, immunisation still remains the preferred route for the isolation of high-affinity antibodies and antibody-like fragments. phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. the selection of binding fragments from phage display libraries has proven significant for routine isolation of invaluable peptides, antibodies, and antibody-like domains for diagnostic and therapeutic applications. here we highlight the many benefits of combining immunisation with phage display in combating infectious diseases, and how our knowledge of antibody engineering has played a crucial role in fully exploiting these platforms in generating therapeutic and diagnostic biologics towards antigenic targets of infectious organisms. the number of monoclonal antibody (mab) based drugs developed by biopharmaceutical companies is at an all-time high with more and more novel anti-infective antibodies gaining regulatory approval. the emergence of multi-drug resistance has reinforced the need to develop novel anti-infective approaches. unlike conventional antibiotics, o. ubah scottish biologics facility, elasmogen ltd, aberdeen, uk s. palliyil (*) scottish biologics facility, university of aberdeen, aberdeen, uk e-mail: soumya.palliyil@abdn.ac.uk monoclonal antibodies exhibit high target specificity and possess the ability to recruit immune system components for effective pathogen removal. modes of action include: specific binding and neutralisation of microbial toxins and virulence factors, opsonising and marking pathogens for cell-death by directing phagocytic cells to the site of infection, antibody mediated bacterial agglutination and clearance, complement activation and direct bacterial lysis [ ] . host effector functions mediated through interactions with the fc region of mab drugs makes them highly effective in treating immunocompromised patients that are unable to generate their own immune response to fight diseases [ ] . certain monoclonal antibodies exhibit synergistic or additive effects with conventional antibiotics providing an attractive therapeutic strategy for treating infections caused by multidrug resistant organisms [ ] . this chapter discusses the advantages and disadvantages of using immunised phage display libraries constructed from mammalian sources for generating monoclonal antibodies against infectious disease targets. several groups have successfully developed monoclonal antibodies specific for bacterial, fungal and viral antigens with potential applications in the detection, diagnosis and treatment of infectious diseases which are reviewed in this chapter. originally mabs were isolated using hybridoma technology, and required the fusion of mouse lymphocyte and myeloma cells to generate specific murine antibodies that were often highly immunogenic if used in a therapeutic setting. a limited number of human mabs were also developed by using human b lymphocytes from naturally infected patients but this proved a technically challenging and generally unreliable approach [ ] . subsequent advances in antibody engineering removed or resolved many of these technology "road-blocks" facilitating the generation of chimeric, humanised or deimmunised mabs and bispecific antibodies with increased potency and reduced immunogenicity. one of the major breakthroughs was the invention of phage display which revolutionised the field of antibody engineering with its robust, easy to use and highly versatile combinatorial display platform. its successful applications include: generation of antibodies with unique functions from immune and non-immune sources, de novo isolation of high affinity binders from non-immune or synthetic sources and the in vitro affinity maturation of antibodies [ ] . phage based selection could be summarised as the display of antibodies (proteins or peptides) on the surface of bacteriophage by fusing the antibody gene to one of the phage coat proteins and selection based on the antigen binding of individual clones. phage antibody libraries are constructed by pcr based cloning of vh and vl repertoires by random pairing into a phage or phagemid vector system and display on the surface of bacteriophage. it has been used widely in the antibody engineering as a technique to mimic b cells, which are self-replicating packaged systems containing antibody genes that encode the antibody displayed on its surface (linked genotype and phenotype) [ ] . phage display libraries can be constructed using antibody variable (v) genes isolated from igm mrna of non-immunised human donor b cells derived from diverse lymphoid sources such as peripheral blood lymphocytes (pbls), spleen cells, tonsils, bone marrow or from nonimmunised animal b cells (naïve antibody libraries). igg mrna from pbls or spleen cells of immunised animals or human patients are used to build immunised libraries. a third class called synthetic and semi-synthetic antibody libraries are constructed using repertoires of rearranged v genes from gene segments using polymerase chain reaction (pcr) and introducing variation into their cdr regions using custom degenerate primers encoding for diversity and length [ ] . immunised libraries are generally constructed using antibody vh and vl genes amplified from the mrna of b cell pools from immunised animals or human donors. post immunisation, the antibody secreting b cells possess a higher percentage of antigen specific heavy and light chain gene transcripts. these b cells would have undergone affinity maturation and isotype switching in germinal centres before entering into the peripheral circulation. typically antibodies isolated from immunised libraries will have a high affinity and tighter specificity for the immunogens used [ ] and can be achieved even from relatively small library diversity [ ] . unlike hybridoma technology, phage display provides researchers with more possibilities to streamlining their monoclonal antibody generation process. the main improvement is the replacement of cell culture screening steps using hybridomas. instead of screening hundreds or thousands of hybridoma monoclonals to find positive clones, phage display, with carefully designed panning strategies, can screen billions of clones and allow the enrichment of small subsets of binders with desirable characteristics that can be further screened to identify individual binders. antibodies recognising specific antigenic conformations and epitopes can be easily isolated by introducing selective or subtractive panning steps during the selection process. it has also been reported that using phage display high specificity antibodies can be isolated from immunised sources which would be missed by traditional immunological methods [ ] . a key feature of this system is the linkage of genotype (phagemid vector) and phenotype (phage coat protein-antibody fragment) which allows immediate access to the corresponding gene sequences of selected antibodies facilitating simple sub-cloning into various antibody formats based on required downstream applications ( fig. . ). over the last years phage display technologies have been successfully used for the development of many therapeutic antibody candidates and approved drugs and with the relaxation of commercial restrictions more and more products are now entering the diagnostic and research markets as well [ ] . the drawbacks of animal immunisation for the construction of phage display libraries include the time period required for the completion of such a process and the requirement to construct separate libraries for each antigen. however, by combining the power of immunisation with phage display, several high affinity monoclonal antibodies against "difficult" antigenic targets have been isolated from relative small antibody libraries and where traditional approaches have failed [ , ] . generating antibodies against self or toxic antigens is limited if immunisation is to be employed. in particular, human autoantigens are highly conserved amongst most routinely used laboratory mammals such as mice or rat. therefore immunisation of non-mammals such as sharks and chickens, which are phylogenetically distant from humans, has been successfully employed for generating an immune response and antibodies and/or antibody like binders (vnar) to epitopes conserved in mammalian species [ ] . chickens are phylogenetically distant from humans and therefore very successful in generating an immune response to mammalian proteins which are highly conserved [ ] , while sharks are even more distant from humans, diverging from a common ancestor approximately million years ago [ , , ] . [ , , , , , , , ] . human monoclonal antibodies from patients which showed protective effects in animal model studies have also been selected against bacterial targets such as anthrax toxins [ ] , botulinum neurotoxin [ ] and the abc transporter of methicillin resistant staphylococcus aureus [ ] . in the case of the west nile virus neutralising mabs were only isolated from the b cell population of convalescent human patients with large phage display libraries constructed from uninfected donors delivering nothing of consequence [ ] . conventional methods of antibody generation such as hybridoma and epstein-barr virus transformation are limited in their abilities to evaluate human monoclonal antibodies from different patients at various stages of their clinical course. technologies like phage display provide a powerful tool that allows pooling of large number of patient immune b cell populations and the selecting of antibodies with distinct specificities and inhibitory activities during different stages of infection [ ] . the ability to improve affinities and broaden specificities post selection is a major attribute of recombinant antibodies. using recombinant dna and protein engineering approaches the pharmacokinetic and pharmacodynamics properties of these molecules can be improved including half-life extension and reduced immunogenicity. for the neutralisation of toxins and viruses, it is often advantageous to generate multiple high affinity antibodies that recognise different epitopes on the same antigen or a variety of antigenic subtypes capable of delivering broad protection against pathogen variants. such 'oligoclonal' antibodies were reported to show strong synergistic activity in neutralising botulinum neurotoxin (bont) assays [ ] . a list of recombinant antibodies generated from phage display libraries constructed using the immune antibody gene repertoires of human patients is given in table . . several research groups have successfully demonstrated the potential of monoclonal antibodies, fragments and single domain antibodies for the prevention and treatment of bacterial infections in animal models. however their translation into the clinic has been somewhat slow. a small number of mab drugs under regulatory review include obiltoxaximab, for the treatment and prevention of inhalational anthrax and bezlotoxumab, which targets clostridium difficile enterotoxin b, developed for the prevention of recurrent c. difficile infection [ ] . with the ever increasing numbers of antibiotic resistant bacterial strains, the need to develop antibody based drugs with novel modes of killing has never been greater especially if their mode of action limits the development of resistance. antibodies developed against bacterial targets fall into two main categories: (i) antibodies that target the bacterial cell surface directly or (ii) those that act indirectly by neutralising bacterial toxins or virulence factors and relying on the host immune system for effective pathogen clearance. monoclonal antibodies and fragments developed against various gram−ve and gram+ve bacterial targets using animal immunisation and phage display based selection are summarised below. infection control using high affinity monoclonal antibodies specifically targeting the quorum sensing (qs) molecules of pseudomonas aerugi-nosa has been reported from our laboratory [ ] . a number of gram negative bacteria, including pathogens like p. aeruginosa, utilise homoserine lactones (hsls) as qs signalling compounds and engage in cell-to-cell communication to coordinate their behaviour. as qs takes a central role in p. aeruginosa infection by regulating the expression of extracellular virulence factors (and also [ ] biofilm formation), immuno-modulation of the hsl molecules by monoclonal antibodies (mabs) can be used as a novel approach to prevent p. aeruginosa infections. sheep immunisation was utilised to develop antibodies with high affinity and sensitivity towards hsl compounds. a mixture of three hsls-n-acyl-c -hsl, -oxo-c -hsl and -oh-c -hsl with different subgroups at the third carbon position were conjugated to the carrier protein thyroglobulin (tg) and used as immunogen to enhance the chance of eliciting an antibody response in sheep. using pbls from immunised sheep as starting material, v h -v λ and v h -v κ anti-hsl phage display libraries were constructed in a scfv format. the panning strategy was designed to drive selection towards the enrichment of high sensitivity and cross-reactivity clones. lead clones were reformatted into sheep-mouse chimeric iggs and had picomolar sensitivities (ic values as determined by competition elisa) for -oxo-c -hsl which is the central qs compound in p. aeruginosa. these values are quite impressive, considering the chemical nature of these lipidlike compounds (average molecular weight da) which possess only a small head like structure and lack critical antigenic features such as aromaticity or charge. modelling of these sensitive anti-hsl antibodies indicated that the level of sensitivity observed was achieved through the generation of a deep and negatively charged binding pocket [ ] . sheep immunisation was chosen for this hsl application as this approach has previously been shown to generate high affinity antibodies against haptenic targets [ ] and sheep polyclonal antibodies have been used as specific high affinity immunologic probes for analytical and clinical purposes for many years [ ] . in contrast to humans and mice, sheep b cell lymphopoiesis occurs predominantly in the ileal peyer's patches (ipp) and v(d)j recombination creates limited diversity in the sheep ig repertoire due to a very few gene segments participating in the rearrangement process. this characteristic feature makes sheep library construction simpler, as the entire heavy and light chain gene repertoire can be amplified using a small number of primers. much of the antibody diversity is achieved through antigen-independent post-rearrangement somatic hypermutation, which diversifies all cdrs as compared to the sole variability of just cdr in humans and mice [ , , , ] . comparison of chicken and sheep responses by hyperimmunisation with the same immunogen revealed the ability of the sheep immune system to produce higher specificity antibodies than chicken [ ] . the overall antibody titre was also much higher in sheep and might be due to the longer half-life (~ days) of sheep immunoglobulins when compared to h in chicken [ , ] . it is also noteworthy that the sensitivities of the sheep polyclonal/mab antibodies described here were far superior to the published mouse monoclonal antibody specific for the same hsl antigens [ ] . the protective effects of the sheep derived, anti-quorum sensing antibodies were demonstrated in vivo. in a slow killing model of the nematode worm caenorhabditis elegans the significant increase in survival rate in the presence of hsl mabs is similar to the defective slow killing observed in lasr mutant strains [ ] . furthermore, in a non-neutropenic lung model of mice infected with p. aeruginosa pa , hsl mab monotherapy demonstrated significant efficacy, prolonging survival by up to %. since no significant reduction in bacterial counts was observed in the lungs of infected mice, it is proposed that hsl specific antibodies protect mice possibly through antibody mediated scavenging of hsl compounds. this mode of action does not necessarily affecting bacterial numbers but probably prevents a switch to a more pathogenic phenotype [ ] . it has been widely accepted that antibodies with sub-nanomolar affinity exhibit improved therapeutic efficacy, if the targets to be bound are present in low concentrations. the above study demonstrates the power of animal immunisation and phage display based selection strategies to isolate high affinity monoclonal antibodies towards non-antigenic targets which inherently lack properties like aromaticity and charge. adp-ribosylating enzymes such as cholera, pertussis, diphtheria toxins and escherichia coli heat-labile (lt) toxins are important virulence factors for a number of extracellular bacterial pathogens. pathogenesis is driven by the secretion of potent toxins that utilise adp-ribosylation as the catalytic mechanism underlying their action. adp-ribosylating toxins comprise a large family, and all produce disease by altering key metabolic processes after transfer of an adpribose moiety from nad to specific host-cell target proteins [ , ] . it was not until the early that the adp-ribosylating enzyme was implicated in intracellular pathogenesis. it was shown that salmonella strains were capable of invading epithelial cells and localising in macrophages during infection [ , ] . the salmonella virulence plasmid factor b (spvb) virulence gene of salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. lesnick et al. provided evidence that spvb encodes an adp-ribosylating enzyme that uses actin as a substrate and depolymerises actin filaments when expressed in cho cells [ ] . a spvb blocking camel vhh single domain antibody (sdab) capable of blocking spvb enzymatic activity at a : molar ratio was isolated from an immune phage display library generated from an spvb immunised llama [ , ] . as an intracellular protein, spvb is inaccessible to conventional antibodies, and small molecule inhibitors of spvb are fraught with potential side effects resulting from the indiscriminate inhibition of endogenous mammalian adp-ribosyltransferases (arts) [ , , ] . the vhh sdab when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric c n-c/spvb toxin, and transfected cells were also protected against cytoskeletal alterations induced by wild-type spvb-expressing strains of salmonella [ ] . this provides evidence to support the development of these sdabs as therapeutic and experimental tools to block mammalian and toxin arts. helicobacter pylori is a gram negative pathogenic bacteria that colonises the human stomach and can cause gastritis, gastric and duodenal ulcers and cancer. the surface proteins in h. pylori mediate several host-pathogen interactions and hence are attractive targets for antimicrobial therapy and vaccination. an antibody phage display library in a scfv format was constructed by hyperimmunising mice with h. pylori total cell lysate and a monoclonal antibody fragment recognising the outer membrane protein hopq was isolated by performing biopanning on whole cells [ ] . in another study a mab generated through mice immunisation and hybridoma technology was engineered using phage-display as a scfv fragment and showed high affinity binding to an h. pylori surface antigen. this phage displayed scfv antibody was shown to inhibit the growth of six different h. pylori strains and offered significant protection in a mouse model of infection. the authors argued that genetically engineered bacteriophage could be used as alternatives to conventional antibiotics in the treatment of bacterial infections [ ] . spleen samples from mice immunised with gamma inactivated brucella melitensis strain m bacteria was used to construct a phage display library and isolate monoclonal antibody fragments that specifically recognise brucella species. brucella can cause long term debilitating illness in humans, can be spread as aerosols and survive extended periods outside their host. the attributes could make brucella species possible biological warfare agents. since many brucella species share their immunodominant lipopolysaccharide (lps) antigen with the closely related yersinia species, a specific lps antibody that can distinguish between these two bacteria is central for rapid detection and diagnosis of brucella infection. specific washing steps were included during bio panning of an immunised b. melitensis library to eliminate phage that might be cross-reactive with strains expressing the same dominant lps epitope on yersinia. the resultant monoclonal antibody fragments was specific for the antigen and not cross-reactive towards yersinia species [ ] . botulinum neurotoxins (bonts), regarded as one of the most toxic substances on earth, are secreted by clostridium botulinum and some other species of clostridium. bont/a which is the most potent among seven serotypes exerts its toxicity by the cleavage of snap- (synaptosomal-associated protein), mediated by its light chain (bont/a-l). this proteolysis causes blockage of nerve impulses and causes flaccid paralysis, including that of respiratory muscles resulting in death [ ] . immunisation of macaques (macaca fascicularis) and construction of hyper immune phage display library resulted in the isolation of nanomolar sensitivity antibody fragments to the light chain fragment of bont/a that are capable of inhibiting bont/a endopeptidase activity in vitro. the variable heavy and light chains of selected clones are highly similar to human germline sequences which predicts good tolerance for clinical use. since immunoglobulin genes of non-human primates are very similar to human abs, the differences in the conserved framework regions of macaque and human iggs are no greater than those between human iggs from different individuals [ ] . sub nanomolar affinity 'nearly human' fab fragments against tetanus toxoid antigen were isolated from a small phage display fab library constructed using the immune antibody repertoire of macaca fascicularis [ ] . similarly high affinity neutralising antibody fragments against the protective antigen (pa) of anthrax toxin was isolated from a macaca immunised phage display library. these fab fragments were shown to bind to a particular region of pa that interacts with cell receptor thereby blocking its binding [ ] . a single chain fv phage display library was constructed from a cynomolgus macaque (macaca fascicularis) immunised with lethal factor (lf) of anthtax toxin. resultant high affinity scfv fragments showed efficient inhibition of anthrax toxin in vitro and in vivo [ ] . in cases where anthrax vaccination is not practical or antibiotic therapy is ineffective, passive immunisation with anthrax neutralising antibodies can be an effective method of treatment. similar to macaques, chimpanzee immunoglobulins are very close to human antibodies and they should be well tolerated in vivo in human therapy [ , ] . lymphocytes from the bone marrow cells of two chimpanzees immunised with anthrax toxin pa, lf and edema factor (ef) were used to construct scfv phage display libraries and neutralising antibodies were isolated against pa and lf proteins. for passive immunotherapy, these fragments were converted into bivalent full length immunoglobulins which showed strong neutralising activity against the cytotoxicity of anthrax toxin in vitro. these high affinity (picomolar range) mabs demonstrated efficient protection in animals from anthrax toxin challenge in vivo, most likely by blocking binding of pa to the cell receptor which suggest their use in the emergency prophylaxis and treatment of anthrax [ , ] . the nature of the antibody-antigen interaction allows for the development of molecules that can target bacterial antigens with a high degree of affinity and specificity. target specificity of mabs can be can be utilised to deliver antimicrobial compounds more effectively [ ] . in addition, by choosing a suitable antibody fc portion, host effector functions can be mediated through the recruitment of elements of the host immune system such as macrophages, nk cells and complement to sites of bacterial infection and accelerating clearance of the infection. despite having a large number of candidates with promising efficacies in preclinical animal models of infection, anti-infective antibodies reaching clinical development and finally into the markets are somewhat slow. in most cases in vivo models can be limited in their ability to predict efficacy in humans, particularly due to differences in the pharmacokinetics of the molecules between humans and model animal species [ ] . geng et al. reported that a large percentage of antibodies for anti-infective indications are ended in the early stage of discovery making them a high risk category in drug development [ ] . challenges in clinical efficacy and pharmacokinetics can be overcome by choosing appropriate format for the indication. for example, igg fc region interacts with the neonatal fc receptor (fcrn) which is important for igg recycling and protection from degradation, contributing towards unique pharmacokinetic properties of the molecule and extending the serum half-life to about days for most igg subclasses [ ] . the variable region (vhh) of heavy chain only antibodies (hcabs) found in camelidae species possess an unusually long complementarity determining region (cdr ) which can form finger-like extensions to penetrate into grooves on the surface of antigens that are usually inaccessible to conventional antibodies. staphylococcus aureus produces several adhesion factors and exotoxins such as hemolysins α, β, γ, δ and panton-valentine leukocidin (pvl) which are important virulence determinants. s. aureus is a leading cause of endophtalmitis which is associated with a poor visual outcome. the expression of pvls and leucotoxins especially luks-pv and lukf-pv induce the activation and permeabilisation of target cells and result in cell lysis. transgenic mice harbouring llama/human hybrid ig heavy chain locus were immunized with recombinant luks-pv and lukf-pv proteins. using phage display technology high affinity vhhs were isolated and reformatted into bivalent and tetravalent camel heavy chain antibodies. anti luks-pv and lukf-pv hcabs were able to inhibit pvl associated disease pathology in a non-infectious model of rabbit intravitreous pvl, when administered prophylactically. the hcabs completely reduced inflammation in the eyes of the treatment group without any apparent damage to vision or behaviour. this shows the possibility of administering toxin neutralising antibodies in combination with an intravitreal antimicrobial strategy for postsurgery endophthalmitis [ ] . camel immunisation using s. aureus exoproteins and successful generation of anti β-hemolysin single domain antibodies was reported by jangra and singh [ ] . in a neutralisation assay, their lead vhh clone completely inhibited five hemolytic units of the toxin in vitro and resisted thermal denaturation up to °c. the cdr loop of vhhs is longer than the average igg and linked with cdr by an interloop disulphide bond which renders additional stability to the protein. in some cases the long cdr loop enables the formation of a convex paratope that can access deep enzymatic clefts and cavities on the surface of an antigen [ ] . the active sites of enzymes such as β-hemolysin are mostly situated in their largest cleft. the long cdr loop of anti β-hemolysin clone is believed to bind this site and able to neutralise enzyme activity by blocking access to the hlb antigen [ ] . vhh fragments specifically binding to tetanus toxoid antigen were generated using camel immunisation and phage display technology. two vhh fragments recognising two different epitopes of the antigen successfully neutralised tetanus toxin in vivo in studies conducted in mice [ , ] . another example where vhhs can inhibit bacterial infection by increasing the bacterial sensitivity to β-lactam antibiotics has been reported [ ] . high affinity β-lactamase inhibiting vhhs were generated from immunised dromedary phage display libraries primed towards the antigens tem- and bcii-lactamases, representatives of class a and class b-lactamases, respectively. in the presence of ampicillin, specific vhh fragments inhibited the growth of e. coli cells expressing a fusion protein of tem- β-lactamase on their outer surface, whereas the cells were able to grown in higher concentrations of ampicillin (> μg/ml) when no vhh fragments were added [ ] . the authors claim that vhhs are less immunogenic than larger murine antibodies as they show a high sequence similarity to human vh families and . vhh nanobody dosed at an immune phage display library. a one-year-old male dromedary (camelus dromedaries) was immunised with purified recombinant bont/e protein [ ] . another high affinity vhh inhibitor of bont/a protease activity was isolated from phage display libraries derived from b cells obtained from the immunised alpaca (lama pacos) [ ] . one of the main advantage of using animal immunised phage display libraries for generating antibodies against bacterial antigens is the easy isolation of high affinity neutralising antibodies without the need for further affinity maturation. table . summarises monoclonal antibodies and antibody fragments against various bacterial targets and their affinities to target antigens. chen et al. [ , ] the eukaryotic nature of fungal pathogens presents an almost insurmountable problem when developing anti-fungal drugs. multiple drug resistance associated with existing chemical antifungal drugs highlight the need for new, more effective antifungal therapies. antibody based antifungal treatments can provide a much needed alternative to the near-exhausted chemical based strategies. due to their high specificity and target selectivity, antifungal antibodies can be successfully employed for targeted killing of fungal pathogens without harming host cells. using hybridoma technology several mabs were generated against various antigenic components of fungal pathogens cryptooccus neoformans, candida albicans, aspergillus fumigatus, pneumocystis jerovecii and histoplasma capsulatum [ , , , , , , ] . the murine monoclonal antibody (mab b ) generated through hybridoma technology directed against the capsular polysaccharide of c. neoformans mediated protection in murine model of infection and was shown safe to use in human patients in a phase i dose escalation study [ ] . in addition to the classical antibody mediated mechanisms of phagocytosis, complement activation and recruitment of inflammatory cells, mab b was shown to increase the stiffness of the capsule by polysaccharide cross linkage which impaired yeast budding by trapping newly emerging buds inside the parental capsule [ ] . opportunistic invasive fungal pathogens cause over million life-threatening infections per year worldwide with mortality ranging from - % [ ] . the most common cause of invasive fungal infections in the intensive care unit are candida spp., and in the usa and western europe, the incidence of candida infections is second only to staphylococcus aureus (including mrsa) [ ] . recent data indicate that the incidence of candidaemia continues to rise unchecked by current clinical practice or drug regimens [ ] . candida mannan specific monoclonal antibodies have been generated through sheep immunisation in our laboratory. a single chain antibody phage display library was constructed using the immune repertoire of a sheep hyperimmunised with candida albicans hyphal cell wall preparation. this antibody library, one of the very first and largest against candida cell wall antigens, has been successfully used to isolate high affinity and specificity binders to candida α-mannan, which are typically poorly immunogenic glycans present on the fungal cell surface. analysis via sem of polyclonal sera derived from immunised sheep confirmed that the library contained a varied collection of antibodies with specificity to both fungal cell wall carbohydrates (mannans and glucans) as well as a number of important cell wall proteins (patent wo (a )). hm- killer toxin protein is secreted by most yeasts to stop the growth of other competing strains by inhibiting the transmembrane enzyme β glucan synthase involved in their cell wall synthesis. a neutralising monoclonal antibody generated against hm- toxin (nmab-hm- ) was used to immunise mice and isolate hm- antiidiotypic antibodies from a scfv phage display library constructed using mice spleenocytes. anti-idiotypic scfv fragments that inhibited β- , -glucan synthase activity were isolated from this library and showed in vitro cell killing in four pathogenic species of candida including c. albicans, c. tropicalis, c. parapsilosis, and c. glabrata. the mic values of these scfvs were in the range . - . μg/ml [ ] . biopanning using aspergillus fumigatus membrane fraction (amf) and employing competitive panning elution methods, scfv clones which showed in vitro antifungal activity against a. fumigatus was isolated from the same library [ ] . the authors argue the potential of these scfvs as candidates for developing as antifungal drugs by either engineering as scfv-drug conjugates or reformatting into whole igg for complement-mediated and antibody-dependent cytotoxic pathways [ ] . a number of human antibody fragments recognising c. albicans cell surface antigens were isolated from human phage display libraries [ , ] which is beyond the scope of this review. the authors have demonstrated antibody mediated opsonisation of c. albicans cells, phagocytosis and killing of the fungus by mouse macrophages and activation of the mouse complement cascade in in vivo models [ ] . combination immunotherapy is another attrac-tive therapeutic strategy where anti-mannan mabs were shown to have protective effect. in combination immunotherapy a chemical antifungal drug is administered along with an antibody. for example, mab b . is synergistic with amphotericin b [ ] and augments the therapeutic effect of fluconazole in a mouse model of disseminated candidiasis [ ] . these examples demonstrate the potential for antifungal mabs in clinical settings and the use of drug combinations as a powerful strategy to enhance antifungal efficacy and abolish drug resistance. the majority of anti-infective monoclonal antibodies developed in the s were antiviral drugs with a main focus on human immunodeficiency virus (hiv) neutralisers. using mice immunisations and sera from infected humans, several mabs were developed against the hiv type- envelop with only a few of them broadly neutralising across all hiv- subtypes. all these neutralising mabs were generated as a result of human infection and antibody responses against a range of epitopes such as gp , gp or various epitopes on gp which are exposed after cd binding [ ] . forsman et al. published the generation of several high affinity and crossreactive vhhs neutralising hiv- primary isolates belonging to subtype b and c. llamas were immunised with recombinant gp belonging to subtype b and the resultant phage display libraries were panned using recombinant gp from subtypes a, b and c. a competitive elution strategy using scd was employed during panning to isolate clones which were shown to inhibit the binding of scd to hiv- gp and gp from selected subtypes. it is proposed that the vhh domains inhibit hiv- infection by interacting with gp prior to its engagement with cd , which makes them potential hiv- inhibitors [ ] . subsequently, a family specific phage display library was constructed using pbls from the same llama immunised with recombinant gp derived from hiv- cn and tailored using degenerate primers based on the nucleotide sequences of the cdr -fr region of the positive hiv- neutralising vhhs described above. resultant picomolar affinity vhhs were shown to neutralise a broad range of hiv- virus belonging to subtypes b and c [ ] . another set of nanobodies that recognise the chemokine receptor cxcr was isolated by llama immunisation and phage display. cxcr belong to gpcr family where therapeutic targeting using conventional antibodies has proved unsuccessful probably because traditional antibodies struggle to access the cryptic and buried antigenic sites that make up the ligand binding pockets on the receptor surface. cxcr plays an important role in stem cell physiology, tissue repair, inflammation, metastatic spread of cancer and also serves as a co-entry receptor for hiv. highly potent nanobodies generated using whole cell immunisation (cxcr -expressing hek t cells), phage display library construction and biopanning behaved as competitive cxcr antagonists by totally blocking ligand cxcl binding and inhibiting chemotaxis and hiv- entry [ ] . two monoclonal antibodies specifically recognising the orf- capsid protein of hepatitis e virus were isolated from a cdna library constructed using the lymphocytes isolated from a chimpanzee bone marrow [ ] . this chimpanzee was experimentally infected with all five recognised hepatitis virus -hepatitis a to e. phage displayed biopanning using recombinant hev orf proteins from human hev strain (pakistani strain sar- ) and a swine hev strain (u.s. strain meng) generated two unique mabs with nanomolar binding affinities as determined by biacore analysis. the γ heavy chains of anti-hev mabs had the most sequence identity with the human vh family of germ line segments ( . % and . % overall identity) and the k light-chain sequences exhibited the most identity with the human vk family of germ line segments. these mabs were able to neutralise sar- strain of hev and offer protection from hev infection in rhesus monkeys. the authors have highlighted the significance of using chimpanzee as a donor for antigen primed antibody repertoire including the possibility of infecting them with viral pathogens of human relevance and reduced immunogenicity when used in human immunotherapy. earlier studies have reported that human antibodies are recognized as self by the chimpanzee immune system thereby generating little immunogenicity compared to other primates and the half-life of a human mab is equivalent to the estimated halflife of igg in humans [ ] . schofield and colleagues also reported the generation of four monoclonal antibodies against hepatitis a virus capsid from the same chimpanzee bone marrow derived cdna library using inactivated whole hav capsid from strain hm- for biopanning [ ] . all four mabs neutralized the strain hm- and two of the four mabs also neutralized the divergent agm- strain. however the actual mechanism of neutralisation remain unclear as the authors noticed that the fab fragments are not inhibiting virus attachment to cells via soluble simian cell receptor for hav, havcr . no further developments for these monoclonal fab fragments are available in the public domain. generating vhhs from immunised llama libraries to hepatitis b surface antigen (hbsag) and hepatitis b core antigen (hbcag) were explored as an approach to inhibit hbv replication thereby tackling viral infection [ , ] . the resultant vhh fragments were expressed as intrabodies to target the antigens which exert their functions in different compartments of the host cell (cytoplasm, er and nucleus). the intrabodies were shown to supress the secretion of hbsag and hbcag in vitro and inhibition of viral release in a mouse model [ ] . other vhh fragments developed using animal immunisation and phage display technology include those binding to rotavirus gp , h hemagglutinin to reduce viral replication in h n influenza virus, vhh recognising the tail of infectious phage in lactococcus bacteria. the list with relevant publications is summarised in table . . also a comprehensive list of antibodies raised against viral targets using non-immune libraries was presented in a recent review publication [ ] . there is currently no effective treatment for respiratory syncytical virus (rsv) lower respiratory tract infection. rsv is a major worldwide cause of morbidity and mortality in infants and young children, immunocompromised patients and the elderly. treatments remain largely supportive and rsv-specific options for prophylaxis are limited to palivizumab [ , ] . alx- (ablynx) is a trivalent nanobody in phase ii clinical trials for the treatment of rsv infections. it is one of the promising candidate biologic drugs in development for the prevention and/or treatment of rsv infections (see mazur et al. [ ] for a full review). alx- was originally isolated from a llama derived immune library as a monovalent domain prior to reformatting into a multivalent construct using genetic fusion. the vhh domain demonstrated high neutralising potency against rsv-a and rsv-b, and demonstrated superiority over palivizumab in blocking rsv replication [ ] . camel vhhs or nanobodies have also been developed to treat diseases such as trypanosomiasis (african sleeping sickness) caused by parasites from the trypanosoma genus. trypanosoma brucei has evolved a range of sophisticated immune evasion mechanisms, which include repeatedly generating variations of the immunogenic variant-specific surface glycoprotein (vsg), efficient internalisation of antibody-vsg complexes from the surface by endocytosis and proteolysis of the immunoglobulin [ ] . due to the dense packaging of vsg protein on the surface of trypanosoma, conventional antibodies are unable to access the more conserved vsg epitopes. high affinity nanobody clones directed towards isotype-specific and conserved epitope on the vsg protein coat, were isolated from an immunised camelid phage display vhh library. these nanobodies were found to be trypanolytic both in vitro and in vivo, which shows the potential of high affinity monovalent fragments to exert a cytotoxic effect on parasites even in the absence of fc domain. the authors have demonstrated the modes of action of these domains which appear to include: rapid arrest of cell mobility, blockage of endocytosis, flagellar swelling, collapse of mitochondrial membrane potential and atp exhaustion [ ] . monoclonal antibodies and novel smaller antigen binding scaffolds presents an attractive option for the development of new therapies and molecular drug targets against a wide range of human diseases and also in diagnostics due to their specific- [ ] ity and flexibility. in this book chapter, we have been able to present the robustness of combining immunisation and phage display technology in raising antibodies specific to antigens relevant in a number of infectious diseases of public health interest, and also those antigens classified as bioweapons. although the limitations associated with animal immunisation, especially those relating to ethical issues surrounding animal welfare and the need to immunise for every antigen of interest may limit access to animal immunisation in the future. however it is the opinion of the authors that the debate regarding animal use for research purposes should be substantially assessed on the basis of a risk-benefit analysis and suitability. antibacterial properties of pseudomonas aeruginosa immunotype lipopolysaccharidespecific monoclonal antibody (mab) in a murine thigh infection model: combined effects of mab and ceftazidime defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction single-domain llama antibodies as specific intracellular inhibitors of spvb, the actin adp-ribosylating toxin of salmonella typhimurium genetic and immunological comparison of anti-botulinum type a antibodies from immune and non-immune human phage libraries in vivo neutralization of botulinum neurotoxins serotype e with heavy-chain camelid antibodies (vhh) shark novel antigen receptors-the next generation of biologic therapeutics? in: pharmaceutical biotechnology antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite mining human antibody repertoires beyond natural antibodies: the power of in vitro display technologies hidden killers: human fungal infections identification of salmonella spi- secretion system components required for spvb-mediated cytotoxicity in macrophages and virulence in mice identification of an immunodominant abc transporter in methicillin-resistant staphylococcus aureus infections a large array of human monoclonal antibodies to type human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals phage-displayed antibody fragments recognizing dengue and dengue viruses as tools for viral serotyping in sera from infected individuals helicobacter pyloriantigen-binding fragments expressed on the filamentous m phage prevent bacterial growth isolation of a nanomolar scfv inhibiting the endopeptidase activity of botulinum toxin a, by single-round panning of an immune phage-displayed library of macaque origin the isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep a high-affinity macaque antibody fab with human-like framework regions obtained from a small phage display immune library monoclonal immunoglobulin g directed against aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis chimpanzee/human mabs to vaccinia virus b protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding ) β-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae functional aspects of protein mono-adp-ribosylation antibody binding to cryptococcus neoformans impairs budding by altering capsular mechanical properties antibody responses against wild-type yellow fever virus and the d vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants generation and characterization of alx- , a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection analysis of the expressed heavy chain variable-region genes of macaca fascicularis and isolation of monoclonal antibodies specific for the ebola virus' soluble glycoprotein a human sars-cov neutralizing antibody against epitope on s protein potential of primate monoclonal antibodies to substitute for human antibodies: nucleotide sequence of chimpanzee fab fragments llama antibody fragments with cross-subtype human immunodeficiency virus type (hiv- )-neutralizing properties and high affinity for hiv- gp isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display efficient production of chicken egg yolk antibodies against a conserved mammalian protein research and development of therapeutic mabs: an analysis based on pipeline projects passive intranasal monoclonal antibody prophylaxis against murine pneumocystis carinii pneumonia a new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks recombinant human antibody single chain variable fragments reactive with candida albicans surface antigens efficacy of combination immunotherapy of igm mab b . and amphotericin b against disseminated candidiasis protection against candidiasis by an immunoglobulin g (igg ) monoclonal antibody specific for the same mannotriose as an igm protective antibody isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis multiple-antigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens overview of antibody phage-display technology and its applications human-like antibodies neutralizing western equine encephalitis virus llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules nanobodies with in vitro neutralizing activity protect mice against h n influenza virus infection nanobodies (vhh-based single variable domains) potently inhibit chemotaxis and hiv- replication and mobilize stem cells human monoclonal antibody production: current status and future prospects staphylococcus aureus β-hemolysin-neutralizing single-domain antibody isolated from phage display library of indian desert camel antibody interference with n-acyl homoserine lactone-mediated bacterial quorum sensing mammalian adpribosyltransferases and adp-ribosylhydrolases generation of a family-specific phage library of llama single chain antibody fragments that neutralize hiv- shark variable new antigen receptor biologics-a novel technology platform for therapeutic drug development the human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to aspergillus fumigatus membrane protein selection of a macaque fab with framework regions like those in humans, high affinity, and ability to neutralize the protective antigen (pa) of bacillus anthracis by binding to the segment of pa between residues and phase i evaluation of the safety and pharmacokinetics of murine-derived anticryptococcal antibody b in subjects with treated cryptococcal meningitis heavy chain-only antibodies and tetravalent bispecific antibody neutralizing staphylococcus aureus leukotoxins combination immunotherapy of mab b . with fluconazole augments therapeutic effect to disseminated candidiasis the best defense is a good offense-salmonella deploys an adpribosylating toxin the salmonella spvb virulence gene encodes an enzyme that adp-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells sheep monoclonal antibody fragments generated using a phage display system a steric antagonism of actin polymerization by a salmonella virulence protein hiv- neutralizing antibodies: understanding nature's pathways lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics detection of antibodies against the four subtypes of ebola virus in sera from any species using a novel antibody-phage indicator assay development and clinical applications of novel antibodies for prevention and treatment of respiratory syncytial virus infection the art of blocking adpribosyltransferases (arts): nanobodies as experimental and therapeutic tools to block mammalian and toxin arts production of recombinant scfv against p of human immunodeficiency virus type by phage display technology a monoclonal antibody directed against a candida albicans cell wall mannoprotein exerts three anti-c. albicans activities nanobodies: natural singledomain antibodies antibody molecules which interact specifically with the active site or cleft of a target molecule a multicentre analysis of epidemiology of the nosocomial bloodstream infections in japanese university hospitals potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody markedly prolonged incubation period of hepatitis b in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis b surface antigen monoclonal antibodies in man that neutralized h n influenza viruses were classified into three groups with distinct strain specificity antibacterial monoclonal antibodies: back to the future? high-sensitivity monoclonal antibodies specific for homoserine lactones protect mice from lethal pseudomonas aeruginosa infections lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea high-affinity, human antibody-like antibody fragment (single-chain variable fragment) neutralizing the lethal factor (lf) of bacillus anthracis by inhibiting protective antigen-lf complex formation novel camelid antibody fragments targeting recombinant nucleoprotein of araucaria hantavirus: a prototype for an early diagnosis of hantavirus pulmonary syndrome an anti-β-glucan monoclonal antibody inhibits growth and capsule formation of cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo antibodies to watch in generation of diversity in mammalian gut-associated lymphoid tissues: restricted v gene usage does not preclude complex v gene organization monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fcrn: the neonatal fc receptor comes of age passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection identification of helicobacter pylori surface proteins by selective proteinase k digestion and antibody phage display directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries monoclonal antibody-based therapies for microbial diseases identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis e virus capsid protein four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus inhibition of fungal β- , -glucan synthase and cell growth by hm- killer toxin singlechain anti-idiotypic antibodies llama-derived single-domain intrabodies inhibit secretion of hepatitis b virions in mice production, characterization and in vitro testing of hbcag-specific vhh intrabodies high affinity nanobodies against the trypanosome brucei vsg are potent trypanolytic agents that block endocytosis design of a peptibody consisting of the antimicrobial peptide dhvar and a llama variable heavy-chain antibody fragment competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response killing of caenorhabditis elegans by pseudomonas aeruginosa used to model mammalian bacterial pathogenesis heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm+ memory b cells camelid single domain antibodies (vhhs) as neuronal cell intrabody binding agents and inhibitors of clostridium botulinum neurotoxin (bont) proteases generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and therapy reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments international study of the prevalence and outcomes of infection in intensive care units human monoclonal antibodies against west nile virus induced by natural infection neutralize at a postattachment step biological half-life of ovine antibody in neonatal lambs and adult sheep following passive immunization human antibodies from immunized donors are protective against anthrax toxin in vivo making antibodies by phage display technology comparison of antibody production to human interleukin- (il- ) by sheep and chickens animal models in the evaluation of antimicrobial agents human combinatorial antibody libraries to hepatitis b surface antigen broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library human recombinant antimannan immunoglobulin g antibody confers resistance to hematogenously disseminated candidiasis in mice key: cord- -yblzopc authors: kuhn, philipp; fühner, viola; unkauf, tobias; moreira, gustavo marcal schmidt garcia; frenzel, andré; miethe, sebastian; hust, michael title: recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display date: - - journal: proteomics clin appl doi: . /prca. sha: doc_id: cord_uid: yblzopc antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. traditionally, these antibodies are generated by hybridoma technology. an alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. this in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. on the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity‐matured antibodies. because the phage packaged dna sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. in this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given. antibodies are essential molecules as tools for basic research [ ], diagnostics [ ], and therapy [ ] . in the past-and still today-polyclonal antibodies were produced as serum in animals like horses [ ] . a milestone in antibody generation was the development of hybridoma technology that allows the production of monoclonal antibodies [ ] . but the hybridoma technology has drawbacks like limited number of candidates, possible instability of the aneuploid cell lines [ ], inability to provide antibodies against highly conserved antigens and most of all its limited application to generate human antibodies [ ] . the hybridoma technology essentially gives murine antibodies that can be used for diagnostic or research purposes. however, their therapeutic applications are limited because repeated administration of murine antibodies can cause human anti-mouse antibody reaction, reducing antibody half-life and leading to severe side effects such as anaphylactic shock [ ] . a strategy to circumvent these problems is antibody humanization or the use of transgenic animals in which the original antibody gene repertoire is replaced with a human gene repertoire [ ] [ ] [ ] [ ] . another strategy is the human hybridoma technology resulting in human antibodies [ , ] , although this technology has the same problems of the murine hybridoma technology regarding the limitation of the immune system. a technology that overcomes the limitation of the immune system is antibody phage display. since this technology involves an in vitro selection process, it is completely independent of any immune system. the display method most commonly used today is based on the work of georg p. smith on filamentous phage, which infect e. coli [ ] . the selection process is called "panning," referring to the gold digger's tool [ ] . m phage display technology was further developed / for antibodies in three places in parallel: heidelberg (germany), cambridge (uk), and la jolla (usa) [ ] [ ] [ ] [ ] . two different genetic systems have been developed for the expression of the antibody::piii (phage protein iii) fusion proteins for phage display. first, the antibody genes can be directly inserted into the phage genome fused upstream of the wild-type piii gene [ ] . second, most successful systems, uncouple antibody expression from phage propagation by providing the genes encoding the antibody::piii fusion proteins on a separate plasmid (called "phagemid") that contains a phage morphogenetic signal for packaging the vector into the phage particles [ ] . no matter the used phage display system, antibody fragments are displayed on the surface of m phage and the corresponding antibody gene is packaged in the phage particle. other phage display technologies using phage lambda [ ] or t [ ] are less suitable for the display of antibody fragments. in addition, these phages are lytic phage that aggravates the practical work. the most common antibody formats used for antibody phage display are the single chain fragment variable (scfv) [ ] [ ] [ ] or fragment antigen binding (fabs) [ , ] . other antibody formats used for phage display are single chain fabs (scfab), human vh domains (dabs), the variable domains of camel heavy chains (vhhs) and immunoglobulins of sharks (ignars) [ ] [ ] [ ] [ ] [ ] [ ] . figure shows an antibody phage and different antibody fragments. for veterinary research, chicken libraries are often used [ , ] . in contrast to what occurs in humans, the diversity of chicken antibody genes is the result of gene conversion, and the n-and c-terminal parts of chicken's vh and vl are always identical, facilitating antibody gene amplification, and library cloning [ , ] . also rabbit phage display was used to generate antibodies against pathogens [ ] . in the selection process (panning), the antigen is immobilized to a solid surface, like column matrixes [ ] , magnetic beads [ ] , or plastic surfaces with high protein-binding capacity such as polystyrene tubes or respectively microtitre wells, which is the most common method [ ] . an other option is the panning in solution using biotinylated antigens followed by a "pull-down" with streptavidin beads [ ] . the vast excess of nonbinding antibody phage will be removed by stringent washing. subsequently, the bound antibody phage will be eluted, e.g. by ph shift or trypsin, and reamplified by infection of e. coli. after infection of the phagemid bearing e. coli with a helperphage, new antibody phage will be produced. the selection cycle will be repeated and the number of antigen-specific antibody phage clones should increase with every panning round. usually - panning rounds are performed. finally, monoclonal antibody phage or monoclonal soluble antibodies can be identified by, e.g. elisa [ ] , immunoblot [ ] , or flow cytometry [ ] . the antibody fragment genes can be subcloned into any other antibody format, e.g. scfv-fc or igg [ , , , , ] . a schema of the selection process is given in fig. . regarding the source of genes, antibodies can be selected from two types of libraries: immune libraries and universal libraries. immune libraries are constructed from immunized/infected donors and typically used in medical research to obtain an antibody against a particular target antigen, e.g. an infectious pathogen like ebola virus [ ] . an advantage of this kind of library is that the v-genes contain hypermutations and are affinity matured, although its development can be restricted to ethical constraints. the alternative are universal or "single pot" libraries, which includes naïve, semisynthetic and synthetic libraries that are designed to isolate antibody fragments binding to every possible antigen, at least in theory [ , ] . naïve libraries are constructed from rearranged v genes from b cells (igm) of nonimmunized donors. examples for this library type are the naïve human fab library constructed by de haard and colleagues [ ] and the hal scfv libraries [ , ] . semisynthetic libraries are constructed from unrearranged v genes from pre-b cells (germline cells) [ ] or from one antibody framework [ ] in which one or several cdrs, but always the cdr h , are randomized. often used semisynthetic libraries are the tomlinson i and j libraries using one defined framework vh - and kappa ikv - with randomized cdr and cdr [ ] . a combination of naïve and synthetic repertoire was used for the fab antibody gene library. in this library, light chains from autoimmune patients were combined with a fd fragment (vh+ch ) containing synthetic cdr and cdr in the human vh - framework and naïve cdr regions, originated from autoimmune patients [ ] . fully synthetic libraries are made of human frameworks with randomized cdr cassettes [ ] [ ] [ ] . the theoretical size of these universal libraries is usually higher than independent clones [ , , [ ] [ ] [ ] . to date, antibodies and antibody conjugates were approved by ema and/or fda (status january ) (http://www.imgt.org/mab-db/query.action, development status: phase m in search field) and about antibodies were under development in [ ] . most approved therapeutic antibodies are for cancer and autoimmune diseases and the annual sales of therapeutic antibodies exceeded billion us$ in [ ] . the mechanisms of therapeutic antibodies are manifold and include neutralization of substances, e.g. toxins [ ] or cytokines like tumor necrosis factor alpha [ ] , blocking of receptors like epidermal growth factor receptor [ ] , binding to cells and modulating the host immune system [ ] , or combinations of these effects [ ] . currently, two recombinant antibodies are approved for the treatment of pathogens or toxins. raxibacumab is a human antibody for anthrax treatment derived from a phage display library from cambridge antibody technology (now medimmune, part of astrazeneca) in cooperation with human genome science (now glaxosmithkline) [ ] . the antibody palivizumab for the treatment of respiratory syncytial virus bronchiolitis is a classical humanized antibody [ ] . a further antibody, but also not derived from phage display, the clostridium difficile antibody bezlotoxumab is in clinical phase [ ] . an overview of recombinant antibodies derived from phage display against bacterial and viral pathogens, eukaryotic pathogens (parasites, fungi), and toxins as well as detailed examples for diagnostic and therapy are given in the next sections. the most therapeutic antibodies against bacterial targets are generated against toxins. these antibodies are described in the section "recombinant antibodies against toxins." the majority of antibodies against bacteria are developed in order to facilitate diagnostics in patients [ , ] and environmental samples [ , ] . in general, cultural and microbial detection of bacteria is regarded as standard in diagnostics for many pathogens, e.g. mycobacterium tuberculosis [ ] or salmonella typhimurium [ ] . since these methods are often time-consuming and require experienced lab personal, high throughput analysis is often limited. real-time pcr measurements have been developed for the diagnostics of many bacteria [ ] , offering high sensitivity and specificity of detection. but sample treatment is still needed in many cases, including expensive and complex laboratory devices [ ] . an other approach is ms for diagnostic, but this technology needs expensive devices [ ] . antibody-based diagnostic like elisa would be more simple and easy to use, also in developing countries. in order to generate antibodies with the desired-binding properties, phage-display has been used to select antibodies on proteins or polysaccharides of chlamydophila psittaci [ ] , chlamydia trachomatis [ ] , haemophilus influenzae [ ] , listeria monocytogenes [ ] , mycobaterium bovis [ ] , mycobacterium tuberculosis [ , , ] , porphyromonas gingivalis [ ] , ralstonia solanacearum [ ] , salmonella typhimurium [ , ] , and yersinia pestis [ ] . but even selections on cell lysate or whole cells were performed on mycobacterium avium [ ] , bacillus anthracis [ , ] , moraxella catarrhalis [ ] , lawsonia intracellularis [ ] , lactobacillus acidophilus [ ] , helicobacter pylori [ ] , brucella melitensis [ ] , and bordetella pertussis [ ] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different bacterial pathogens. tuberculosis (tb) is a bacterial infection caused by mycobacterium tuberculosis (mtb), a pathogen with particular cell wall and membrane characteristics that is able to infect and multiply within alveolar macrophages leading to cough, weakness, and fever [ ] . in , million people were infected and . million died worldwide [ ] . in order to improve treatment and control disease spread, tb must be diagnosed as early as possible. one prominent target for this purpose is the bacterial antigen (ag ) that is the most abundant protein secreted by mtb [ ] . it is a complex of three highly homolog proteins (ag a, ag b, and ag c) with a molecular size of - kda each [ ] . in a novel approach by ferrara et al. [ ] monoclonal antibodies were selected against the ag complex by combining phage-and yeast-display. first, a naïve scfv-library was preselected against ag by phage display, reducing the diversity from ß unique scfv clones to ß colony forming units. second, the enriched sublibrary was cloned into a yeast-display system. each clone of the selection output was screened individually for antigen binding by facs. those with the highest antigenbinding signals were sorted and further analyzed. in total clones were sequenced revealing genetically unique scfv clones. further screening assays identified seven antibody pairs, which can detect ag down to a concentration of . nm in the absence of serum and . nm in its presence. interestingly, none of the antibodies were absolutely specific for one of the ag subunits of the complex. all three subunits a, b, and c were bound although not equally. in contrast, fuchs et al. selected five human antibodies against the recombinant ag b protein from the naïve hal / libraries [ ] . three of them bound specifically to the b protein in elisa (lod: ng/ml). even sandwich detection of recombinant b in elisa (lod: ng/ml) and integration into lateral flow immuno assay (lod: < ng/ml) was shown. but antigen detection in mtb cell extracts or culture filtrates was restricted to direct elisa and immunoblot assay. the authors of both papers argued that the selected antibodies must be affinity matured in order to reach required detection limits. successful implementation of this concept was demonstrated by sixholo et al. [ ] . chicken antibodies were selected from the semisynthetic nkuku library against a kda recombinant antigen of m. tuberculosis, also known as heat shock protein x (hspx). after four rounds of panning, three scfvs were identified in elisa. the clone with the lowest elisa signal was selected for in vitro engineering using two different approaches. first, a mutant library was generated from the parental scfv by error-prone pcr with a diversity of ß × unique scfv. this library was selected again on the hspx antigen under more stringent conditions, leading to identification of three mutant scfvs. these contained one or three individual amino acid exchanges, occuring both in cdr and framework regions. all mutants showed increased elisa-binding signals, reaching a signal ß times higher when compared to the parental scfv. furthermore, the mutants showed improved association and dissociation kinetics in elisa and spr analysis. in the other approach, the length of the scfv linker was reduced from amino acids to a single glycine residue, forcing tetramerization of antibodies. due to cooperative binding, the apparent functional affinity was improved. in elisa, tetrameric scfv generated a ß times higher elisa signal, and spr proved increased association and decreased dissociation rates. porphyromonas gingivalis is one of the major pathogens involved in periodontitis [ ] . periodontitis is an inflammatory disease, which causes the loosening and loss of teeth. secreted cysteine proteases like rgpb contribute to disease pathology and represent potential biomarkers for disease detection and progression [ ] . skottrup et al. [ ] generated a naïve antibody library from camels with a diversity of ß × clones. a special feature is that the antibodies consist of a single monomeric vhh domain, also called nanobodies. advantages of this format are the small size (ß kda), the ease production in e. coli, and the convex paratope that is formed which enables the targeting of cryptic epitopes. the library was selected on immobilized rgpb. one clone was isolated that binds specifically to cell surface displayed and soluble rgpb with an affinity of pm. a detection limit of ß × cells/ml of saliva was reached when tested by subtractive inhibition elisa. but catalytic activity of rgpb was not inhibited by antibody binding. salmonella typhimurium, is one of the most important pathogens of foodborne gastrointestinal infections [ ] . meyer et al. [ ] first identified novel immunogenic proteins of s. typhimurium from a genomic library using oligopeptide phage display. in a second step human antibodies were selected against these targets from the naïve hal gene libraries. for diva (differentiating infected from vaccinated animals) vaccine development, s. typhimurium strains lacking a marker protein, e.g. ompd (outer membrane protein d) were developed (diva vaccines) [ ] . to allow the discrimination between vaccinated animals and infected animals a diagnostic of this specific marker is necessary. here, meyer et al. [ ] generated scfvs against ompd for diagnostics purposes. only some of the generated antibodies were suitable for a competitive elisa using swine serum showing also the difficulties when developing diagnostic assays that are often hampered by the complexity of serum samples. an overview of recombinant antibodies generated by phage display against bacterial pathogens is given in table . up to now, a large panel of antibodies against various viruses has been generated from either naïve or immune libraries using phage display technology. panning against peptides, recombinant viral proteins, or complete virus particles has led to the identification of antibodies directed against human pathogenic viruses such as sin nombre virus [ ] , dengue virus [ , ] , influenza virus [ , ] , veev [ ] , norovirus [ ] , sars coronavirus [ ] , or hepatitis c [ ] from naïve antibody gene libraries. other antibodies were selected from immune antibody gene libraries targeting, e.g. western equine encephalitis virus (weev) [ ] , hiv [ , ] , sars [ ] , yellow fever virus [ ] , or influenza virus [ , ] . semisynthetic libraries were also used to generate antibodies specific for influenza virus [ ] . beside human and animal viruses, antibodies were also generated against plant viruses [ , , ] . libraries originated from different species have been successfully employed to isolate virus-specific antibodies in the past such as those from macaque [ ] , mouse [ ] , chimpanzee [ ] , llama [ ] , chicken [ ] , and human origin [ ] . most of the virus-specific antibodies have been isolated from libraries in scfv format [ , ] , although fab [ , ] and vhh libraries [ ] were also successfully used. an interesting approach was used by xiao et al. using the antibody ch domain as scaffold to generate binders against gp of hiv [ ] . different antibody characteristics have an influence on virus binding and neutralization. neutralizing antibodies prevent cell binding of the virus. the anti-gp antibody hk has a higher neutralizaton rate as scfv or fab compared to igg showing, that the epitope is less accessable for the igg format [ ] . another interesting example is the anti-gp vhh h . here, a tryptophan in the cdr is not relevant for epitope binding, but essential for virus neutralization [ ] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different virus groups. vaccinia virus is the prototype virus in the genus of orthopoxvirus. it is a relatively large dna virus with a genome of about kbp [ ] . the genus orthopoxvirus includes various species such as monkeypox virus, cowpox virus, and especially variola virus that is the causative agent of smallpox in humans. naturally occurring smallpox has been eradicated in because of a massive who vaccination program that began in . however, no vaccination of the civilian population is conducted nowadays. the potential threat of intentional release has renewed the search for safe and effective smallpox vaccines as case fatality rates of % or more among unvaccinated subjects are reported [ ] . because orthopoxviruses are highly related, it is assumed, that immunity against one poxvirus goes along with immunity against most members of the entire virus family [ , ] . using an immune scfv phage display library constructed from vaccinia virus immunized patients, a panel of human vaccinia-specific antibodies was selected. plaque-reduction neutralization tests revealed that seven of these antibodies neutralized vaccinia as well as cowpox virus in vitro. five of those antibodies additionally neutralized monkeypox virus [ ] . other antibodies were generated from a fab immune library derived from vaccinia virus immunized chimpanzee. converted into a chimeric chimpanzee/human igg format, two antibodies displayed high affinities to vaccinia protein b (k d of . and . nm). antibody ah al was neutralizing in vitro for vaccinia and smallpox virus and proofed to be protective in mice challenged with vaccinia virus even when administered days after challenge. in this model ah al proved to provide significantly greater protection than that of the previously isolated rat anti-b antibody c [ ] . vaccinia had to be used as model, because the final confirmation of protection against smallpox is not possible. ebola virus and marburg virus, two filoviruses, cause severe hemorrhagic fever and possess high mortality of up to % in humans. in addition to public health concerns associated with natural outbreaks, ebola virus might be a potential agent of biological warfare and bio-terrorism [ ] . human antibodies directed against ebola virus were selected from a library originated from patients that recovered from infection in the ebola virus outbreak in kikwit, democratic republic of congo [ ] . several antibodies against various viral proteins such as nucleoprotein, envelope glycoprotein, and secreted envelope glycoprotein have been isolated in this study. one antibody (kz ), specific for envelope glycoprotein, was able to neutralize in vitro as fab ( % neutralization at . g/ml) and as full igg ( % neutralization at . g/ml) [ ] . follow-up studies were showing effective protection in vivo in a guinea pig ebola challenge model when the antibody was administered up to one hour postviral challenge [ ] . interestingly, kz was not protective in macaques challenged with ebola even if the antibody was given as a two-dose treatment with the first dose one day prior viral challenge and the second dose days postchallenge [ ] . a murine scfv and two shark ignar v immune libraries were generated against inactivated zaire ebola virus to yield various antibodies specific for the viral matrix protein vp and the viral nucleoprotein [ ] . interestingly, this work represents the first example of a successful targeted ignar v isolation from a shark immune response library. dengue virus (denv), a member of the flaviviridae family, is responsible for at least million symptomatic infections each year and became a major health and economic burden in over countries worldwide [ ] [ ] [ ] . it is a positive strand rna virus with a ß kb genome, that compromise a single open reading frame. the four circulating serotypes of dengue virus show approximately % sequence homology [ , ] . fab monoclonal antibodies to dengue type virus were isolated from a chimpanzee immune library. two fab, namely h and d neutralized denv- efficiently with a titer of . - . g/ml by plague reduction neutralization test [ ] . another study selected human scfv antibodies specific to dengue virus envelope protein by panning against recombinant full-length envelope protein and its domain iii [ ] . because denv envelope protein is an essential molecule for virion assembly and virus entry, scfvs selected in this study were shown to exhibit inhibitory effects on denv infection in vitro [ ] . dengue nonstructural protein (ns ) is essential for viral replication and host immune response modulation, making it an excellent target for dengue-inhibiting antibodies. a naïve human fab-phage library was screened for ns -specific antibody fragments using various ns variants from dengue virus serotypes - as antigens for panning and characterization [ ] . using ns from alternating dengue serotypes for each round of panning, this strategy resulted in the identification of two clones that are cross-reactive against all four dengue serotypes. another study selected antibodies using phage display by panning with dengue virus particles directly captured from supernatant of infected vero cells. here, highly serotype-specific antibodies were generated. from a total of nine antibodies, seven were shown to be specific to only one serotype. one dengue- selected clone cross-reacted with dengue , whereas another clone showed cross-reactivity with all serotypes despite being selected solely on dengue particles. interestingly, all of the obtained antibodies recognized several strains of distinct genotypes within the corresponding serotype [ ] . panning against dengue envelope protein resulted in the identification of an antibody (c ) from a mouse/human chimeric fab library that cross-reacts with denv - and neutralizes denv in cell-based assays after conversion into full-length igg [ ] . besides scfv and fab, variable domain heavy-chain antibodies (vhh antibodies) were also selected using phage display technology. after four rounds of panning on recombinant denv ns protein, positive clones were selected. affinity measurements with ns revealed a k d value of . × − m for the best vhh antibody p [ ] . venezuelan equine encephalitis virus (veev), an alphavirus of the togoviridae family, causes equine epidemics but can also cause encephalitis in humans [ , ] . because this virus is classified as category b agent by the centers for disease control and prevention (cdc), much research has been done to generate neutralizing antibodies against it. antibodies were generated from an immune library from human donors targeting both veev envelope glycoproteins e and e [ ] . the isolated fabs l a and f were neutralizing in vitro, with f being times more effective than l a . subsequently, f was converted into full igg format and was employed to generate neutralization-escape variants of veev for epitope mapping. within another study, an immune macaque library was used to generate human-like antibodies [ ] . one of these antibodies, scfv-fc tor - b , was protective in mice when administered h postviral challenge with veev trinidiad strains, showing - % survival after a challenge with -fold ld . however, scfv-fc tor - b was not able to neutralize trinidad strain in vitro, but other veev strains instead, showing that neutralization is not mandatory for an in vivo protective antibody [ ] . another study describes the selection of antibodies from a human naïve scfv gene library using complete, active veev particles as antigen. in this case, specific detection of the veev strains tc , h / , and by the selected antibodies was proven. remarkably, none of the selected scfv phage clones showed cross-reactivity with alphavirus species of the eastern equine encephalitis virus and weev antigenic complex or with chikungunya virus, making them ideal tools for the immunological detection and diagnostic of alphavirus species [ ] . two different scfv antibody libraries were constructed from weev immunized macaques. subcloned as scfv-fc, three antibodies from these libraries specifically bound weev in elisa with little or no cross-reactivity with other alphaviruses and were found to be neutralizing in vitro. in this study, the first antibodies against weev, that were shown to be neutralizing in vitro, were developed. about ng/ml of the best antibody (tor - a ) neutralized % of × tcid /ml weev [ ] . flu is a disease caused by influenza viruses. in the last years, the bird flu (h n ) and the pandemic swine flu (a variant of h n ) moved in the research focus. due to many genetic events, such as antigenic drift and shift, new influenza variants will occur in the future and will challenge vaccine and diagnostic development [ , ] . different groups developed antibodies by phage display against influenza viruses. sui et al. [ ] selected antibodies from a nonimmune scfv library against the h hemagglutinin ectodomain. hemagglutinin is a trimer and the extracelluar part consists of a stalk domain and a globular head domain [ ] . they identified ten antibodies binding to the trimeric h , but not monomeric h . interestingly, nine of these antibodies shared the same germline framework (vh - ). these antibodies were converted into igg and were protective in mice with or mg/kg in a prophylactic or therapeutic challenge model, respectively. very remarkably, some antibodies crossneutralized h , h , h , h , h , and h influenza strains. these are phage display derived antibodies that are candidates for broad-spectrum influenza immunotherapy [ ] . rabies is caused by the rabies virus that infects the central nervous system. before louis pasteur developed the rabies vaccination, the disease was always fatal. the current postexposure therapy is based on vaccination-as performed by pasteur in -and polyclonal anti-rabbies immunoglobulins [ ] [ ] [ ] . one hundred forty-seven unique recombinant antibodies against the rabies glycoprotein were selected from two immune scfv libraries [ ] . the neutralization of street rabies viruses was tested in vitro and the best neutralizing antibodies were tested in vivo in a hamster rabies infection model. here, the antibody cr showed % postexposure protection with iu/kg [ ] . this antibody was further analyzed in combination with an other human antibody cr , derived by somatic cell hybridization technique [ ] , in in vitro and in vivo models [ ] . the safety of this mab cocktail named cl was tested in a clinical phase study [ ] and subsequently in phase studies. the antibodies were named foravirumab (cr ) and rafivirumab (cr ). an overview of recombinant antibodies generated by phage display against viruses is given in table . using phage display technology, a large panel of antibodies against a broad range of eukaryotic pathogens has been generated. these antibodies are directed against pathogenic animals, e.g. taenia solium [ ] , protozoa, e.g. cryptosporidium parvum [ , ] , plasmodium falciparum [ , ] , or toxoplasma gondii [ ] and fungi such as aspergillus fumigatus [ ] . not only human pathogens have been in the focus of antibody generation, but also veterinary pathogens like myxobolus rotundus [ ] (a fish pathogen) or babesia gibsoni (a dog pathogen) [ ] and plant pathogens like aspergillus niger [ ] , fusarium verticilloides [ ] , or sclerotinia sclerotiorum [ ] . most antibodies generated in these studies derive from human antibody gene libraries, but libraries from mouse [ ] , chicken [ ] , camel [ ] , or macaque [ ] origin have been also successfully applied in phage display to generate monoclonal antibodies against eukaryotic pathogens. in the following paragraphs detailed examples for the use of phage display for the generation of antibodies against different eukaryotic pathogens are given. aspergillus fumigates is the causative pathogen of allergic bronchopulmonary aspergillosis, chronic necrotizing aspergillosis, saprophytic aspergilloma, and highly lethal invasive aspergillosis, the most important aspergillus-related disease [ , ] . invasive aspergillosis occurs in immunocompromised individuals for example after hematopoietic stem cell transplantation or solid organ transplantation [ ] . due to its severity, an early diagnosis is crucial for a successful treatment. in , schütte et al. [ ] reported the generation of several antibodies binding specifically to crf , an a. fumigates antigen of the glycosyl hydrolase family that is located in the cell wall of the growing hyphae. these antibodies could serve as tools for new diagnostic assays such as serum elisa or histopathological immunofluorescence microscopy. the antibodies described in this study were isolated from two different phage-displayed scfv libraries: one was a macaque immune library with a theoretical diversity of . × independent clones; while the other was human naïve antibody library hal / [ ] . both libraries were used in two different panning strategies: the first was performed on recombinant antigen immobilized on immunostrips; the second was carried out in solution with biotinylated antigen. finally, individual scfv clones were selected, with six clones deriving from the naïve library and ten from the immune library. interestingly, all antibodies generated on immobilized antigen bind to linear epitopes, whereas all antibodies generated by panning in solution bind to conformational epitopes. seven of these antibodies bound to their native antigen on growing hyphae of aspergillus fumigatus and did not show crossreactions to other aspergillus species or candida albicans. malaria is a life-threatening protozoal infection of the red blood cells and one of the most common mosquito-borne diseases. currently, an estimated . billion people in tropical and subtropical countries live at risk of malaria [ , ] . in humans malaria is elicited by at least five different species of plasmodium, p. falciparum, p. vivax, p. ovale, p. malariae, and p. knowlesi [ ] . p. falciparum is responsible for most malaria-related deaths globally, while p. vivax is the most widespread parasite [ ] . three different approaches to generate antibodies against p. falciparum using phage display are described. in these studies various structures of different developmental stages of the parasite served as antigens. in a first study roeffen et al. [ ] used two scfv immune libraries, constructed from b-lymphocytes from p. falciparum patients with transmission-blocking immunity, to generate antibodies against pfs / , a surface protein of p. falciparum that is expressed during zygote-and macrogamete stages. pfs / is a potential vaccine candidate since it is a target of transmissionblocking antibodies that are taken up by the mosquito together with the blood meal and block within the mosquito's intestinal tract the oocyte development [ ] [ ] [ ] . the panning was performed on an extract of gametocytes immobilized in immunotubes and the elution was performed by competition with a mixture of four rat monoclonal mabs, which recognize distinct epitopes on pfs / (epitopes i, iib, iii, and v). interestingly, all selected antibody clones bound to epitope iii of pfs / . in , sowa et al. [ ] reported the isolation of a human monoclonal antibody against the block region of plasmodium falciparum merozoite surface protein- (pfmsp- ) by phage display from a malaria patient derived scfv library. lundquist et al. [ ] generated a fab-immune library from leukocytes of adults with acquired immunity to malaria. three individual fabs designated ram , ram , and ram were isolated from this immune library by panning on a recombinant fragment of the merozite surface protein (msp- ). msp- is involved in heme binding, and antibodies against this protein promote eradication of the parasite by monocytes [ , ] . a synthetic peptide of the n-terminal fragment of msp- is even used in human clinical trials as vaccine [ ] . the antibodies were subcloned into igg and igg format for further analysis. binding of the antibodies to native parasite protein was demonstrated for all three antibodies in immuno blot and immunofluorescence microscopy. ram and ram also bound to their antigen in fixed and permeabilized cells in flow cytometry. the igg format of ram showed in a functional assay (antibody-dependent cellular inhibition assay, acdi) an inhibition rate that is comparable to affinity-purified polyclonal anti-msp- - antibodies derived from immune donors. the igg format also showed inhibition in the acdi, but lower compared to igg [ ] . an overview of recombinant antibodies generated by phage display against eukaryotic pathogens is given in table . several toxins are classified by the cdc as category a or b agents (for definition see: www.niaid.nih.gov/topics/ biodefenserelated/biodefense/pages/cata.aspx) that are relevant for diagnostics and therapeutics. they can easily be disseminated and result in high or moderate mortality rates [ ] . in this context, the antibody phage display offers a powerful tool for antibody selection and allows the isolation of neutralizing antibodies against complete active toxins or special domains by using different human naïve antibody libraries with high diversity [ ] [ ] [ ] . for toxins, the aim is to find antibodies binding to the cell binding domain of the toxin and neutralize the interaction of this domain with the corresponding cellular target, mainly cell surface proteins. but also antibodies directed against the translocation domain or the enzymatic domain can neutralize the toxicitiy. for the isolation of high-affinity antibodies against specific targets, animals are often immunized with toxoids, nontoxic subunits or selected toxin domains. well suited are macaques for the construction of immune libraries, because macaque v-genes are very similar to their human counterparts [ , , [ ] [ ] [ ] [ ] . in the following paragraphs, we give detailed examples for antibody generation using phage display and antibody engineering against different toxins. so far, antibody phage display was successfully used for antibody selection against a panel of toxins classified as category a agents, such as from clostridium botulinum (botulism) [ , [ ] [ ] [ ] and bacillus anthracis (anthrax) [ ] and also against different category b agents, such as staphylococcal enterotoxin b [ , ] and ricin toxin from ricinus communis [ , ] . an example for a high-risk microorganism that [ , ] . especially, serotype a is recognized as the most toxic substance known, showing ld of about ng/kg by intravenous route, about ng/kg by the pulmonary route and about g/kg for the oral route [ ] . bonts are composed of a disulfide bond-linked kda light chain and a kda heavy chain. the heavy chain contains two functional domains (hc and hn) that are responsible for toxin uptake into nerve cells by receptor-mediated endocytosis and for the translocation of the light chain across the membrane into the neuronal cytosol. whereas the catalytic domain of the light chain is responsible for the bont toxicity. the current approach for treatment of botulism includes the application of human anti-botulism immunoglobulins, such as babybig, or equine anti-toxin serum. but the human serum stock of babybig is limited and the equine anti-toxin may cause hypersensitivity and serum sickness. in these situations, antibody phage display provides a technology to generate toxinneutralizing antibodies against each serotype. for instance, a macaque immune library was used to isolate neutralizing scfv with nm affinities against the light chain of bont/a [ , , ] , but also antibodies against the heavy chain or other relevant serotypes of bont are of therapeutic interest. phage display technology was also used for isolation of single domain antibodies (vhh) after immunization of a llama with a cocktail of bont toxoids (a-f) [ ] . another approach was the generation of a human antibody gene library after inducing a bont/a-specific immune response by in vitro immunization [ ] . furthermore, antibody phage display was used to generate antibodies against other clostridial toxins such as those from clostridium tetani or clostridium difficile [ , ] . anthrax, another serious infectious disease is caused by bacillus anthracis, an aerobic, gram-positive, spore-forming bacterium that is found in soils around the world. bacillus anthracis secrets two toxins: the lethal toxin (lt) and the edema toxin (et) [ ] . both are composed of two subunits: the lt consists of the lethal factor (lf), and the protective antigen (pa); while the et is formed by the edema factor, and pa. it was demonstrated that only lt has an essential role in the pathogenesis of anthrax [ ] . the subunit pa is the basis of current vaccines and induces the generation of neutralizing antibodies. in combination with antibiotics, commercial monoclonal antibodies against pa, such as raxibacumab, are commonly used for treatment [ ] . in , the fda approved raxibacumab to treat inhalational anthrax. due to security issues the use of anti-pa antibodies alone is questionable, since pa could be modified and lose the recognized epitopes while retaining biological activity. an alternative to anti-pa antibodies are antibodies targeting the lf, such as lf, which was isolated from an immune library via antibody phage display technology [ ] . a combination of an anti-pa antibody with an anti-lf antibody could lead to a synergistic effect and improve the efficacy of the therapy. an example for bacterial toxins classified as category b agent is staphylococcal enterotoxin b (seb) from staphylococcus aureus. this bacterium is a potential causative agent for food-borne illness and produces twenty-one types of staphylococcal enterotoxins that cause symptoms of food poisoning such as abdominal cramps, vomiting, and diarrhea [ , ] . seb is a single polypeptide of kda and is the most potent toxin secreted by s. aureus. as a superantigen, it stimulates t cells and leads to an overproduction of cytokines, causing clinical symptoms such as fever, hypertension, and in some cases death. phage display was used to generate recombinant antibodies from a murine immune library [ ] and to identify the epitope of a seb-specific monoclonal antibody using a peptide phage library [ ] . furthermore, a human monoclonal antibody against seb was isolated from a synthetic human antibody gene library that inhibited seb binding to mhc-ii [ ] . the phage display technology was also used to isolate antibodies against ricin, which is also classified as category b agent by cdc. ricin is a -kda glycoprotein from the castor bean plant (ricinus communis), which consists of two distinct subunits (rta and rtb). rtb is a galactose-and n-acetylgalactosamine-specific lectin that binds to specific sugar residues on the cell surface, allowing internalization of the toxic rta by endocytosis [ ] . rta is an rna n-glycosidase that irreversibly inactivates eukaryotic ribosomes, leading to the inhibition of protein synthesis [ ] . human-like antibodies were selected by phage display from a macaque immunized with rta. one antibody, rca, had a picomolar affinity and neutralized the biological activity of ricin in vitro [ ] . furthermore, neutralizing vhh with high affinity was selected from a llama immune library. the best antibody c was able to neutralize % ricin activity in an in vitro assay using g/ml vhh [ ] . in addition to the different toxins that are classified by the cdc as category a or b agents, the number of toxins is almost endless. different animals are known to produce high potential toxins containing a complex composition. for example, tityus serrulatus, known as brazilian yellow scorpion and the most dangerous scorpion in brazil, produces a -amino acid peptide, called gamma toxin, which is the major toxic component in the venom [ , ] . regarding this toxin, a neutralizing antibody was isolated from a human library via phage display and was capable of protecting mice [ ] . the same procedure was used for bothrops jararacussu, a venomous pit viper species endemic in south america. by using a human antibody gene library, different antibodies were selected to inhibit the phospholipase activity of the venom in vitro and reduce the myotoxicity in vivo [ ] . marine organisms can also produce toxins, e.g. the tetrodotoxin (ttx) from pufferfish. elisa, enzyme-linked immunosorbent assays. here, scfv were selected from a human naïve antibody gene library neutralizing % of ttx activity in vitro [ ] . an overview of recombinant antibodies generated by phage display against toxins is given in table . antibody phage display allows the generation of recombinant antibodies from different species, including human, llama, camel, macaque, shark, or mice. these antibodies are mainly derived from two types of sources: immune, or naïve libraries. immune libraries should be preferred when immunized animals or convalescent patients are available, offering the chance to directly isolate affinity matured antibodies. if immunization is not possible or ethically not feasible, naïve antibody gene libraries are an alternative. in such an approach, the antibody generation process is not limited by the immune system. using antibody phage display a variety of recombinant antibodies was generated for diagnostics and therapy against bacterial, viral pathogens, and eukaryotic pathogens as well as toxins. man-made antibodies development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms high-avidity human igg kappa monoclonal antibodies from a novel strain of minilocus transgenic mice production of fully human antibodies by transgenic mice human antibodies from transgenic mice development trends for human monoclonal antibody therapeutics human monoclonal antibodies accessing the human repertoire for broadly neutralizing hiv antibodies. mabs filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface antibody-selectable filamentous fd phage vectors: affinity purification of target genes assembly of combinatorial antibody libraries on phage surfaces: the gene iii site a surface expression vector for antibody screening making antibody fragments using phage display libraries phage antibodies: filamentous phage displaying antibody variable domains identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda a human scfv antibody generation pipeline for proteome research application of phage display to high throughput antibody generation and characterization human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library a large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies generation of high-affinity human antibodies by combining donor-derived and synthetic complementaritydetermining-region diversity domain antibodies: proteins for therapy single chain fab (scfab) fragment single domain camel antibodies: current status camelid immunoglobulins and nanobody technology selection and affinity maturation of ignar variable domains targeting plasmodium falciparum ama isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scfvs in inhibition elisas chicken scfvs and bivalent scfv-c(h) fusions directed against hsp of mycobacterium bovis chicken immunoglobulin gamma-heavy chains: limited vh gene repertoire, combinatorial diversification by d gene segments and evolution of the heavy chain locus singlechain antibody fragments from a display library derived from chickens immunized with a mixture of parasite and viral antigens recombinant rabbit single-chain antibodies bind to the catalytic and cterminal domains of hiv- integrase protein and strongly inhibit hiv- replication methodology for selection of human antibodies to membrane proteins from a phage-display library the production of a genus-specific recombinant antibody (scfv) using a recombinant potyvirus protease identification of a putative crf splice variant and generation of recombinant antibodies for the specific detection of aspergillus fumigatus construction of human antibody gene libraries and selection of antibodies by phage display highthroughput screening of single-chain antibodies using multiplexed flow cytometry generating recombinant antibodies to the complete human proteome high level transient production of recombinant antibodies and antibody fusion proteins in hek cells recombinant human monoclonal antibodies to ebola virus making antibodies by phage display technology which are the antibodies to watch in ? mabs the therapeutic monoclonal antibody market highaffinity, human antibody-like antibody fragment (singlechain variable fragment) neutralizing the lethal factor (lf) of bacillus anthracis by inhibiting protective antigen-lf complex formation tumor necrosis factor alpha drugs in rheumatoid arthritis: systematic review and metaanalysis of efficacy and safety anti-epidermal growth factor receptor monotherapy in the treatment of metastatic colorectal cancer: where are we today? the oncologist cd -specific antibodies: a portal to the treatment of autoimmunity monoclonal antibody therapy of cancer reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to rsv f protein mechanisms of protection against clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab diagnostic evaluation of a nanobody with picomolar affinity toward the protease rgpb from porphyromonas gingivalis using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen , a tuberculosis biomarker development of specific recombinant monoclonal antibodies against the lipopolysaccharide of ralstonia solanacearum race development and implementation of a single-chain fv antibody for specific detection of bacillus anthracis spores challenges and perspectives in the diagnosis of extrapulmonary tuberculosis detecting non-typhoid salmonella in humans by elisas: a literature review real-time pcr as a diagnostic tool for bacterial diseases sample preparation: the forgotten beginning a systematic review of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry compared to routine microbiological methods for the time taken to identify microbial organisms from positive blood cultures analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from chlamydophila psittaci efficient construction of a large nonimmune phage antibody library: the production of high-affinity human singlechain antibodies to protein antigens human fab fragments specific for the haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression high affinity anti-internalin b vhh antibody fragments isolated from naturally and artificially immunized repertoires improving the characteristics of a mycobacterial kda-specific chicken scfv novel human recombinant antibodies against mycobacterium tuberculosis antigen b identification of immunogenic proteins and generation of antibodies against salmonella typhimurium using phage display development of phage-based single chain fv antibody reagents for detection of yersinia pestis nimotuzumab and cetuximab block ligand-independent egf receptor signaling efficiently at different concentrations rugged single domain antibody detection elements for bacillus anthracis spores and vegetative cells phage antibodies obtained by competitive selection on complement-resistant moraxella (branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein isolation of lawsonia intracellularis specific single-chain fv antibody fragments from phage display library using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes generation and characterization of human monoclonal scfv antibodies against helicobacter pylori antigens isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis construction and characterization of single-chain variable fragment antibodies directed against the bordetella pertussis surface adhesins filamentous hemagglutinin and pertactin is mycobacterium tuberculosis a closer relative to gram-positive or gram-negative bacterial pathogens? tuberc the antigen complex: a major secretion product of mycobacterium tuberculosis. microbiol a family of cross-reacting proteins secreted by mycobacterium tuberculosis microbial complexes in subgingival plaque the role of gingipains in the pathogenesis of periodontal disease the salmonella enterica pan-genome immunization of pigs to prevent disease in humans: construction and protective efficacy of a salmonella enterica serovar typhimurium live negative-marker vaccine selection and characterization of scfv antibodies against the sin nombre hantavirus nucleocapsid protein selection of phage-displayed human antibody fragments on dengue virus particles captured by a monoclonal antibody: application to the four serotypes human monoclonal single-chain antibodies specific to dengue virus envelope protein neutralizing human monoclonal antibody against h n influenza ha selected from a fab-phage display library structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses development of human antibody fragments using antibody phage display for the detection and diagnosis of venezuelan equine encephalitis virus (veev) isolation of cross-reactive human monoclonal antibodies that prevent binding of human noroviruses to histoblood group antigens potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association genetically engineered singlechain fvs of human immunoglobulin against hepatitis c virus nucleocapsid protein derived from universal phage display library. asian pac human-like antibodies neutralizing western equine encephalitis virus production of recombinant scfv against p of human immunodeficiency virus type by phage display technology functional characterization of two scfv-fc antibodies from an hiv controller selected on soluble hiv- env complexes: a neutralizing v -and a trimer-specific gp antibody human neutralizing fab molecules against severe acute respiratory syndrome coronavirus generated by phage display antibody responses against wild-type yellow fever virus and the d vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza a h n viruses heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm+ memory b cells molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza a viruses cucumber mosaic cucumovirus antibodies from a synthetic phage display library generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus isolation and characterisation of a human-like antibody fragment (scfv) that inactivates veev in vitro and in vivo selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library humanized monoclonal antibodies derived from chimpanzee fabs protect against japanese encephalitis virus in vitro and in vivo development of vhh antibodies against dengue virus type ns and comparison with monoclonal antibodies for use in immunological diagnosis a large semi-synthetic single-chain fv phage display library based on chicken immunoglobulin genes the neutralizing human recombinant antibodies to pathogenic orthopoxviruses derived from a phage display immune library protective and therapeutic capacity of human single-chain fv-fc fusion proteins against west nile virus novel phage display-derived h n -specific scfvs with potential use in rapid avian flu diagnosis alternative recognition of the conserved stem epitope in influenza a virus hemagglutinin by a vh - -encoded heterosubtypic antibody identification and molecular characterization of human antibody fragments specific for dengue ns protein a large library based on a novel (ch ) scaffold: identification of hiv- inhibitors crystal structure and size-dependent neutralization properties of hk , a human monoclonal antibody binding to the highly conserved heptad repeat of gp a gp mper-specific llama vhh requires a hydrophobic cdr for neutralization but not for antigen recognition vaccination of balb/c mice with escherichia coli-expressed vaccinia virus proteins a l, b r, and d l protects mice from lethal vaccinia virus challenge smallpox as a biological weapon: medical and public health management. working group on civilian biodefense immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox chimpanzee/human mabs to vaccinia virus b protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus isolation and characterisation of ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries ebola virus can be effectively neutralized by antibody produced in natural human infection pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody neutralizing antibody fails to impact the course of ebola virus infection in monkeys identification of human neutralizing antibodies that bind to complex epitopes on dengue virions phage display approaches for the isolation of monoclonal antibodies against dengue virus envelope domain iii from human and mouse derived libraries high affinity human antibody fragments to dengue virus non-structural protein identification of chimpanzee fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin g antibody that is highly efficient for neutralization of dengue type virus venezuelan equine encephalitis venezuelan equine encephalitis the first human epitope map of the alphaviral e and e proteins reveals a new e epitope with significant virus neutralizing activity continuing challenges in influenza emerging influenza strains in the last two decades influenza virus hemagglutinin stalkbased antibodies and vaccines the role of vaccination in rabies prevention the prevention and management of rabies comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin the human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants biological characterization of human monoclonal antibodies to rabies virus first administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity development of specific scfv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen p of cryptosporidium parvum single chain variable fragment antibodies selected by phage display against the sporozoite surface antigen s of cryptosporidium parvum recombinant human antibodies specific for the pfs / protein of the malaria parasite plasmodium falciparum human recombinant antibodies against plasmodium falciparum merozoite surface protein cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties construction and characterization of recombinant single-chain variable fragment antibodies against toxoplasma gondii mic protein. parasitology a combined phage display scfv library against myxobolus rotundus infecting crucian carp, carassius auratus auratus (l.), in china detection of babesia gibsoni by reaction of phage display single chain antibodies with p proteins isolation and characterization of the human monoclonal antibodies c in single-chain fragment variable (scfv) format to glucose oxidase from aspergillus niger generation of a highly reactive chicken-derived single-chain variable fragment against fusarium verticillioides by phage display isolation, expression and characterization of two single-chain variable fragment antibodies against an endopolygalacturonase secreted by sclerotinia sclerotiorum isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen- parallel selection of multiple anti-infectome nanobodies without access to purified antigens pulmonary aspergillosis: a spectrum of disease aspergillus fumigatus and aspergillosis patients at high risk of invasive fungal infections: when and how to treat mosquito-borne diseases detection of plasmodium falciparum, p. vivax, p. ovale, and p. malariae merozoite surface protein -p antibodies in human malaria patients and experimentally infected nonhuman primates plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening challenges in antimalarial drug treatment for vivax malaria control target antigens of transmission-blocking immunity on gametes of plasmodium falciparum biosynthesis of the target antigens of antibodies blocking transmission of plasmodium falciparum sequential expression of antigens on sexual stages of plasmodium falciparum accessible to transmissionblocking antibodies in the mosquito isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the block region of plasmodium falciparum merozoite surface protein- merozoite surface protein- : a malaria protein inducing antibodies that promote plasmodium falciparum killing by cooperation with blood monocytes plasmodium falciparum merozoite surface protein : oligomerization, self-assembly, and heme complex formation phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein antigen. infect. immun antibodies for biodefense isolation and characterization of a human antibody fragment specific for ts toxin from tityus serrulatus scorpion human monoclonal scfv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin. asian pac single chain variable fragment antibodies against shiga toxins isolated from a human antibody phage display library a highaffinity macaque antibody fab with human-like framework regions obtained from a small phage display immune library development of human-like scfv-fc neutralizing botulinum neurotoxin e development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library isolation of a human-like antibody fragment (scfv) that neutralizes ricin biological activity genetic and immunological comparison of anti-botulinum type a antibodies from immune and non-immune human phage libraries llama single domain antibodies specific for the botulinum neurotoxin serotypes as heptaplex immunoreagents generation of a recombinant full-length human antibody binding to botulinum neurotoxin a inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin b construction of a single-chain variable-fragment antibody against the superantigen staphylococcal enterotoxin b development of antiricin single domain antibodies toward detection and therapeutic reagents botulinum toxin as a biological weapon: medical and public health management a novel strain of clostridium botulinum that produces type b and type h botulinum toxins botulism: cause, effects, diagnosis, clinical and laboratory identification, and treatment modalities isolation of a nanomolar scfv inhibiting the endopeptidase activity of botulinum toxin a, by single-round panning of an immune phage-displayed library of macaque origin isolation of nanomolar scfvs of non-human primate origin, cross-neutralizing botulinum neurotoxins a and a by targeting their heavy chain neutralization of clostridium difficile toxin a with single-domain antibodies targeting the cell receptor binding domain anthrax lethal and edema toxins in anthrax pathogenesis anthrax as a biological weapon, : updated recommendations for management raxibacumab: potential role in the treatment of inhalational anthrax identification and characterization of two novel staphylococcal enterotoxins, types s and t staphylococcal enterotoxin-like toxins u and v, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster generation of fab fragment-like molecular recognition proteins against staphylococcal enterotoxin b by phage display technology mechanism of cell entry and toxicity of an affinity-purified lectin from ricinus communis and its differential effects on normal and virus-transformed fibroblasts the mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. the site and the characteristics of the modification in s ribosomal rna caused by the toxins modification of na channel gating by an alpha scorpion toxin from tityus serrulatus scorpion toxins from centruroides noxius and tityus serrulatus. primary structures and sequence comparison by metric analysis human antibody fragments specific for bothrops jararacussu venom reduce the toxicity of other bothrops sp. venoms human scfv that block sodium ion channel activity of tetrodotoxin protein targeting compounds phage-display antibody detection of chlamydia trachomatis-associated antigens isolation of recombinant antibodies directed against surface proteins of clostridium difficile isolation of high-affinity single-chain antibodies against mycobacterium avium subsp. paratuberculosis surface proteins from sheep with johne's disease generation and characterization of a novel recombinant antibody against lmp -tes of epstein-barr virus isolated by phage display production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein abc isolation of recombinant antibodies (scfvs) to grapevine virus b novel camelid antibody fragments targeting recombinant nucleoprotein of araucaria hantavirus: a prototype for an early diagnosis of hantavirus pulmonary syndrome neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gb and gh potent neutralization of hendra and nipah viruses by human monoclonal antibodies four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus screening and evaluation of human single-chain fragment variable antibody against hepatitis b virus surface antigen interference of hcv replication by cell penetrable human monoclonal scfv specific to ns b polymerase human recombinant antibodies specific for hepatitis c virus core and envelope e peptides from an immune phage display library identification of a human epitope in hepatitis c virus (hcv) core protein using a molecularly cloned antibody repertoire from a non-symptomatic, anti-hcvpositive patient nonneutralizing human antibody fragments against hepatitis c virus e glycoprotein modulate neutralization of binding activity of human recombinant fabs identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis e virus capsid protein recombinant human fab to glycoprotein d neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses and in vitro directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria cross-reactive hiv- neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp ) isolated from a patient (r ) with broadly hiv- neutralizing antibodies. virology llama antibody fragments recognizing various epitopes of the cd bs neutralize a broad range of hiv- subtypes a, b and c a monoclonal fab derived from a human nonimmune phage library reveals a new epitope on gp and neutralizes diverse human immunodeficiency virus type strains a human monoclonal antibody neutralizes diverse hiv- isolates by binding a critical gp epitope human neutralizing human immunodeficiency virus type -specific fab molecules generated by phage display a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza a virus subtype h n fab mabs specific to ha of influenza virus with h n neutralizing activity selected from immunized chicken phage library isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus human monoclonal scfv specific to ns protein inhibits replication of influenza viruses across types and subtypes construction of human fab (gamma /kappa) library and identification of human monoclonal fab possessing neutralizing potency against japanese encephalitis virus neutralizing human fab fragments against measles virus recovered by phage display identification of human single-chain antibodies with broad reactivity for noroviruses development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis human recombinant puumala virus antibodies: crossreaction with other hantaviruses and use in diagnostics a neutralizing recombinant human antibody fab fragment against puumala hantavirus selection of single chain variable fragments (scfv) against the glycoprotein antigen of the rabies virus from a human synthetic scfv phage display library and their fusion with the fc region of human igg the analysis of vh and vl genes repertoires of fab library built from peripheral b cells of human rabies virus vaccinated donors generation and characterization of neutralizing human recombinant antibodies against antigenic site ii of rabies virus glycoprotein single domain antibody multimers confer protection against rabies infection single-chain variable fragment (scfv) antibodies against rotavirus nsp enterotoxin generated by phage display selection of single-chain antibodies against the vp * subunit of rotavirus vp outer capsid protein and their expression in lactobacillus casei sars patientsderived human recombinant antibodies to s and m proteins efficiently neutralize sars-coronavirus infectivity chicken single-chain variable fragments against the sars-cov spike protein simian immunodeficiency virus (siv) envelopespecific fabs with high-level homologous neutralizing activity: recovery from a long-term-nonprogressor sivinfected macaque isolation of single-chain antibody fragments against venezuelan equine encephalomyelitis virus from two different immune sources human monoclonal fab antibodies against west nile virus and its neutralizing activity analyzed in vitro and in vivo identification of a wssv neutralizing scfv antibody by phage display technology and in vitro screening recombinant human antibody single chain variable fragments reactive with candida albicans surface antigens differentiation of candida albicans and candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae inhibition of candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for als p a phagedisplayed chicken single-chain antibody fused to alkaline phosphatase detects fusarium pathogens and their presence in cereal grains singlechain antibodies produced by phage display against the c-terminal kda region of merozoite surface protein- of plasmodium yoelii reduce parasite growth following challenge structural and functional characterization of a novel scfv anti-hsp of strongyloides sp nanobodies, a promising tool for species-specific diagnosis of taenia solium cysticercosis vhh, bivalent domains and chimeric heavy chainonly antibodies with high neutralizing efficacy for scorpion toxin aahi isolation of single chain variable fragment (scfv) specific for cry c toxin from human single fold scfv libraries production of human antibody fragments binding to melittin and phospholipase a in africanised bee venom: minimising venom toxicity monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice a v h h that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type a molecular characterization of murine humoral immune response to botulinum neurotoxin type a binding domain as assessed by using phage antibody libraries development of human-like scfv-fc antibodies neutralizing botulinum toxin serotype b. mabs in vivo neutralization of botulinum neurotoxins serotype e with heavy-chain camelid antibodies (vhh) a novel multivalent, single-domain antibody targeting tcda and tcdb prevents fulminant clostridium difficile infection in mice selection of nanobodies that block the enzymatic and cytotoxic activities of the binary clostridium difficile toxin phagedisplay derived single-chain fragment variable (scfv) antibodies recognizing conformational epitopes of escherichia coli heat-labile enterotoxin b-subunit production of a single-chain variable fragment antibody against fumonisin b a strategy for the generation of specific human antibodies by directed evolution and phage display. an example of a single-chain antibody fragment that neutralizes a major component of scorpion venom detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library single chain antibody fragment with serine protease inhibitory property capable of neutralizing toxicity of trimeresurus mucrosquamatus venom development and characterization of recombinant antibody fragments that recognize and neutralize in vitro stx toxin from shiga toxin-producing escherichia coli a single vhh-based toxin-neutralizing agent and an effector antibody protect mice against challenge with shiga toxins and construction of a single chain variable fragment antibody (scfv) against tetrodotoxin (ttx) and its interaction with ttx. toxicon off human monoclonal scfv neutralize lethal thai cobra, naja kaouthia, neurotoxin screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the vibrio parahaemolyticus thermolabile hemolysin a human single-chain variable fragment targeting to vibrio vulnificus rtxa toxin key: cord- -h itv authors: mok, darren z. l.; chan, kuan rong title: the effects of pre-existing antibodies on live-attenuated viral vaccines date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: h itv live-attenuated vaccines (lavs) have achieved remarkable successes in controlling virus spread, as well as for other applications such as cancer immunotherapy. however, with rapid increases in international travel, globalization, geographic spread of viral vectors, and widespread use of vaccines, there is an increasing need to consider how pre-exposure to viruses which share similar antigenic regions can impact vaccine efficacy. pre-existing antibodies, derived from either from maternal–fetal transmission, or by previous infection or vaccination, have been demonstrated to interfere with vaccine immunogenicity of measles, adenovirus, and influenza lavs. immune interference of lavs can be caused by the formation of virus–antibody complexes that neutralize virus infection in antigen-presenting cells, or by the cross-linking of the b-cell receptor with the inhibitory receptor, fcγriib. on the other hand, pre-existing antibodies can augment flaviviral lav efficacy such as that of dengue and yellow fever virus, especially when pre-existing antibodies are present at sub-neutralizing levels. the increased vaccine immunogenicity can be facilitated by antibody-dependent enhancement of virus infection, enhancing virus uptake in antigen-presenting cells, and robust induction of innate immune responses that promote vaccine immunogenicity. this review examines the literature on this topic and examines the circumstances where pre-existing antibodies can inhibit or enhance lav efficacy. a better knowledge of the underlying mechanisms involved could allow us to better manage immunization in seropositive individuals and even identify possibilities that could allow us to exploit pre-existing antibodies to boost vaccine-induced responses for improved vaccine efficacy. "it's time to close the book on infectious diseases, declare the war against pestilence won, and shift national resources to such chronic problems as cancer and heart disease" [ ] . contrary to this infamous statement, long misattributed to the former us surgeon general dr. william h. stewart, and despite advances in healthcare and technology, we remain extremely vulnerable to the threat of communicable diseases. in the last ten years alone, we have experienced the pandemic spread of swine-origin h n influenza, the west african ebola epidemic, the resurgence of yellow fever, the zika virus emergency, and the return of a global coronavirus threat [ ] [ ] [ ] [ ] [ ] . with the continued emergence and re-emergence of new and current viral pathogens, it is imperative that we continue to design new strategies to combat their spread and prevent human disease. among the various methods to impede viral transmission, vaccines are widely heralded as one of the most effective medical interventions. indeed, since the pivotal discovery by edward jenner over two hundred years ago, vaccination has seen tremendous success in reducing the burden of viral diseases against wild-type virus infections and not cause worse disease outcomes. for vaccines that are administered to neonates, the presence of passively acquired maternal antibodies within the first six months may also interfere with vaccination [ , ] . the potential effects of pre-existing antibodies on lav and virally vectored antigens will hence be the focus of this review. in this review, we examine the scenarios in which pre-existing antibodies can either enhance or inhibit lav efficacy, as well as the underlying mechanisms involved. a better understanding will allow us to tailor our vaccination schedules or vaccine doses, to ensure that lav efficacy will not be compromised by the presence of pre-existing immunity. prior to the advent of vaccination, the incidence rate of measles was so high that infection by the measles virus was basically considered an inevitability [ , ] . the virus, which is spread by the respiratory route, is highly contagious and infects over % of individuals by age in the pre-vaccination era [ , ] . about seven to eight million children were estimated to have died from measles infection each year during this period, with many others suffering from disease complications [ ] . however, in , society experienced a turning point in the fight against measles with the development of the first live-attenuated measles vaccine (mv) [ ] . the live-attenuated mv is one example of a highly successful lav, and its introduction has transformed measles from a complicated disease into a triviality in most developed countries, although measles still forms a significant disease burden in developing nations [ ] [ ] [ ] . the wild-type virus was first isolated in by dr. john f. enders and his team from the blood of an -year-old boy named david edmonston, who became the namesake of the viral strain that would eventually become the first measles vaccine [ ] . serial passage of the wild-type edmonston strain in human and chicken embryo fibroblast tissue culture resulted in a virus with reduced virulence. however, the ability of the parental strain to induce protective immunity is retained. the mv is highly immunogenic, inducing both humoral and cellular immunity at magnitudes comparable to that of a natural infection, although antibody titers induced are often lower [ , ] . investigators have demonstrated that this protection is highly robust, and could last for as long as years after vaccine administration [ ] . its excellent safety profile, highly immunogenic nature, and low possibility of reversion to virulence has also placed it as a promising candidate for use as a viral vector to deliver heterologous antigens [ ] . some of the first evidence that describes the role of pre-existing immunity on live vaccines comes from the measles vaccine. the efficacy of live mv was found to be often hampered by pre-existing immunity at the time of vaccination, and this effect is best illustrated in infants who are born to measles-immune mothers. during gestation, infants acquire protective antibodies as a result of transplacental transfer of maternal igg antibodies [ ] . these antibodies, while protective against infection, can also suppress infant responses to immunization. indeed, studies have shown that vaccinating infants born to measles-immune mothers before or at the age of six months often results in seroconversion failure [ , ] . by contrast, immunization campaigns with mv between to months of age are relatively successful [ , ] . this is likely explained by waning maternal antibody levels over the period of to months, where antibody titers fall below the inhibitory threshold required for successful vaccination [ ] . moreover, the claims that pre-existing antibody titers can impact mv efficacy are further supported by animal studies. for instance, following the intravenous transfer of varying titers of mv neutralizing antibodies, immunization of mice with a recombinant measles vaccine (rmv) expressing simian immunodeficiency virus (siv) gag protein was significantly inhibited when pre-existing antibody titers were above miu/ml of serum [ ] . likewise, investigations in cynomolgus macaques by van binnendijk et al. revealed that pre-existing antibody titers as low as . iu/ml abrogated the development of antibodies following vaccination with mv or a recombinant vaccinia virus vector expressing measles antigens [ ] . interestingly, while antibody induction by mv is negatively impacted by pre-existing immunity, the effect on cellular responses seem unaffected. for example, infants vaccinated with mv at age or months followed by the measles-mumps-rubella (mmr) vaccine at months generated equivalent t-cell responses to control infants given only mmr at age months [ ] . thus, experimental models and clinical data support that the inhibition of mv vaccination in neonates is more likely due to interference from maternal antibodies rather than the immaturity of the neonatal system. adenoviruses (adv) and adeno-associated viruses have been widely studied as a potential viral vector for cancer gene therapy and infectious diseases. this double-stranded dna virus holds several advantages as a vector, including but not limited to the ability to induce robust cellular and humoral responses, high expression of transgenes, favorable safety profiles, and no risk of integration into the host genome. the most commonly used adenoviral vector is adv serotype (ad ) and has been tested in more than gene therapy trials. however, the large majority of the human population having pre-existing immunity to adv limits its widespread use in the clinics. indeed, a recent international cross-sectional serological survey demonstrated that greater than % of the study participants possessed neutralizing antibodies against ad [ ] . in a similar line of investigation, mast et al. found that . % of their study participants from the us, europe, thailand, africa, and brazil were seropositive for anti-ad neutralizing antibodies [ ] . these pre-existing antibodies have been shown to neutralize the ad vectors after administration, thereby lowering their efficacy and transgene expression. the most convincing evidence is from the large-scale clinical trial (step) that tested an ad -based hiv- vaccine, where reduced efficacy of ad was found to be associated with subjects who had pre-existing immunity to ad [ ] . in support of this theory, the clinical trial for the ad -based ebola vaccine showed that the low-dose vaccine of . × viral particles was weakened by pre-existing immunity, whereas immunogenicity was enhanced at a higher dose of . × - . × viral particles. in a mouse model, pre-existing anti-ad neutralizing antibodies were observed to severely hamper the induction of both cellular and humoral responses by a recombinant ad -ebolavirus glycoprotein vaccine [ ] . likewise, a recombinant ad vector expressing the human immunodeficiency virus- (hiv) gag gene showed diminished, but not complete, abrogation of gag-specific cellular immune responses following the vaccination of rhesus monkeys pre-exposed to an empty ad vector [ ] . nonetheless, there exists conflicting data that pre-existing anti-ad may not affect the induction of cytotoxic t-cell responses, indicating that more studies may be required to resolve these discrepancies [ ] . the high seroprevalence of anti-ad immunity has pivoted the development of genetically-modified adv and the search of rarer adv serotypes for use as vectors, in hopes that these viruses may circumvent the effects of pre-existing anti-ad immunity [ , ] . however, there remains the possibility of cross-reactivity between antibodies against the different adv serotypes, which could in turn potentially influence the efficacy of these rarer adv as vaccine vectors. indeed, investigations by heemskerk et al. have shown that ad -specific cd + t-cells could cross-react with other adv serotypes including but not limited to ad , ad , ad , and ad , suggesting that the different adv serotypes share similar t-cell epitopes [ ] . several studies have attempted to further explore this school of thought, and their results have provided greater insight into this particular topic. ad is one of the rarest human adv serotypes, with a seroprevalence of less than % in the population. in an attempt to determine its potential as an alternative vector to ad , barouch et al. compared the immunogenicity of a rad vaccine expressing simian immunodeficiency virus (siv)-gag versus a rad siv-gag vaccine in ad immune c /bl mice [ ] . indeed, the efficacy of the rad -gag vaccine was unaffected even by high levels of pre-existing ad immunity, indicating the absence of a cross-reactive response between ad and ad . likewise, the presence of anti-ad immunity did not affect the efficacy of a rad -gag vector vaccine in mice. interestingly, pre-existing anti-ad immunity could suppress cellular immune responses elicited by the rad -gag vaccine, suggesting some cross-reactivity between ad and ad immune responses [ ] . the inhibitory effect of pre-existing antibodies to adv remains a challenge for their use as viral vectors, and the development of alternative serotypes remains to be one of the best strategies to circumvent this limitation. the history of mankind is intricately intertwined with that of influenza, and the virus remains one of the top threats to global health. estimates indicate that there are approximately billion cases globally each year, of which to million are severe cases and up to , eventually succumb to the disease [ ] . combined with the economic impact from the loss of work productivity, a proper and robust framework is required to counter this threat. the global influenza strategies recommend vaccination as the most effective intervention to mitigate the impact of influenza [ ] . three types of influenza vaccines are licensed for use: ( ) recombinant, ( ) inactivated, and ( ) live-attenuated. the three vaccines are multivalent and provide protection against selected influenza type a and type b strains that are predicted to spread in the upcoming season [ ] . the former two are capable of inducing only igg responses [ , ] . by contrast, the live-attenuated influenza vaccine (laiv) can generate strain-specific igg antibodies as well as mucosal iga immunity and t-cell responses that are associated with protection from influenza illness [ , ] . indeed, a meta-analysis by ambrose et al. on the efficacy of laiv in children showed that recipients of the live vaccine demonstrated a % reduction in influenza cases compared to those who received the trivalent inactivated vaccine (tiv) [ ] . put together, these studies demonstrate the potential of laiv as a highly efficacious vaccine. yet, efforts to develop such a vaccine have been stymied by the presence of pre-existing immunity gained over a lifetime of exposure to different viral strains either from natural infection or vaccination. indeed, several observational studies point to the possibility that pre-existing immunity can reduce the efficacy of both inactivated and life-attenuated influenza vaccine [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, saito et al. found that children who received tiv for a previous season had reduced vaccine effectiveness for the current seasonal vaccine compared to unvaccinated children [ ] . likewise, sasaki et al. showed lower antibody induction by a laiv in individuals who had prior year tiv vaccination [ ] . furthermore, coelingh et al. demonstrated that younger children aged two to eight as well as baseline seronegative adults had higher fold-induction of serum hemagglutinin inhibition (hai) antibody titers post-laiv vaccination [ ] . however, given how complicated it is to trace the history of an individual's exposure to influenza strains, it is difficult to tease out the exact impact that pre-existing immunity has on vaccine efficacy in human samples. perhaps then, by establishing a good animal model with controlled infection histories, we will be able to better understand these complexities. flaviviruses include a number of clinically important pathogens that are either transmitted by mosquitoes (dengue, zika, yellow fever, west nile, and japanese encephalitis virus) or by ticks (tick-borne encephalitis, powassan virus) [ ] . the recent zika pandemic witnessed more than cases of congenital birth defects linked to zika virus infection in the americas and was declared by the world health organization in february to be a public health emergency [ ] . dengue infections, on the other hand, account for million infections annually, of which million infections are symptomatic [ ] . the antigenic closeness between flaviviruses is such that infection with one flavivirus induces species-specific immunity, as well as cross-reactive antibodies against related serocomplexes [ , ] . however, these cross-reactive antibodies do not necessarily cross-protect. initial insights come from human dengue challenge studies by albert sabin, who provided evidence that a homologous challenge in humans with the same dengue virus (denv) serotype protects against re-infection, but only short-term protection against the heterologous denv serotype challenge for up to six months [ , ] . moreover, when cross-reactive antibodies decline to sub-neutralizing levels, these antibodies can opsonize dengue virus infection, resulting in enhanced virus burden and risk of severe dengue in patients experiencing secondary infection [ ] . indeed, recent cohort studies conducted in nicaragua and thailand provide clinical evidence that a specific range of antibody titers is associated with an increased risk of severe dengue [ , ] . the presence of waning maternal anti-dengue antibodies can also predispose children to dengue hemorrhagic fever, reinforcing the concept that pre-existing antibodies can promote disease pathogenesis [ ] . consistent with the notion that sub-neutralizing cross-reactive antibodies can promote viral infection, the presence of pre-existing cross-reactive antibodies can also increase the immunogenicity of flaviviral lavs. as demonstrated in both dengvaxia ® and takaeda ® dengue vaccine trials, seropositive individuals produced greater neutralizing antibody responses and protection against the wild-type denv infection compared to seronegative vaccinees [ , , ] . these studies were also supported by in vivo studies showing that sequential immunization for flaviviruses with shared cd epitopes that could enhance protection during subsequent heterologous infection [ ] . however, in another study, pre-existing antibodies from yellow fever vaccination can cause impairment of neutralizing antibody responses to tick-borne encephalitis vaccination [ ] . the clinical trial finding that subjects with a limited range of cross-reactive antibodies from a prior japanese encephalitis vaccine were able to enhance yellow fever vaccination, by prolonging vaccine viremia duration that leads to higher antibody titers, thus hints at the possibility that whether pre-existing antibodies inhibit or augment flavivirus infection will depend on both antibody titers and the type/specificity of antibodies produced [ ] . the plausible mechanisms involved are as elaborated below. the primary role of antibodies is antigen binding and interacting with fc-gamma receptors (fcγrs) to modulate subsequent immune responses. the integration of both activating and inhibiting signals is critical for the generation of an effective immune response. in this aspect, fcγrs are an archetype of how such signals influence both innate and adaptive immune functions. functionally, fcγrs can be classified into either activating or inhibiting receptors depending on the pathway they initiate [ ] . activating fcγrs possess an immunoreceptor tyrosine-based activation (itam) motif in their cytosolic domain, or in the case of fcγri and fcγriiia, associate with an itam-containing γ-chain. engagement of activating fcγrs by immune complexes results in the phosphorylation of the γ-chain by src-family kinases in order to create a docking site for spleen tyrosine kinase (syk). subsequent activation of syk results in a signaling cascade, leading to the induction of pro-inflammatory responses and activation of innate immune effector cells [ , ] . by contrast, the inhibitory fcγriib receptor contains an immunoreceptor tyrosine-based inhibition (itim) motif within its intracellular domain. cross-linking of fcγriib enables the recruitment of sh domain-containing inositol polyphosphate phosphatase (ship) and sh domain-containing protein tyrosine phosphatase (shp ) to modulate signals generated by activating fcγrs, thereby regulating the magnitude of inflammatory responses. furthermore, fcγriib is important for controlling b-cell development [ ] [ ] [ ] [ ] [ ] [ ] . indeed, b-cells express fcγriib as the only fcγr on their cell surface, and cross-linking of fcγriib on naïve b-cells could inhibit their proliferation and differentiation into plasma cells [ ] . likewise, cross-linking of fcγriib induces apoptosis in bone marrow plasma cells, suggesting that fcγriib may influence the lifespan of these antibody-producing cells [ ] . the signaling responses triggered by virus-antibody complexes are thus highly dependent on the type of fcγrs with which the immune complexes interact, which can result in either virus inhibition or enhancement of virus infection. virus neutralization occurs when virions are bound by antibodies with stoichiometry exceeding a required threshold. hence, one of the popular explanations to explain the lack of lav efficacy in the presence of pre-existing antibodies is the neutralization of the lav, which could consequently decrease the amount of viral antigens to levels that are below the threshold for immune detection and recognition. antibody concentrations, affinity, and epitope accessibility are critical determinants for virus neutralization [ ] [ ] [ ] . antibody affinity, defined as the fraction of epitopes that are bound by antibodies at non-saturating concentrations, has been shown to correlate with neutralizing activity in vitro. on the other hand, epitope accessibility is defined as the number of epitopes on viruses that are available for binding and can be affected by virus structure, structural dynamics of virus, and virus maturation states [ ] . the epitope availability would thus affect the fraction of epitope occupancy that will be required for virus neutralization. taken together, cross-reactive antibodies that can neutralize virus infection are likely those that can bind to accessible epitopes with considerable affinity. conversely, antibodies that bind weakly and target epitopes with reduced accessibility are unlikely to neutralize viruses, and may instead enhance viral infection via fcγr-mediated uptake. antibodies can neutralize lav strains in a variety of ways, as summarized in figure . they may block virus attachment and entry by either binding to epitopes that are directly involved in virus-receptor interactions or by imposing steric hindrance that prevent virus interaction with host receptors. as most virus structures are dynamic and can change structural conformations at different temperatures, it is thought that antibody binding to these dynamic structures may cause structural changes that can impair virus attachment, thereby causing virus neutralization [ ] . however, the blockade of virus-receptor interactions alone may not be able to completely neutralize the viruses, especially in fcγr-bearing cells, as activating fcγrs can enable entry of virus-antibody complexes by fcγr-mediated uptake. thus, pre-existing antibodies that can block viral fusion and uncoating will likely be more efficient in virus neutralization. in situations where pre-existing cross-reactive antibodies are unable to inhibit viral fusion processes intracellularly, high concentrations of antibodies may enable the formation of viral immune aggregates that influence the types of fcγrs engaged. these large viral aggregates can then inhibit phagocytosis by co-ligating the lowly expressed inhibitory receptor fcγriib that inhibit phagocytosis [ , ] . finally, there have been theories suggesting that fcγr cross-linking by virus immune complexes may increase the production of il- that abolishes innate immune responses [ ] . however, more experimental evidence will be required to support this theory. viruses , , x for peer review of virus maturation states [ ] . the epitope availability would thus affect the fraction of epitope occupancy that will be required for virus neutralization. taken together, cross-reactive antibodies that can neutralize virus infection are likely those that can bind to accessible epitopes with considerable affinity. conversely, antibodies that bind weakly and target epitopes with reduced accessibility are unlikely to neutralize viruses, and may instead enhance viral infection via fcγrmediated uptake. antibodies can neutralize lav strains in a variety of ways, as summarized in figure . they may block virus attachment and entry by either binding to epitopes that are directly involved in virus-receptor interactions or by imposing steric hindrance that prevent virus interaction with host receptors. as most virus structures are dynamic and can change structural conformations at different temperatures, it is thought that antibody binding to these dynamic structures may cause structural changes that can impair virus attachment, thereby causing virus neutralization [ ] . however, the blockade of virus-receptor interactions alone may not be able to completely neutralize the viruses, especially in fcγr-bearing cells, as activating fcγrs can enable entry of virus-antibody complexes by fcγr-mediated uptake. thus, pre-existing antibodies that can block viral fusion and uncoating will likely be more efficient in virus neutralization. in situations where pre-existing cross-reactive antibodies are unable to inhibit viral fusion processes intracellularly, high concentrations of antibodies may enable the formation of viral immune aggregates that influence the types of fcγrs engaged. these large viral aggregates can then inhibit phagocytosis by co-ligating the lowly expressed inhibitory receptor fcγriib that inhibit phagocytosis [ , ] . finally, there have been theories suggesting that fcγr cross-linking by virus immune complexes may increase the production of il- that abolishes innate immune responses [ ] . however, more experimental evidence will be required to support this theory. unlike myeloid cells, b-cells exclusively express fcγriib but not the activating fcγrs [ ] . therefore, cross-linking of the b-cell receptor (bcr) with fcγriib mediated by virus immune complexes can lead to the inhibition of b-cell activation (figure a) . indeed, by adding sheep red blood cell-specific (sbrc) igg to srbcs, b-cell antibody secretion is reduced [ ] . similarly, using the cotton rat model of mv vaccination, maternal antibodies were demonstrated to inhibit b-cells by the cross-linking of bcr and fcγriib [ ] . however, this mechanism of inhibition has not been demonstrated for other viruses. it is conceivable that this mode of inhibition is dependent on the size of the virus-antibody immune complexes, as immunization of small polypeptides can escape maternal antibody inhibition [ ] . more mechanistic studies will hence be required to evaluate the conditions that need to be satisfied to cause b-cell inhibition. viruses , , x for peer review of unlike myeloid cells, b-cells exclusively express fcγriib but not the activating fcγrs [ ] . therefore, cross-linking of the b-cell receptor (bcr) with fcγriib mediated by virus immune complexes can lead to the inhibition of b-cell activation (figure a) . indeed, by adding sheep red blood cell-specific (sbrc) igg to srbcs, b-cell antibody secretion is reduced [ ] . similarly, using the cotton rat model of mv vaccination, maternal antibodies were demonstrated to inhibit b-cells by the cross-linking of bcr and fcγriib [ ] . however, this mechanism of inhibition has not been demonstrated for other viruses. it is conceivable that this mode of inhibition is dependent on the size of the virus-antibody immune complexes, as immunization of small polypeptides can escape maternal antibody inhibition [ ] . more mechanistic studies will hence be required to evaluate the conditions that need to be satisfied to cause b-cell inhibition. another way in which antibodies can inhibit b-cell responses is through epitope masking. this hypothesis postulates that the presence of pre-existing antibodies can mask the exposure of epitopes, thereby prohibiting recognition by the b-cell. this is also termed as epitope-specific suppression, whereby epitopes covered by these antibodies are unable to be recognized by b-cells (figure b) . interestingly, there have been reports of epitope unspecific suppression, where monoclonal antibodies that target only one specific epitope can suppress b-cell recognition of a whole antigen, suggesting the possibility that steric hindrance or obstruction by high concentrations of antibodies can also lead to overall suppression b-cell recognition [ , ] . antibody-dependent enhancement (ade) of viral infection has been documented for many viruses, including flaviviruses, influenza, mv, ross river viruses, hiv, and coronaviruses [ ] . ade can occur when sub-neutralizing levels of cross-reactive antibodies form immune complexes with viruses, opsonizing viral infection in fcγr-bearing myeloid cells including monocytes, macrophages, and dendritic cells via activating fcγr-mediated uptake. the majority of the mechanistic insights about ade of viral infection are gathered from dengue, as waning cross-reactive antibodies that are acquired from a heterotypic denv infection or through maternal-fetal transmission can result in a heightened risk of severe dengue that can be life-threatening [ ] [ ] [ ] . both activating fcγri and fcγriia have been shown to be involved in ade-mediated infection, although increasing studies indicate that fcγriia could be more important than fcγri in enhancing viral infection [ , ] . while the precise mechanisms involved remain unclear, it is possible that the trafficking of immune complexes through fcγriia-mediated uptake is slower, thereby allowing more viral fusion and infection [ , ] . the activation of fcγri, however, may aid to further enhance immunogenic responses to viral antigens by targeting virus immune complexes to the late endosomes or lysosomes for enhanced antigen processing and antigen presentation to the cd + t-cells, thereby increasing b-cell responses [ ] . overall, at sub-neutralizing antibody levels, the presence of pre-existing antibodies can activate both fcγri and fcγriia, which promotes virus uptake, replication, and antigen presentation that consequently augments vaccine immunogenicity. besides promoting viral entry and antigen presentation, the cross-linking of fcγrs may also modulate cellular and host responses that promote viral replication and lav immunogenicity ( figure ). some insights can be obtained from our clinical trial, where subjects were sequentially vaccinated with the inactivated japanese encephalitis virus vaccine followed by the yellow fever vaccine. subjects within a restricted range of cross-reactive antibodies resulted in increased antibody responses, whereas too many or too few antibodies resulted in reduced antibody responses, indicating the possible role of ade in augmented vaccine antibody responses [ ] . in addition to the extended duration of viremia observed in these subjects, enhancing titers of cross-reactive antibodies provoked greater pro-inflammatory responses, including increased innate immune responses and the production of pro-inflammatory metabolites such as arachidonic acid, linoleic acid, and -hete that promote phagocytosis and adaptive immune responses. the co-ligation of both fcγri and fcγriia by virus-antibody immune complexes or cross-linking can also upregulate immune semaphorins such as sema a, sema a, and sema a which are critical for antigen-presenting cell and t-cell interactions [ ] . while the mechanisms of whether upregulation of immune semaphorins by immune complexes leads to increased t-cell proliferation and activation in humans remains to be evaluated, previous studies have shown that sema a can enhance t-cell activation through interaction with tim- , thereby increasing antigen-specific t-cell and antibody responses against t-cell dependent antigens [ , ] . however, it is also noticeable that not all subjects within that specific window of cross-reactive antibody levels exhibited increased vaccine immunogenicity, suggesting that baseline variations, such as genetics, dietary or environmental factors may also influence the outcome of lav immunogenicity [ ] . some recent studies hinted at the possibility that baseline variations in b-cell signatures and gene regulation could influence lav reactogenicity and immunogenicity, which can be potential avenues for future studies [ , ] . viruses , , x for peer review of cell dependent antigens [ , ] . however, it is also noticeable that not all subjects within that specific window of cross-reactive antibody levels exhibited increased vaccine immunogenicity, suggesting that baseline variations, such as genetics, dietary or environmental factors may also influence the outcome of lav immunogenicity [ ] . some recent studies hinted at the possibility that baseline variations in b-cell signatures and gene regulation could influence lav reactogenicity and immunogenicity, which can be potential avenues for future studies [ , ] . immune complexes formed by pre-existing antibodies and viruses can engage fcγrs, resulting in increased viral uptake and fusion through the process of antibody-dependent enhancement that leads to increased vaccine viremia and antigen presentation. activating fcγr-signaling, on the other hand, provokes greater innate immune responses and production of pro-inflammatory metabolites that can enhance innate and adaptive immune responses. in addition, the cross-linking of fcγrs causes increased expression of immune semaphorins, which are critical for antigen-presenting cell and t-cell interaction. overall, this leads to increased t-cell proliferation and activation, which consequently improves lav immunogenicity. in this review, we have highlighted several studies that have shown that the efficacy of some commonly used lavs, such as measles, adenovirus, influenza, and flaviviral vaccines, can be affected by these pre-existing adaptive immune responses. this knowledge will be critical to understanding the limitations of administering lavs in seropositive individuals to reduce the incidence of vaccine failures, as well as in future designs of clinical trials to evaluate the efficacy of lavs. whether preexisting antibodies can inhibit or augment lav immunogenicity depends on the concentration and the type of antibodies that are present (figure ). with high antibody levels or potently neutralizing antibodies, pre-existing antibodies can inhibit lav efficacy by virus neutralization or inhibition of bcell responses. by contrast, at sub-neutralizing titers, pre-existing antibodies can enable viruses to better infect cells and increase innate immune responses that augment lav immunogenicity. moreover, pre-existing immunity can prime dendritic cells and memory t-cells to enhance protection during secondary infection with an antigenically related virus [ ] . we believe that a deeper understanding of the underlying mechanisms involved will help us better understand the circumstances that can allow us to manage immunization in the presence of pre-existing antibodies, and even explore the possibilities of exploiting pre-existing antibodies to promote vaccine immunogenicity and efficacy. it would also be interesting to determine if cross-reactive antibodies immune complexes formed by pre-existing antibodies and viruses can engage fcγrs, resulting in increased viral uptake and fusion through the process of antibody-dependent enhancement that leads to increased vaccine viremia and antigen presentation. activating fcγr-signaling, on the other hand, provokes greater innate immune responses and production of pro-inflammatory metabolites that can enhance innate and adaptive immune responses. in addition, the cross-linking of fcγrs causes increased expression of immune semaphorins, which are critical for antigen-presenting cell and t-cell interaction. overall, this leads to increased t-cell proliferation and activation, which consequently improves lav immunogenicity. in this review, we have highlighted several studies that have shown that the efficacy of some commonly used lavs, such as measles, adenovirus, influenza, and flaviviral vaccines, can be affected by these pre-existing adaptive immune responses. this knowledge will be critical to understanding the limitations of administering lavs in seropositive individuals to reduce the incidence of vaccine failures, as well as in future designs of clinical trials to evaluate the efficacy of lavs. whether pre-existing antibodies can inhibit or augment lav immunogenicity depends on the concentration and the type of antibodies that are present (figure ). with high antibody levels or potently neutralizing antibodies, pre-existing antibodies can inhibit lav efficacy by virus neutralization or inhibition of b-cell responses. by contrast, at sub-neutralizing titers, pre-existing antibodies can enable viruses to better infect cells and increase innate immune responses that augment lav immunogenicity. moreover, pre-existing immunity can prime dendritic cells and memory t-cells to enhance protection during secondary infection with an antigenically related virus [ ] . we believe that a deeper understanding of the underlying mechanisms involved will help us better understand the circumstances that can allow us to manage immunization in the presence of pre-existing antibodies, and even explore the possibilities of exploiting pre-existing antibodies to promote vaccine immunogenicity and efficacy. it would also be interesting to determine if cross-reactive antibodies can impact future development of lavs against newly emerging pandemic viruses, including ebola, severe acute respiratory syndrome coronavirus- (sars cov- ), and zika viruses. can impact future development of lavs against newly emerging pandemic viruses, including ebola, severe acute respiratory syndrome coronavirus- (sars cov- ), and zika viruses. in the presence of high levels of pre-existing antibodies or with potently neutralizing antibodies, lav immunogenicity is hindered due to immune interference from virus neutralization or inhibition of b-cell responses. however, at sub-neutralizing antibody titers, there is a window of augmented vaccine immunogenicity due to increased uptake into fc receptor-bearing cells and greater induction of innate immune responses. the horizontal dotted line indicates lav immunogenicity in the absence of pre-existing antibodies. the first pandemic of the st century: review of the pandemic variant influenza a (h n ) virus resurgence of yellow fever in angola transmission dynamics and control of ebola virus disease (evd): a review zika virus in the americas-yet another arbovirus threat outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle epidemiology and ecology of yellow fever virus a decade of vaccines: integrating immunology and vaccinology for rational vaccine design declaration of global eradication of smallpox state of 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conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- - bccxgbd authors: saxena, latika; khanna, madhu title: production and characterization of human monoclonal antibodies from the cells of a(h n )pdm influenza virus infected indian donors date: - - journal: procedia in vaccinology doi: . /j.provac. . . sha: doc_id: cord_uid: bccxgbd abstract analysis of human monoclonal antibodies (mabs) developed from influenza infected donors have enormously contributed to the identification of neutralization sensitive epitopes of influenza virus. the ha protein is a crucial target of neutralizing antibodies and at monoclonal level only abs binding to ha have been able to neutralize the virus. in this study, eight a (h n )pdm seropositive patients within the age range of - years (median= years) were recruited. two anti-ha mabs secreting stable clones, d and f were established under optimized conditions from the peripheral blood mononuclear cells (pbmcs) of the volunteers. these antibodies efficiently neutralized the homologous laboratory isolated strain of the pandemic virus as well as the reference strain. our study suggests that the anti-ha antibodies derived from infected indian patients display neutralization potential against the a(h n )pdm virus. this is the first ever study of generation of mabs against the pandemic influenza virus involving the immune repertoire if indian patients. molecular characterization of the target regions will help in identifying potential immunogens in the indian pandemic isolates and confer protective immunity against this virus. influenza a virus is a member of the orthomyxoviridae family and it is one of the main causes of prevalent infection of the respiratory tract in humans. the susceptibility to influenza illness is most common in immunologically naive infants, immunocompromised individuals and elderly . the virulence of influenza a virus is because of its easy spread by aerosol; the frequent changes in the viral antigens by antigenic drift and antigenic shift that enables it to escape from protective immunity. influenza infection in humans is self-limiting, but the virus is known to cause substantial mortality and morbidity worldwide . during the most devastating influenza pandemic of the global mortality reached to million individuals in one year. in a stark reminder to the pandemic, the population that is considered fittest was the most vulnerable during the recent h n pandemic . despite intensive efforts, the threat to influenza persists as there are many limitations to the use of existing vaccines and antiviral therapies. the protection provided by vaccines that contain killed or recombinant viral glycoprotein is weak and may last as little as months. moreover, the vaccine efficacy in immunocompromised and elderly individuals is only % . above all the vaccines have to be formulated annually as the existing year's vaccine may not provide protection to the newly emerging strains. currently four antiviral drugs are approved for use against influenza virus but, their use is restricted due to possible side effects and rapid emergence of resistant strains in the recent years. hence, the development of new therapeutic targets and strategies to control pandemic and seasonal influenza virus infection in humans is urgently needed. passive immunotherapy against influenza has been reported since the influenza pandemic, where convalescent sera was used for treating patients . however, use of convalescent plasma is being largely replaced with monoclonal antibody preparations owing to the recent advances in monoclonal antibody engineering. there are several reports where antibody therapy using polyclonal and monoclonal antibodies has been used effectively as prophylaxis against varicella, hepatitis a, hepatitis b, rabies, and respiratory syncytial virus infections . the use of human mab or humanized mab to key epitopes of infectious pathogens may help in defining the humoral responses with significant therapeutic potential. the use of human mabs may also lead to more effective post exposure prophylaxis including their use intranasally in viral diseases . human monoclonal antibody technology has seen various advances recently, like the use of epstein barr virus (ebv) to transform human b cells, development of several new heteromyeloma cell lines and cpg oligonucleotides that further enhance the efficiency of b cell transformation . in this study, we generated strongly neutralizing novel human monoclonal antibodies that were selected from the immune repertoire of influenza infected seropositive patients. we generated two stable monoclonal antibody secreting fusion clones that produced antibodies specific against a(h n )pdm virus. we further investigated the in vivo prophylactic and therapeutic efficacy of these monoclonal antibodies against influenza a virus infection in the in vivo model. this study supports the fact that fully human mabs with neutralization activity can be successfully generated from the peripheral blood of convalescent patients under optimized conditions. madine darby canine kidney (mdck) cells were procured from the national centre for cell science (nccs), pune, india. ebv transformed marmoset leukocyte cell line (b - ) was a kind gift, from dr. rahul pal, nii, delhi. the hmma . human mouse heteromyeloma was provided by dr. lisa cavacini, beth israel deaconess medical centre, boston, usa. pandemic virus reference strain a/cal/ / (h n ) was procured from victoria infectious disease reference laboratory (vidrl), australia. eight a(h n )pdm seropositive patients ( males and females) within the age range of - years (median = years) were recruited in this study. rna was isolated from the nasal and throat swab samples and influenza virus infection was detected by real time rt pcr. all the volunteers had low ct (cycle threshold) values (below ) for influenza approximately - weeks after onset of the disease. ml blood samples were collected in heparanized vacutainers from these donors. - ml plasma samples were collected from each sample and stored at - °c for serological examination. the remaining blood was used for pbmc isolation. plasma samples were treated with receptor destroying enzyme (rde) for the destruction of non-specific inhibitors. serial two fold dilution of the plasma samples was prepared in pbs in a microtiter plate.the plasma samples (diluted : ) were added in equal volume ( μl) of ha units of a/cal/ / (h n ). plates were covered and incubated at room temperature (rt) for min. the contents of the plate were mixed by gently agitating the plates manually. μl of the . % guinea pig rbcs were added to each well. plates were covered and incubated at rt for min. the hai titre was the highest dilution of the serum that showed hemagglutination activity. the monoclonal antibodies were generated from the pbmcs of infected patients by the method as per gorny m, , with minor modifications. mononuclear cells were isolated from the blood of the seropositive patients by density gradient centrifugation and were resuspended in the ebv transformation medium (b - culture supernatant that was diluted : with complete medium (rpmi+ % fcs) to a concentration of x cells/ml in the presence of cpg odn- ( μg/ml) and were cultured overnight at °c. after three weeks of culture, proliferating transformed colonies of b lymphocytes were observed using an inverted microscope . the positive clones were screened by microneutralization and expanded to t- tissue culture flasks. the positive clones were fused with hmma . (at a ratio of : ) cell lines by adding ml of warm peg/dmso solution dropwise for min. the cell culture supernatant of the stable clones was collected and the monoclonal antibodies were subsequently purified by affinity chromatography by protein g columns (sigma aldrich, usa). madin darby canine kidney (mdck) cells were grown in wells tissue culture plates, at a density of . x cells per well for h.cells were infected with tcid units of a (h n ) pdm virus. h after infection, cells were fixed with acetone: methanol ( : ) for min, followed by permeabilization with triton x . fixed cells were blocked with % bsa in pbs for h at °c and washed twice with pbs. monoclonal antibodies were added at a concentration of μg/ml and the plates were incubated for h at °c. plates were washed thrice with pbs. bound antibodies were detected with fitc labeled goat anti-human igg and observed under an immunofluorescent microscope . hemagglutination inhibition assay of the purified monoclonal antibodies was performed by the end point dilution method . two fold serial dilutions of the purified monoclonal antibodies were taken, with the maximum concentration of . μg/ml. the hai titers were expressed as the lowest concentration of the antibody that completely inhibited ha units of the pandemic virus. microneutralization assay of the purified antibodies was performed by the end point dilution method . briefly, two fold serial dilutions of the antibodies were prepared in pbs starting with the highest dilution of . μg/ml in well tissue culture plates. tcid of the pandemic isolate as well as reference strain were incubated with equal volume of the antibody for h at °c. μl mdck cells ( . x cells/ml) were added to each well. the plates were incubated for - days at °c in % co and examined for cpe. the residual infectivity was tested in four wells per dilution. the neutralizing titer was determined as the lowest concentration of the antibody at which the infectivity of tcid of the respective virus for mdck cells was completely neutralized in % of the wells. infectivity was identified by the presence of cytopathic effect on day and the titer was calculated by the reed-muench method . the infected cell lysates were first resolved on % sds-page and then transferred to pvdf membrane using trans-blot sd semi-dry transfer cell (bio-rad laboratories, ca, usa). the membrane was blocked with % non-fat milk in tbst for h at room temperature and was washed thrice with tbst. the blot was then cut into strips and denatured antigens were probed by incubating each strip with a single purified monoclonal ab at a concentration of μg/ml. anti-human igg was taken as the negative control. the strips were then washed thoroughly with x tbst for three times, min each, followed by incubation with : dilution of horse-radish peroxidase (hrp) conjugated goat anti-human igg as secondary antibody (diluted in % milk-tbst) for h at room temperature. after washing, the immuno-reactive bands were visualized by the addition of pbs containing μl/ml of % h o and . mg/ml of dab (sigma aldrich, usa). to evaluate the degree of protection of monoclonal antibodies, - weeks old female balb/c mice (n= per group) were intraperitoneally injected with purified monoclonal antibodies in three different concentrations ( μg, μg, μg), h before (pre), h after ( h post) and h after ( h post) challenge with x pfu a/cal/ / (h n ) virus. mice were monitored daily for sickness, weight loss and death for days. significance of the differences between means was determined by student t test using graph pad prism software version . (san diego, ca, usa). the results were presented as mean + s.d. plasma samples of all the donors showed serologic reactivity with the ha of the reference pandemic strain as was evident with the hemagglutination inhibition assay and showed a titer of < : .the individual titers of the plasma samples are mentioned in table i. pbmcs from all the eight seropositive volunteers were transformed with ebv and the supernatants of the transformed colonies were screened by microneutralization assay for the influenza virus specific antibodies (table ii) . the positive clones were fused with the hmma . heteromyeloma cells. after fusion only two stable fusion clones secreting neutralizing monoclonal antibodies against the pandemic virus were established. pbmcs of all patients were successfully transformed with ebv. the mean of pbmcs isolated from patients was . million per individual (range . - . ). pbmcs were plated in well tissue culture plates at , - , cells per well. approximately . % wells ( / ) were positive for neutralizing antibodies against their autologus virus. both the clones d and f clones were derived from separate donors (donor and donor respectively). most of the transformed colonies lost reactivity in subsequent rounds of screening due to possible out-growth of nonsecretors and instability in antibody generation. the mabs were purified by affinity chromatography on protein g columns. the monoclonal antibodies were analyzed in vitro by a series of assays. d and f bound to the pandemic virus a/cal/ / on elisa in a dose dependent manner. f showed lesser binding compared to the d antibody ( figure ). similar results were obtained when the two monoclonal antibodies were tested for their binding to the ha of a panel of viruses selected for the hai assay. the mabs showed binding to the reference pandemic strain as well as the laboratory isolate but did not show binding to any other h n or h n virus they were tested against. >indicates that hai activity was not detected at any concentration that was tested up to . μg/ml. the immunofluorescence data further supported the above findings that d bound more strongly to influenza virus infected mdck cells compared to the f mab ( figure ). the gel was run in the denaturing condition, to allow ha protein to denature to ha and ha . antibody d reacted with the denatured viral ha protein as was evident by a band at kda (figure ). in contrast antibody f showed no binding activity to the denatured ha .it may be hypothesised that f that did not show binding to any region in the denatured ha protein, as it might be recognizing a conformational epitope. mice were treated with graded doses ( μg, μg, and μg) of the two mabs ( d and f ), h pre, h post and h post infection with the respective strains of influenza viruses as mentioned in the methods section. the antibodies were evaluated for their prophylactic or therapeutic efficacy by the assessment of morbidity (loss of weight), mortality, viral load and histopathologic examination of the lungs. the survival and weight loss experiments suggested that the mab d achieved considerable protection at the doses μg and μg when administered prophylactically and therapeutically (table iv) . the f mab was more effective at the dose of μg. viral load analysis was performed in the subgroups h pre and h post challenge with μg mab. the lung virus titers of the treated mice confirm that upon treatment with the mabs, the virus titer considerably reduces in the lungs (table v) . the production of human monoclonal antibodies is a much more complex procedure than the production of murine monoclonal antibodies. scarcity of antibody producing cells in the peripheral blood and the difficulty to obtain the blood from naturally infected patients at the right time is one of the major concerns. moreover, ebv transformed b cells are hard to grow and secrete very low amount of antibodies . the instability of antibody production by epstein-barr virus transformed cells or fused cells is another concern. these factors result in the decreased fusion efficiency and the chances of obtaining a human hybridoma against an antigen of interest is only of the order of - to - . the human hybridoma technology is based on two main methods: fusion of ab producing b cells to myeloma cells and transformation of b lymphocytes with ebv . combination of both the methods has proved to be more beneficial than either of them alone . in this study, we report the generation of two stable fusion clones, secreting monoclonal antibodies against a(h n )pdm influenza viruses. the conditions of ebv transformation and fusion with the heteromyeloma cell line were optimised by overcoming the limitations of the current technologies. the convalescent phase plasma of the eight influenza positive patients was screened for influenza virus specific antibodies, as the serum/plasma antibody titre is a critical factor for the generation of monoclonal antibodies from pbmcs. the pbmcs were ebv transformed in the presence of cpg oligonucleotides as it has been reported to increase the rate of ebv transformation . an overall . % transformation efficiency was achieved and the cells that were positive for antibodies against the pandemic h n virus were fused with hmma . cells. the best antibody secreting hybrids were cloned at one cell /well to ensure monoclonal antibody generation and stable fusion clones were obtained- d and f that secreted mabs against a(h n )pdm and showed neutralization in the in vitro as well as in vivo conditions. monoclonal antibody d showed the maximum binding in the in vitro assays and neutralized the human isolate of the pandemic strain as well as the reference strain at lowest concentrations when compared to the f antibody. mab f showed the lesser binding in elisa and immunofluorescence assay and although it showed hai and neutralizing activity, but at a higher concentration compared to the d antibody. the western blot analysis of the monoclonal antibodies revealed that d bound to the ha region in the denatured ha protein. f did not show binding to any of the region in the denatured ha protein. hence, it is hypothesised that f may be binding a conformational epitope in the ha region. however, the binding properties of f need to be investigated further to confirm the hypothesis. the monoclonal antibodies did not show binding to the seasonal h n or any other h n or h n virus against which its hai activity was determined. the reference strains available in the laboratory were picked randomly for the hai studies. further studies regarding the mapping of the epitope of these antibodies can reveal more information on whether or not they exhibit cross reactivity to other influenza virus strains. the antibodies however, showed comparative neutralization and hai activity between the laboratory isolates of the pandemic virus and the reference strain a/cal/ / (h n ). this data reflects the fact that these indian pandemic isolates have not shown much deviation from the prototype pandemic strain in terms of antibody binding sites. the prophylactic and therapeutic efficacy of these antibodies was determined in mice. the antibody treatment was given h before, h after and h after challenge with the pandemic strain. the in vivo results were consistent with the in vitro study and all the antibodies were able to considerably protect mice from lethal influenza challenge. both the antibodies were protective prophylactically but, f could achieve only % protection in terms of survival assay at μg, but achieved % survival rate at μg. the antibodies were less protective h post infection as compared to h, the fact that is supported by the survival assays and weight loss percentage. the histopathologic examination of the lungs was done in the group of mice that were treated therapeutically at h the hisptopathology data further supported the survival and morbidity results, as the lungs from the mice that were given therapeutic doses of the antibodies, showed considerably less signs of alveolar damage and neutrophilic infiltrates resulting from virus infection. the antibodies d and f may be employed as therapeutic agents for at least h after influenza infection as is indicated by the studies in mice. to, the best of our knowledge, these antibodies are the first fully human monoclonal antibodies generated from the immune repertoire of indian patients infected with a(h n )pdm virus. molecular characterization of their epitopes with indian pandemic isolates will reveal more information of their cross-protectivity and binding characteristics. the strength of our approach for antibody generation lies in the fact that it has used human immune repertoire rather than animals and the antibodies have been generated in response to a natural infection of influenza virus. since, the antibodies have originated from humans, self -reactivity against self-antigens is minimised in comparison to antibodies that have been generated in mice or through the technique of phage display. new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription broadly protective monoclonal antibodies against h influenza viruses following sequential immunization with different hemagglutinins human serum in influenza antibodies for the prevention and treatment of viral diseases intranasal antibody prophylaxis for protection against viral disease production of human monoclonal antibodies via fusion of epstein-barr virus-transformed lymphocytes with heteromyeloma generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune b cells an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus detection of antibody to avian influenza a (h n ) virus in human serum by using a combination of serologic assays a simple method of estimating fifty percent endpoints human monoclonals from antigen specific selection of b lymphocytes and transformation by ebv continuous cultures of fused cells secreting antibodies of pre defined specificity eb virus induced b lymphocyte cells lines producing specific antibody an optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies the author saxena l, acknowledges the senior research fellowship granted to her by the council of scientific and industrial research, government of india, without which this work would not have been possible. key: cord- -ulb d fe authors: bramstedt, katrina a. title: antibodies as currency: covid- ’s golden passport date: - - journal: j bioeth inq doi: . /s - - - sha: doc_id: cord_uid: ulb d fe due to covid- , the fragile economy, travel restrictions, and generalized anxieties, the concept of antibodies as a “declaration of immunity” or “passport” is sweeping the world. numerous scientific and ethical issues confound the concept of an antibody passport; nonetheless, antibodies can be seen as a potential currency to allow movement of people and resuscitation of global economics. just as financial currency can be forged, so too is the potential for fraudulent antibody passports. this paper explores matters of science, ethics, and identity theft, as well as the problems of bias and discrimination that could promulgate a world of pandemic “golden passports.” the concept of a "passport" is changing due to covid- . conventionally, a passport is an official document issued by a government agency to certify identity and citizenship, as well as authorizing travel. "golden passports" have been sought after by the wealthy as a method to gain citizenship-by-investment in regions of the world that have advantageous tax structures (shachar ) . in the era of covid- , antibodies (proteins made by the immune system to fight infection) are posed as a form of currency that can permit "certified" individuals to return to work or travel (see, for example, bartlett ) . in this way, antibodies for sars-cov- (severe acute respiratory syndrome coronavirus type -the covid- coronavirus) are potentially the new golden passport, but the concept is a moving target with clinical unknowns, as well as legal and ethical complexity (phelan ; persad and emanuel ) . research and innovation normally move at the pace of a marathon, but during a pandemic the pace is more like a sprint, creating an environment which is ripe for human error, as well as research and business misconduct, amid a sea of scientific uncertainty, anxious patients and families, and economic frailty (bramstedt ) . in the united states, at least types of antibody test kits have been available; however, due to limited regulatory assessment in the race to release product, these have questionable performance (burton ) . sensitivity and specificity performance thresholds are now set (u.s. food and drug administration a), and the results are catalogued in an open access format (u.s. food and drug administration b). yet even with a robust quality standard and improved regulatory screening of test kits, there remains more questions to address. home testing for the identification of sars-cov- rna [active infection] is already available in the united states (u.s. food and drug administration c). this permits symptomatic people [patients] to selfswab with a home collection kit, sending their nasal swab sample via priority shipping to the approved laboratory for analysis. kits are registered online with a twelve-digit barcode that links the patient to the collection tube. positive results are reported to the patient and the public health authority. positive results can facilitate a clinical treatment plan, but what is the role of negative results? how long does the negative result last and when should such patients be retested? these questions about negative results are important because there can be a window of time during which patients (with and without symptoms) are infected and potential vectors of the disease. also, knowing the true source of the sample is vital; this is not necessarily the name of the person linked to the barcode. there are many ways to fake an identity, but genetic linking can give true identity. however, the associated patient documents for the rna home test do not mention any provision for genetic testing to identify the sample owner (labcorp a, b). currently, covid- antibody testing requires an in-person visit for blood sampling in the inpatient or outpatient setting. this in-person visit allows for personal identification verification of the sample provider. if covid- antibody testing evolves to the home setting using a finger-stick blood sample, the issue of identity verification emerges (brown ) . this is because the perceived social and economic value of a positive antibody result is significant, and fraudulent sampling can occur with ease (for example, submission of a sample from someone already confirmed to be covid- antibody positive). without verification of the true owner of the antibody specimen, identify theft could be a new problem of this pandemic. with the covid- pandemic causing a fragile worldwide economy and millions of people unemployed (congressional research service ), there is a risk of antibody certificates being viewed as the "golden passport" to return to work and travel. the who (world health organization ) has stated the presence of sars-cov- antibodies is not a guarantee that a person cannot be reinfected (fu, chen, and wang ) . also, some people might have false positive tests for sars-cov- antibodies and have zero protection against their first infection (ismail ) . conversely, another potential situation which has been observed is patients have prior tested positive for sars-cov- rna (active infection with or without symptoms) and yet test negative for sars-cov- antibodies (centers for disease control and prevention ). those with negative antibody test results could use various forms of identity theft to confer themselves a positive test result. even if a positive result were scientifically confirmed to confer immunity, there would be the additional matter of a validity date/expiration date. also, there is the matter of creating a globally recognized standard for certification [test method, titres], robust certification agencies, and the logistical issues of documentation language so that certificates could be readable across countries. attempting to rescue the economy with a scientifically unproven antibody passport is problematic for many reasons, including a false sense of health security. but if a covid- antibody passport could be scientifically validated and distributed via a system with globalized recognition, there is the potential for a caste system to result; namely the haves and the have nots of "golden passports." those holding such a passport could benefit from work and movement rights similar to the rights generated by customary immigration visas and passports. systems to prevent fraud with the antibody passport could be implemented using biometrics; however, there could remain a group of people for whom work and travel could have restrictions. this could create a coercion for people to intentionally expose themselves to the virus (kates ) in order to "earn" a passport that could provide them valuable privileges. ethically, work and travel restrictions are appropriate for people who are actively infected. work and travel restrictions could also be appropriate for others as well, but care must be taken to not psychologically or otherwise damage those who are "not immune" from covid- . stigma as well as other harms could potentially negatively impact a person's employability, promotability, insurance rates, access to housing, etc. these ethical concerns heighten the need for policy advisors to reflect beyond the science when they consider enacting antibody passports. in the future there might be a call for covid- vaccination certificates (phelan ) ; however, this too will generate legal and ethical debate. there will be issues of cost, access, safety, and consent, but also many facets of the current antibody issue will be present as well; namely, antibody titres, re-infectivity, and expiry/ boosters. the ethical call is to proactively reflect on these antibody-related matters to create systems which are clinically safe and fraud resistant and avoid discrimination. funding information no funding was received for this work. conflicts of interest nothing to declare. chile's "immunity passport" will allow recovered coronavirus patients to break free from lockdown, get back to work ethical and robust research during pandemics: how? health-care data protection and biometric authentication policies: comparative culture and technology acceptance in china and in the united states fda sets standards for coronavirus antibody tests in crackdown on fraud global economic effects of covid- letter to the editor: three cases of redetectable positive sars-cov- rna in recovered covid- patients with antibodies serological tests for covid- antibodies: limitations must be recognized show me your passport: ethical concerns about covid- antibody testing as key to reopening public life fact sheet for patients: labcorp's covid- rt-pcr test the ethics of covid- immunity-based licenses covid- immunity passports and vaccination certificates: scientific, equitable, and legal challenges citizenship for sale? insight-fdas-revised-policy-antibody-testsprioritizing-access-and-accuracy key: cord- -e k gcse authors: may, jori e.; siniard, rance c.; marques, marisa title: the challenges of diagnosing heparin‐induced thrombocytopenia in patients with covid‐ date: - - journal: res pract thromb haemost doi: . /rth . sha: doc_id: cord_uid: e k gcse nan to the editor, riker eias are sensitive, but not specific, for hit diagnosis because they detect all anti-platelet factor (pf )/heparin antibodies, including those that are nonpathogenic. in contrast, functional assays (including sra) identify only antibodies with the pathogenic ability to activate platelets and therefore have increased specificity. given that severe covid- is a hyperinflammatory state, it is plausible that the increased immunoreactivity also increases production of anti-pf /heparin antibodies; however, they may not result in clinical hit but may instead increase potential for false-positive eias. herein, we report our experience with hospitalized patients with the authors have no conflicts of interest to disclose. heparin-induced thrombocytopenia with thrombosis in covid- adult respiratory distress syndrome covid- and its implications for thrombosis and anticoagulation testing for heparin-induced thrombocytopenia antibodies key: cord- -zpi uis authors: roberts, anjeanette; wood, john; subbarao, kanta; ferguson, morag; wood, david; cherian, thomas title: animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting – august , london, uk date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: zpi uis abstract severe acute respiratory syndrome (sars) emerged in the guangdong province of china in late and spread to countries. by the end of the outbreak in july , the cdc and who reported cases with a . % case fatality rate. the disease was caused by a previously unrecognized coronavirus, sars-cov. drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. available data suggest that vaccines should be based on the the kda viral spike protein, s, the only significant neutralization antigen capable of inducing protective immune responses in animals. in the absence of clinical cases of sars, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the united states food and drug administration's “animal rule” would apply to licensure of a sars vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. this report summarizes the recommendations from a who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on – august in south mimms, uk, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate sars vaccines. severe acute respiratory syndrome (sars) is a severe respiratory illness caused by the sars coronavirus (sars-cov) [ ] . the disease emerged in the guangdong province of china in late [ ] and spread to countries mostly within asia, although europe and north america were also affected, notably toronto, canada. the epidemic was finally controlled by july through strict implementation of prevalence for sars-cov antibody, although they had no history of sars-like disease. sars-cov-like viruses that were isolated from civets and raccoon dogs had more than % homology with human sars-cov, with major differences found in orf , whose deletion has been suggested to represent a sign of adaptation to humans [ ] . only four amino acid residues in the receptor glycoprotein ace binding domain of the viral spike protein differ between the human epidemic sars-cov strains and civet strains, but they cause more than a -fold difference in binding affinity to the ace molecule [ , ] . although a high prevalence of sars-like coronaviruses were found in chinese horseshoe bats [ , ] , their great genetic diversity makes it difficult to identify which one might be the ancestor of sars-cov and to decide with certainty whether bats indeed are the animal reservoir of the virus. sars-cov infection exhibits a wide clinical course characterized mostly by fever, dyspnea, lymphopenia and lower respiratory infection, often with concurrent gastrointestinal symptoms including diarrhea [ , ] . pathology in sars patients has been associated with diffuse alveolar damage, epithelial cell proliferation and multinucleated giant cell infiltrates of epithelial or macrophage origin, suggestive of syncytium-like formation in the lung. the virus can be recovered from peripheral blood mononuclear cells, respiratory secretions, stools, urine and even sweat (for a review, see [ ] ). sars vaccine development efforts were initiated very rapidly after the identification of the etiologic agent, even though the immune correlates of protection were not known. research efforts to identify protective antigens and to develop animal models were undertaken in parallel with efforts to develop candidate vaccines [ ] , drawing on experience with animal coronavirus vaccines and using several vaccine strategies, including inactivated virus vaccines, purified subunit vaccines, plasmid dna and viral vector-based vaccines as well as virus-like particles. much effort has been made to identify appropriate animal models for sars-cov replication and pathogenesis. several research groups have shown that mice [ , ] , ferrets [ ] , hamsters [ ] and nonhuman primates [ ] [ ] [ ] [ ] [ ] [ ] support replication of sars-cov with varying degrees of associated disease. these animal models were used for the evaluation of candidate vaccines, and the common conclusion that has emerged from the evaluation of several vaccines is that the kda viral spike protein, s, is the only significant neutralization antigen [ ] [ ] [ ] [ ] and the only one to elicit protective immunity in animal models [ , [ ] [ ] [ ] [ ] [ ] [ ] . the s protein can be divided into two domains by analogy with other coronavirus spike proteins: an n-terminal s domain, which contains the receptor-binding site and neutralization epitopes and a cterminal s domain which forms the membrane-anchored stalk region and contains a putative fusion peptide followed by two heptad repeats predicted to form a six-helix coiled-coil bundle [ ] . in the absence of clinical cases of sars, candidate vaccines will have to be evaluated for efficacy in animal models. the united states fda "animal rule" states that, when efficacy studies in humans are not feasible, vaccines may be approved based on animal data alone, provided the pathophysiological mechanism of the disease is reasonably wellunderstood as is its prevention or reduction by the vaccine. moreover, the protective effect of the vaccine should be demonstrated in more than one animal species expected to react with a response predictive for humans. the endpoint of animal studies should be clearly related to the desired benefit in humans (i.e. enhancement of survival or reduction in major morbidity), and the data generated should allow selection of an effective dose in humans. at the present time it is uncertain whether the "animal rule" would apply to licensure of a sars vaccine. however, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. scientists at the who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on - august in south mimms, uk, discussed many aspects of research pertaining to the use of animal models in vaccine development including available animal models, suitability of the various models, correlates of protection, critical components of potential vaccines, and the potential for disease enhancement in vaccinated animals following exposure to sars-cov. in addition, standardization of antibody assays and the establishment of a who international standard for sars-cov antibody were also discussed. this report endeavors to summarize the recommendations from this meeting, based on consensus agreement. recommendations for use of each animal model are given in section below. correlates of protection, an overview of vaccine development, and observations pertaining to potential disease enhancement are summarized in the following sections - . in selecting animal models for vaccine evaluation, it is important to remember the principle underlying the so called "animal rule", where data from more than one animal species is often required: each animal species should contribute something different to our understanding of disease and protection. at this time, no single model offers a direct reproduction of what was seen in humans with sars. pathology (including pneumonitis, alveolar edema, and diffuse alveolar damage (dad)) in humans is probably the most difficult element to reproduce in an animal model. attention also should be given to the reduction of viral shedding because this would likely correlate with decreased risk of further spread of the disease among humans. in using animal models to study aspects of sars-cov infection, it must be emphasized that the kinetics of viral replication and appearance and resolu-tion of pathological findings are much more rapid in animal models than in humans. whichever animal model is employed, special consideration should be given to the presence of co-existing pathogens, the age of the animals and the route(s) of infection. a sufficient number of time points and large enough number of animals should be used to allow statistical evaluation. the strain of sars-cov used also could be of importance. it should be emphasized that different species may prove useful for studying different aspects of sars-cov. whereas vaccines or antivirals may be addressed in many models, pathogenesis is best evaluated in those animal models for which immunological tools and reagents are available for detailed analysis of the immune response to the vaccine. this includes inbred mice and rhesus and cynomolgus macaques. it may actually be worthwhile to enhance the virulence of a sars-cov isolate by serial passages in an animal model to produce a challenge virus stock for vaccine studies that would elicit more reproducible disease in the animals. if a highly virulent host-adapted virus were to become available, such as a mouse-adapted or a monkey-adapted sars-cov strain, demonstration of the capacity of vaccines to protect against challenge with these more virulent strains would provide an almost ideal animal model. different models may also need to be employed to evaluate pathogenesis versus immunogenicity. for pathogenesis studies in animal models, mortality is not required as a readout. it would be ideal to develop animal models with comparable levels of mortality to that seen in humans (∼ % overall), including the increased mortality at increased age (∼ % > age ). the optimal result would be to demonstrate efficacy of vaccines or antivirals in sars-cov animal models that mimic human morbidity and mortality and that show protection without vaccine-associated immunopathology. inbred mouse strains (balb/c, c bl/ , svev, stat −/−) support sars-cov replication and can develop pneumonitis ( s), but pneumonia and clinical symptoms are only observed in older balb/c mice [ ] . the mouse model is suitable for immunogenicity and efficacy studies of vaccines. prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in stat −/− mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs. specific pathogen-free (spf) animals must be used. their age can be either - weeks or over months and should be specified. the number of animals included must be sufficient for statistical analysis, and should include mock-infected controls. a variety of sars-cov strains has been tested in mice, including urbani, frankfurt, hku- , tor- , and the mouse-adapted sars-cov strain ma- . these were inoculated by the intranasal (in) route under light anesthesia [ , ] , using a dose of tcid in l/mouse. critical time points for specimen collections are days (peak titer) and post-infection (p.i.) for quantitative virology, days and p.i. for the study of interstitial pneumonitis and dad in aged mice (inflate lungs with % neutral-buffered formalin), and day for resolution. golden syrian hamsters are an excellent animal model because they demonstrate high levels of sars-cov replication and develop pneumonitis. hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies [ ] . in contrast with balb/c mice, in which the virus is detected only in the respiratory tract and is cleared by day p.i., hamsters demonstrate a longer duration of viral shedding from the upper respiratory tract, some transient viremia, spread of the virus to liver and spleen and, most significantly, inflammation of respiratory tissues [ , ] . spf animals older than weeks of age should be used in sufficient number for statistical analysis and study design should include mock-infected animals. the animals can be housed in pairs or by three if the experiment is to last less than - weeks. reserve space should be available to separate the animals in case of fights. males and littermates tend to fight less. virus strains that have been tested in hamsters are urbani, frankfurt and hku- . virus should be administered by the in route under light anesthesia, using a dose of tcid in l/hamster. as an outcome of efficacy studies, quantitative virology should be preferred over quantitative pcr. for pathology studies, one can grade pathology as none, mild, moderate or severe as per roberts et al. [ ] . critical time points for specimen collection are days or p.i. (peak viral titer) and (clearance) for quantitative virology studies; and days p.i. (consolidation and pneumonitis) and or (resolution) for pathology studies (lung). there is evidence from one study that ferrets support sars-cov replication and develop pulmonary lesions [ ] but according to another study, the animals remain asymptomatic, in the presence of sars-cov replication [ ] . in view of these conflicting data, the ferret model needs to be further studied to determine its optimal utility for vaccine efficacy and immune prophylaxis studies. additional studies are needed to define the extent of biological variability of the model and the possible role of co-pathogens that may contribute to the variability observed between different laboratories. animals aged months or older should be used. although not well documented, more consistent viral replication, pathology and clinical symptoms seem to be observed in older animals. the animals should be screened for viral co-pathogens: aleutian mink disease, respiratory viruses, hepatitis viruses, and others. the route of inoculation may be in or it, but not iv. the dose of virus (strains tor- , hku- ) sufficient to ensure reproducibility of infection in all animals is likely to be pfu or more/ferret. again, quantitative virology is preferred over qrt-pcr. for pathology studies, the same recommendations apply as for nonhuman primate studies (see below): slides should be shared between pathologists to develop a scoring system. regarding specimen collection of respiratory tissues, further studies are needed to establish how much variation occurs in samples from different lobes of the lungs. critical time points are day p.i. for quantitative virology and days - p.i. for pathology studies (pneumonitis). nhps support sars-cov replication and develop pneumonitis with a variable degree of clinical symptoms depending upon the species employed. no single nhp species is preferred at this time. the number of animals in a given study needs to be large enough to account for animal-toanimal variability: a sample of or animals is not sufficient. in view of the cost of the experiments, challenge studies should be limited to those vaccine candidates that are most promising, using larger sample sizes ( - animals/group) and avoiding animals with free-range periods in life if possible. immunological responses are best studied in species for which microarrays and reagents for identifying immune components are available, such as rhesus or cynomolgus macaques. however, the limited viral replication observed in cynomolgus macaques might be a disadvantage in selecting this species for studies. other recommended nhp species are the common marmoset and african green monkeys (agms, chlorocebus aethiops sabeus). the country of origin may play an important role and should be specified, e.g. philippines (cynomolgus macaques, macaca fascicularis); china (rhesus, macaca mulatta); brazil (marmosets, callithrix jacchus), etc. prior to the experiment, the animals should be housed indoor to limit exposure to potential co-pathogens. they should be screened for parasites (strongyloides, pneumonyssus simicola (lung mites)) and for possible viral co-pathogens (retroviruses, respiratory viruses, adenoviruses). the sars-cov strains tested in nhp models are hku- (cynos), pumc (rhesus) and urbani (marmosets and agms). these were inoculated by the respiratory route (in, it) at a dose of pfu or more/nhp. here again, quantitative virology is preferred over qrt-pcr. for pathology studies, it would be an obvious advantage that laboratories share pathology slides for review by different pathologists in order to develop a scoring system. specimens of respiratory tissues should be collected, but further studies are needed to establish how much variation occurs in samples from different lobes of the lungs, as was done and reported for african green monkeys (agms) [ ] . critical time points are days - p.i. for quantitative virology in cynomolgus macaques and agms, and later than day p.i. for rhesus macaques of chinese origin. for collection of tissues for histopathological analyses, days - p.i. are optimal for cynomolgus macaques and agms, and later than day p.i. for rhesus macaques. due to limitations of immunological reagents (including microarray assays now available), research may be limited to rhesus and cynomolgus macaques. data on other animal models are insufficient for consideration for use in sars-cov vaccine and antiviral evaluations [ ] [ ] [ ] . any additional model other than the four listed above (section . . to section . . ) would require thorough characterization including viral replication data and histopathological analysis of sars-cov-infected and mockinfected animals of the same age and gender. viral replication and histopathological data in any new animal model should be reminiscent of at least some aspect of sars in humans. although all the correlates of protection from sars associated disease have not been identified in human infections, neutralizing antibodies are present in convalescent human serum. antibodies to sars-cov spike (s) protein have been shown to prevent virus entry and neutralize virus infectivity in vitro [ , ] . prophylactically administered monoclonal antibodies and passively transferred sars-cov hyper-immune sera have been shown to prevent sars-cov infection and associated disease following sars-cov challenge of naive mice and hamsters [ , , [ ] [ ] [ ] . monoclonal antibodies administered therapeutically (i.e. post-infection) also have been shown to limit viral replication and reduce associated disease in hamsters [ ] . although cell mediated immunity may have a protective role in viral clearance or resolution of disease, work in animal models shows that antibody alone is effective for prevention and treatment of sars. thus, mice immunized with live-recombinant vaccines expressing the sars-cov spike protein, using rabies virus [ ] , vesicular stomatitis virus (vsv) [ ] , adenovirus (ad ) [ , ] or attenuated vaccinia virus mva [ , ] as a vector, as well as mice immunized with dna vaccines expressing the s gene [ , ] , developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. similar findings were reported after mucosal immunization of hamsters and agms using a bovine parainfluenza virus type (bpiv ) vector expressing the sars cov s gene [ , ] . several whole inactivated virus and recombinant protein candidate vaccines also have been developed and shown to elicit a neutralizing antibody response that provided protection against infectious challenge [ ] [ ] [ ] [ ] [ ] [ ] . in addition, passively administered sera from vaccinated animals prevented sars-cov infection upon subsequent challenge of naïve mice, demonstrating that antibodies induced by these vaccines did confer protection [ , ] . the neutralizing antibody titer that is necessary to achieve protection in humans exposed to sars-cov is, however, still not known. it was recommended that when evaluating vaccine efficacy in future animal experiments, the challenge virus should be administered at two different time-points, once when postimmunization neutralizing antibody titers are high, and later when neutralizing antibody titers have waned or are low. it also was suggested that viral titers and pathology should be evaluated at two different time points. specific times points for sample collection are given for each animal model in section above. previous observations of disease enhancement have been reported for human viral pathogens and shown to be due to antibody-mediated enhancement of virus entry (for reviews see [ , ] ). enhanced disease and mortality have been observed in kittens immunized against or infected with a type-i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection [ ] [ ] [ ] . aggravated fip is apparently a result of enhanced viral entry into macrophages mediated by sub-neutralizing antibody levels [ ] . children vaccinated with inactivated respiratory syncytial virus (rsv) vaccines developed serious disease on subsequent exposure to rsv [ ] [ ] [ ] . individuals exposed to one of the four serotypes of dengue virus developed severe disease when subsequently infected with a second, different serotype [ , ] . enhanced disease following rsv vaccine or dengue infection occur by different mechanisms than fipv. in view of such examples of enhanced disease following infection in a vaccinated host, there has been heightened concern that a similar phenomenon could occur with sars-cov vaccines. it was highly recommended, therefore, that known mechanisms of disease enhancement observed with other viruses and especially with other coronaviruses should be examined in sars-cov infections, especially in vaccinated animals. although none of the studies to date have shown enhanced respiratory disease following sars-cov challenge in previously immunized animals, further studies in this area are warranted in view of some of the available in vitro data. antibodies against human sars-cov isolates were shown to enhance the entry of pseudo-typed viruses expressing the civet sars-like cov-spike protein into a human renal adenocarcinoma cell line ( -o) . enhancement was only demonstrated at the level of entry, but not of replication [ ] . this phenomenon was seen with pseudo-typed lentiviruses expressing sars-cov spike protein of civet sequence specificity, but not with pseudo-typed viruses expressing spike proteins of human sars-cov isolates. it also was not observed with human isolates of sars-cov. the role of enhanced viral entry, as observed in these in vitro studies, has not been related to any known component of human disease or infection in vivo. however, given that sars-cov may replicate, albeit poorly, in human pbmcs [ , ] , in vitro experiments looking for antibody-enhancement of sars-cov replication in human cells (e.g. macrophages and b-cells) should be performed. several groups have studied sars-cov infection in animals in the presence of neutralizing and sub-neutralizing levels of sars-cov anti-sera or anti sars-cov s-protein monoclonal antibodies, but no evidence of enhanced respiratory disease has been observed. however, foci of hepatic necrosis were noted following sars-cov challenge in mva-sars-s immunized ferrets [ ] . although these findings are worrisome, several questions were raised regarding the significance of the observation. the mva-sars-s vaccine used in these experiments was poorly immunogenic in the ferrets. the question of whether there could have been any co-pathogen in the animals was raised. it also would be important to know if the observed phenomenon depends on the mva vector or on the animal model. it was strongly urged, therefore, that the experiment be repeated in ferrets. additional experiments, in nonhuman primates and hamsters, looking for evidence of enhanced respiratory and hepatic diseases upon vaccination and challenge were also encouraged. several candidate sars vaccines that are at various stages of pre-clinical and clinical development are being developed worldwide. in china alone, three companies have been given regulatory approval for the clinical evaluation of a candidate sars vaccine. it is important, therefore, to be able to compare data from each of the candidate vaccines, which, in turn, requires international standardization of the immunological assays used for the evaluation of these vaccines. the accepted method of international standardization is to employ a who international standard (is), which allows comparison of results from different laboratories [ ] . this is essential for establishing international requirements for vaccines, diagnostics or therapeutics. an is is prepared from material bearing a close resemblance to the samples being assayed; the material is distributed in glass ampoules with high precision and reproducibility and then freeze dried. it is important that a sufficient number of ampoules ( - ) be prepared so as to provide for about years of use, and that the activity of the contents remain stable over this period. the process of establishing a who is involves an international collaborative study, in which the candidate is is compared with other samples. if the results of the tests are suitable, the candidate is assigned a provisional arbitrary unitage, a report is distributed for approval by study participants and for eventual approval by the who expert committee on biological standardization (ecbs). the preparation, storage and distribution of over % of is have been undertaken by the national institute for biological standards and control (nibsc) at south mimms (uk), which is a who laboratory for biological standards. nibsc has developed in the past several who iss for calibration of the antibody response against virus vaccines, including iss for antibodies against dengue, hepatitis a virus (hav), [ ] hepatitis b virus (hbv), measles, [ ] polio, rabies, [ ] rubella, and smallpox. a candidate standard against human papilloma virus- (hpv- ) is under evaluation. the corresponding is's were used for a variety of antibody assays including virus neutralisation (vn), haemagglutination-inhibition, single radial diffusion, enzyme-linked immunoassay and radio-immunoassay. antibody iss are most useful in epidemiological studies and in clinical trials. their use allows correlates of immunity and potency requirements of prophylactic and therapeutic products to be expressed in international units (ius). data from several collaborative studies demonstrate that use of an is generally reduces the level of variability between assay results. however, there may be problems in using iss due to the complex array of antibody populations in each serum and the different sensitivity of different assay systems. examples of potential problems can be found in hbv and parvovirus b studies [ ] , which showed that different assay kits gave different results even when the is was included in the assays. another issue is the degree of antigenic homology between the viral antigen used for the preparation of the is and the virus used in the assays. in a jev collaborative study, a candidate antibody is, which had been prepared from the sera of vaccinees immunized with an inactivated vaccine that was antigenically different from some of the viruses used in vn assays, demonstrated that the response to at least some inactivated vaccine is strain specific and the candidate is was consequently not established by the who ecbs. whether a panel of monoclonal antibodies to sars-cov could be used to prepare an is is an attractive alternative which should be explored. significant progress with standardisation of sars-cov antibody assays had been made in china with the development of a national antibody standard. in order to develop the national standard, sera were collected from convalescent sars patients who were found to have sars-cov vn antibody titers ranging from undetectable to : . one serum sample was selected for further evaluation based on crossreactivity with four sars-cov strains and on western blot analysis. this serum was freeze dried in . ml aliquots and was then assessed for stability by vn assays. the chinese standard was assigned a vn titer of . with % confidence limits of . - . . a further important development in china was the preparation of human immunoglobulin for treatment of sars patients. national guidelines have been prepared for collec-tion of plasma, quality control testing and standardisation of assays. three chinese manufacturers have been licensed for preparation of sars immunoglobulin. the source material was plasma from convalescent patients at more than days after infection. all were in good health and their plasma tested negative for blood-borne agents. plasma samples were processed by a combination of cold ethanol fractionation and ultrafiltration. in september , three lots of igg were produced and assayed by nationally-agreed procedures. the stock of immunoglobulin currently available is sufficient to treat patients. monoclonal antibodies have not yet been considered for treatment purposes in china. the importance of assessing immunogenicity of candidate sars-cov vaccines using vn assays is well acknowledged, but the variety of vn tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. it is therefore desirable to establish an antibody is to serve as a basis of comparison in all vn assays. the most important activity at this time is to obtain a suitable source of antibody. a number of options can be considered, such as convalescent human sera, post-immunization human sera, monoclonal antibodies or hyperimmune animal sera. as an example, the availability of a suitable source of serum from convalescent patients in hong kong needs to be explored, although antibody levels in these individuals are probably quite low by now. it also would be important that other assays than vn be included in the collaborative study, and that the impact of sars-cov strain variation be examined by using different sars-cov strains and/or sera with different specificities. the centralized facility for aids reagents (cfar), which is based at nibsc, could be a suitable model for a sars-cov repository [ ] . the cfar was established in to support aids vaccine research and it is now eu-funded [ ] . there are currently reagents available including peptides, recombinant proteins, human sera/plasma, monoclonal and polyclonal antibodies, expression systems, cdna clones and viruses. a comparison can be drawn between sars-cov and hiv vn assays. currently, there are several different hiv neutralization assays formats under consideration and a lack of agreement on the most suitable assay. the cfar is supporting a joint who/eu project (neunet) to evaluate and standardize hiv vn assays in an international collaborative study. in the usa, a sars-cov repository has been established on behalf of the american type culture collection (atcc) in order to meet the needs of biodefence and the threat of emerging infections [ ] . the type of reagents stored includes viruses, peptide arrays, monoclonal antibodies and proteins. it is hoped that an active collaboration can be established between niaid and nibsc in order to meet the expanding needs of the sars research community. based on the discussion at the meeting, the following recommendations were made with respect to standardization of the immunological assays for sars vaccine evaluation: . a who repository for sars-cov reagents ought to be developed. collaboration between niaid and nibsc is recommended to achieve this goal. . consensus must be reached for the reagents to be given priority in the repository. needed. . the most suitable source of antibody for the is is convalescent human sera, but post-vaccination human sera could also be used. . a protocol for an international collaborative study aimed at validating the is should be developed and distributed to prospective participants. . collaborative study participants should be asked about their assay capabilities, e.g. number of sera, virus strains handled, etc. . . . the proposed is collaborative study should include a core set of antibody preparations to be distributed and assayed in each laboratory (e.g. monoclonal antibodies, animal sera, other human sera). . tests should be conducted using the same strain of sars-cov in each laboratory, but the different genetic lineages of sars-cov should also be represented in the study. biosafety issues associated with sars-cov vaccine development stem from the reports of laboratory-acquired infections in china. sanofi pasteur has adopted bsl practices and bsl equipment (e.g. class or microbiological safety cabinets with respiratory protective equipment) for the preparation of sars-cov vaccines. of note is the fact that sars-cov appears to be quite resistant to normal methods of virus decontamination (jf saluzzo, personal communication). who has developed guidance, both general [ ] , and specific for handling sars specimens [ ] . the rapid success in the development of immunogenic and protective vaccines against sars using a variety of platforms is encouraging, but should be tempered with concerns about the possibility of enhanced disease following exposure in vaccinated individuals [ ] . concerns mainly stem from reports of enhanced disease in fipv-immunized or -infected kittens [ , ] , from observations that antibodies elicited against certain coronaviruses mediate antibody-dependent enhancement of viral entry [ ] , and from the observation of inflammatory foci in liver tissue following sars-cov challenge in mva-sars-s vaccinated ferrets [ ] . candidate sars vaccines will need to be evaluated in more than one animal model. they also will need to be thoroughly evaluated for the duration of the antibody response they induce, as well as for the breadth of their protective effi-cacy against different strains of sars-cov. the implications of the sequence heterogeneity among sars-cov strains are difficult to test at this time because the most divergent strains (civet sars-like viruses) have not been recovered in culture. validation and international standardization of immunological assays for the evaluation of candidate sars vaccines are essential to compare data across different trials. this requires the establishment of international standards for sars-cov antibody and a repository for sars-cov reagents, with an international collaborative study to validate the iss. the establishment of the repository by who in collaboration with nibsc and niaid was recommended. severe acute respiratory syndrome who investigates china's fall in sars cases who says sars outbreak is over, but fight should go on summary of probable sars cases with onset of illness from identification of a novel coronavirus in patients 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standard for rabies immunoglobulin report of a collaborative study to calibrate the second international standard for parvovirus b antibody the importance of standardisation of laboratory evaluations in hiv vaccine trials biodefense and emerging infections research resources repository (bei resources world health organization. laboratory biosafety manual who biosafety guidelines for handling of sars specimens caution urged on sars vaccines the authors thank dr. marc p. girard and dr. marie-paule kieny for their invaluable assistance in preparing the manuscript. the contributions of anjeanette roberts and kanta subbarao were supported in part by the intramural research program of nih/niaid. key: cord- -pcsuu authors: chan, kuan rong; ong, eugenia z; mok, darren zl; ooi, eng eong title: fc receptors and their influence on efficacy of therapeutic antibodies for treatment of viral diseases date: - - journal: expert rev anti infect ther doi: . / . . sha: doc_id: cord_uid: pcsuu the lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. in recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, ebola virus and hendra virus. the binding affinity of these antibodies can directly impact their therapeutic efficacy. however, we and others have also demonstrated that the subtype of fc-gamma receptors (fcγrs) engaged influences the stoichiometric requirement for virus neutralization. hence, the development of therapeutic antibodies against infectious diseases should consider the fcγrs engaged and fc-effector functions involved. this review highlights the current state of knowledge about fcγrs and fcγr effector functions involved in virus neutralization, with emphasis on factors that can affect fcγr engagement. a better understanding of fc-fcγr interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. the lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. in recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, ebola virus and hendra virus. the binding affinity of these antibodies can directly impact their therapeutic efficacy. however, we and others have also demonstrated that the subtype of fc-gamma receptors (fcgrs) engaged influences the stoichiometric requirement for virus neutralization. hence, the development of therapeutic antibodies against infectious diseases should consider the fcgrs engaged and fc-effector functions involved. this review highlights the current state of knowledge about fcgrs and fcgr effector functions involved in virus neutralization, with emphasis on factors that can affect fcgr engagement. a better understanding of fc-fcgr interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. while vaccination now protects against several important infectious diseases, many others still lack effective vaccines. the ability of viruses to rapidly mutate and alter their antigenic makeup (e.g., influenza and hiv), as well as the potential interference between different virus serotypes (e.g., dengue virus) has made vaccine development a challenging task. given that infection with such viruses can result in a significant number of cases and high mortality rates, rapid development of potent therapeutic measures can potentially save lives and limit the magnitude of outbreaks. with technological advances in the field of immunoglobulin g (igg) research and generally good safety profiles observed during antibody-based therapies, therapeutic antibodies have become attractive candidates for treating infectious diseases. the recent ebola outbreak in west africa is a good example of the potential of mab cocktails as an anti-infective strategy [ ] . other examples include the use of mab m . against hendra virus [ ] and palivizumab that targets respiratory syncytial virus (rsv) [ ] , resulting in significant reduction in the incidence of hospitalization. given the need for new antiviral therapies and considerable advances in igg research, there is a growing interest in developing therapeutic antibodies against infectious diseases. the binding affinity of antibodies to viruses can directly impact the efficacy of mabs [ ] , suggesting that target-specific mechanisms likely account for much of the efficacy of therapeutic mabs. however, many studies have also highlighted the contribution of fc-mediated immune effector functions in modulating the efficacy of these mabs [ ] . antigen-presenting cells such as dendritic cells, monocytes and macrophages can internalize antibody-opsonized viruses via fc-receptor-mediated phagocytosis. this results in antigen processing and presentation to effector cells of the adaptive immune system, eventually leading to virus clearance. on the other hand, neutrophils, monocytes, macrophages, mast cells and natural killer (nk) cells kill infected cells opsonized with antibodies in a process described as antibody-dependent cell-mediated cytotoxicity (adcc). while immune complexes are expected to be phagocytosed by fcreceptor bearing cells and cleared from the blood circulation, delivering viruses to monocytes or macrophages at subneutralizing antibody levels can result in antibody-enhanced infection due to their ability to serve as a host for viral replication. in support of this theory, subneutralizing levels of antibodies have been shown to result in increased infection of viruses such as dengue virus (denv), ross river virus, influenza, hiv- and epstein-barr virus (ebv) [ ] [ ] [ ] [ ] [ ] . hence, therapeutic antibodies ought to be delivered at doses high enough to prevent antibodyenhanced virus infection and avoid the cytokine storm that can result from excessive stimulation of fc-gamma receptors (fcgrs). given the delicate balance between antibody efficacy and adverse side effects, there is a need to develop methods that can better assess fc-mediated effector functions triggered by therapeutic antibodies. this could be beneficial in the future development of antibodies engineered to possess enhanced therapeutic activity [ , ] . here, we review the significance of the various aspects of fc-mediated effector functions involved in virus neutralization and factors that can potentially affect fcgr engagement. an improved understanding of these processes would allow identification of predictive factors to better assess the efficacy of therapeutic antibodies and facilitate development of engineered therapeutic antibodies with improved efficacy. among the different classes of immunoglobulins (iga, igd, ige, igg and igm), igg is the most commonly used and hence, will be the focus of this review. in general, antibodies consist of two distinct functional units: the fab and the fc regions (figure ). the fab portion of the antibody contains the variable region comprising three hypervariable complementarity-determining regions that bind specific antigenic epitopes. the binding affinity of the fab to viruses can be determined by binding assays such as elisa or by more specialized and precise methods such as biacore surface plasmon resonance. fab binding to viruses can block receptor interaction with the host, thereby hindering attachment and subsequent virus entry. in addition, several studies have suggested that antibodies that target regions other than the receptor-binding sites can also be neutralizing. for instance, antibodies that bind gp , a hiv- fusogenic transmembrane protein, did not inhibit virus-cell binding but was able to neutralize hiv- infection by recognizing fusionintermediate forms of gp at a postattachment step [ , ] . similarly, neutralizing antibodies that bind to west nile virus and denv have been shown to block infection at the postattachment step [ , ] . altogether, these investigations suggest that the fab portion of neutralizing antibodies can function by either blocking virus attachment or inhibiting virus fusion. by contrast, the fc portion of the antibody binds to fcgrbearing immune cells of the mononuclear phagocyte system such as monocytes, macrophages and dendritic cells. fcgrs have been shown to be important in modulating the efficacy of therapeutic mabs [ ] due to their involvement in fcgrmediated phagocytosis, cytokine production, adcc and complement-dependent cytotoxicity (cdc) that aids in virus neutralization (figure ). however, whether these fc effector functions are activated or inhibited depends on the subtypes of fcgrs engaged. among the human-activating fcgrs, fcgria has the highest affinity for both monomeric igg and immune complexes. fcgriia and fcgriiia, on the other hand, bind strongly to immune complexes but not to monomeric igg [ ] . both fcgria and fcgriiia require an accessory immunoreceptor tyrosine-based activation motif (itam) bearing gammachain for signal transduction. by contrast, the itam motif of fcgriia is located within its cytoplasmic tail (figure ). when activating fcgrs are clustered, src-related tyrosine kinases (e.g., src, fyn, fgr, hck and lyn) are activated and phosphorylated, resulting in itam phosphorylation that mediates recruitment and activation of protein kinases of the spleen tyrosine kinase (syk) and zap- family kinases [ ] . these activated kinases then catalyze the activation of downstream kinases, initiating a signaling cascade in several pathways to trigger immune effector functions. on the other hand, clustering of the inhibitory receptor fcgriib with activating fcgrs results in phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (itims) that recruit and activate phosphatases such as src homology (sh )-domain-containing inositol phosphatases ship- , ship- and sh -containing tyrosine phosphatases shp- [ , , ] . these phosphatases then dephosphorylate the kinases downstream of itam-mediated signal transduction to inhibit activating fcgr signaling (figure ). thus, coligation of inhibitory fcgrs with activating fcgrs results in abrogation of itam-mediated immune effector functions [ , ] . interaction of activating fcgrs with antibodies can directly affect efficacy of therapeutic antibodies. for instance, the efficacy of clinically approved anticancer mabs such as rituximab (anti-cd mab) and trastuzumab (humanized anti-her mab) was significantly reduced in mice deficient in activating fcgrs compared to wild-type mice [ , ] . this is in contrast to mice deficient in the inhibitory fcgrs, in which increased antibody-mediated cytolytic activity was observed [ ] . in addition, common single-nucleotide polymorphisms in fcgriia gene at position (rs ) and in fcgriiia gene at position (rs ) can lead to significant reduction in binding to igg [ ] and response to clinically approved mabs such as trastuzumab, rituximab and cetuximab (anti-egf mab) [ ] [ ] [ ] [ ] [ ] . furthermore, fc modifications and the use of f (ab) that alter the interaction between fc and fcgrs can directly affect fc-effector functions and hence therapeutic outcome [ ] . for example, hiatt et al. demonstrated that different glycan variants of palivizumab mediated different levels of adcc, affecting in vivo efficacy of palivizumab against rsv [ ] . overall, these studies suggest that therapeutic mabs directed against similar antigenic targets may differ in their clinical profile depending on fc-fcgr interaction. the neonatal fc receptor (fcrn) is a structurally distinct fc-receptor expressed in various cell types (e.g., vascular endothelium cells, monocytes and macrophages) [ , ] . fcrn binds to igg within the acidic environment of endocytic vacuoles but not at physiological ph. immune complexes that bind to fcrn are sorted to antigen-processing endosomes or recycling endosomes. however, monomeric iggs are sorted into recycling endosomes, which recycle iggs back to the cell surface following endocytosis, thus preventing intracellular degradation of iggs. this allows antibodies or immune complexes to persist in the blood circulation for up to several weeks after treatment. mutations at positions (thr gln) and (met leu) can significantly increase binding to fcrn and half-life of antibodies without affecting antigen-binding and fc-mediated effector functions [ , ] . while improved affinity to fcrn can extend the half-life of antibodies [ ] ; this has never been linked with increased clinical efficacy. however, in vivo studies with a hfcrn/rag knockout mouse model suggest that the antitumor activity of bevacizumab (anti-vegf) and cetuximab (anti-egf) can be significantly improved when binding to fcrn was increased [ ] . furthermore, recent studies have shown that administration of a broadly neutralizing antibody with enhanced fcrn affinity can improve protection against hiv- infection in nonhuman primates, not only by increasing serum half-life but also by enhancing transcytosis of antibodies to mucosal surfaces [ , ] . with slower clearance of antibodies and improved protection, less frequent dosing schedules would be expected, potentially reducing compliance issues. moreover, the extended half-life would ensure that the levels of circulating antibodies are kept sufficiently high for virus neutralization. this would also minimize the risk of antibody-dependent enhancement (ade) of virus infection, which would be particularly useful for viruses such as denv, where ade is hypothesized to result in increased viremia and increased risk of severe dengue [ , [ ] [ ] [ ] . however, the effects of improved half-life of these antibodies should be tested to avoid any potential toxic adverse effects. given the importance of fcgrs in mediating virus neutralization and fc effector functions, a better understanding of how therapeutic antibodies neutralize virus infections in fcgrbearing cells will impact implementation of dosing regiments and allow development of improved therapeutic antibodies against infectious diseases. here, we discuss the approaches that can be used to test for various fc-mediated effector functions and describe how these processes could contribute to efficient neutralization of viruses. virus neutralization in the presence of fcgr-mediated phagocytosis fcgr-mediated phagocytosis plays a critical role in clearing igg-opsonized pathogens from the systemic circulation and augmenting antigen presentation [ ] . cross-linking of fcgrs on phagocytes initiates downstream signaling that results in phosphorylation and activation of kinases such as phosphatidyl inositol -kinase, p s kinase and akt [ , ] , which are directly involved in reorganization of the actin cytoskeleton and membrane remodeling [ ] . antibody-opsonized viruses are then directed to lysosomes, where the antigens are processed into peptides. in human monocytes, fcgri has been shown to be involved in neutralization of virus immune complexes. for instance, reduction of antibody-opsonized hiv- infection in monocyte-derived macrophages was observed upon inhibition of fcgri [ ] . reducing fcgri but not fcgriia expression using small interfering rna also resulted in reduced antibody required for denv neutralization in monocytes, reinforcing the role of fcgri in denv neutralization [ ] . while the exact mechanism involved remains to be fully elucidated, activation of fcgri has been demonstrated to induce expression of proinflammatory mediators and is associated with trafficking of immune complexes that directly targets virus immune complexes to the late endosomes or lysosomes for enhanced antigen processing and antigen presentation to cd + t-cells [ ] . this can occur as early as min after fcgri cross-linking. in contrast, cross-linking of fcgriia resulted in slower trafficking of immune complexes, as trafficking to late endosomes or lysosomal compartments was not observed during the -min time interval [ ] . in the late endosomes or lysosomes, the viral proteins are degraded to peptide fragments that bind to mhc class ii molecules and presented to the plasma membrane for cd + t-cell activation. besides cd + responses, immune complexes have also been shown to increase cd + responses [ , ] as immune complexes can be shuttled to antigen-processing endosomes to stimulate the mhc class i cross-presentation machinery [ , ] . at present, the precise activating fcgrs involved in mhc class i cross-presentation still remain speculative. besides neutralization in antigen-processing endosomes, antibody-opsonized viruses internalized into the cell may also bind to tripartite motif-containing protein , a cytosolic igg receptor that targets antibody-opsonized viruses for proteasomal degradation via the e ubiquitin ligase activity [ , ] . of interest, this mode of neutralization has been shown to be particularly effective against nonenveloped viruses such as adenovirus [ , ] . the significance of antigen-presentation in the presence of antigen uptake has also been demonstrated in vivo, where protection mediated by antibodies was eliminated in mice with dendritic cells deprived of b- microglobulin, transporter associated with antigen processing (tap) or mhc-ii [ ] . however, igg-mediated uptake of viruses is not always favorable for the host. monocytes and macrophages are important sites of replication for viruses such as denv, ross river virus, influenza, hiv- and epstein-barr virus (ebv). increased internalization of antibody-opsonized viruses via fcgrs may, therefore, result in enhanced infection if these antibodies are not present at sufficient levels required for virus neutralization. this can occur via extrinsic or intrinsic ade [ ] . in extrinsic ade, immune complexes formed between viruses and subneutralizing levels of antibodies can lead to an increased number of infected cells due to increased uptake by fcgr-mediated phagocytosis. besides increased uptake, fcgr-mediated signaling can also increase production of anti-inflammatory cytokines such as il- that acts via the suppressor of cytokine signaling system to suppresses innate immunity, resulting in increased virus replication and output by infected cells [ ] . in addition, some viruses can also directly ligate other inhibitory receptors to suppress innate immunity. for instance, we have recently demonstrated that antibody-opsonized dengue can coligate the inhibitory receptor, leukocyte immunoglobulin-like receptor subfamily b member (lilrb ), to activate the phosphatase shp- that inhibits activation of signal transducer and activator of transcription (stat- ) and subsequent interferonstimulated gene (isg) expression [ ] . lilrb has also been shown to interact with other viral antigens such as human cytomegalovirus glycoprotein ul- [ , ] . although both fcgri and fcgriia can mediate ade of virus infection, fcgriia appears to do so more effectively [ , ] . hence, for pathogens that can replicate efficiently in monocytes and macrophages, it is important to ensure that therapeutic antibodies can neutralize these viruses even in the presence of fcgr-mediated phagocytosis to minimize the risk of ade. fcgr-mediated phagocytosis is affected by the size of immune aggregates, as immune complex size can affect the types of fc-receptors engaged [ ] . aggregation refers to the self-association of a number of molecules to form dimers, oligomers or even submicron to micron-sized complexes, which can be assessed by dynamic light scattering or nanoparticle tracking analysis [ ] . antibody concentration, affinity and stability can influence the size of immune complexes. our previous observations indicate that while small immune complexes coligate activating fcgrs, increased antibody concentrations can result in the formation of large immune aggregates that coligate the lowly expressed inhibitory receptor fcgriib to inhibit phagocytosis (figure ) [ ] . in line with our observations, morefield et al. observed that dendritic cells preferentially phagocytose smaller aggregates of~ mm compared with aggregates greater than mm [ ] . in another study by fifis et al. that used model polystyrene particles, smaller particles of < . um (especially - nm) were delivered more efficiently to dec + dendritic cells, triggered the production of higher antibody titers and promoted better type i cd and cd t cell responses when compared with larger particles [ ] . finally, antibody-mediated protection against cryptococcus neoformans was shown to be protective at optimal doses, but administration of large amounts of antibodies abrogates protection as phagocytic index was shown to decline at higher antibody concentrations [ ] . as such, determining whether the virus immune complexes can be neutralized intracellularly could potentially predict efficacy of the therapeutic antibody. these antibodies should preferably neutralize virus infections at levels that do not form viral aggregates, as this would ensure that virus neutralization occurs in the presence of fcgrmediated entry to result in enhanced antigen presentation and activation of adaptive immune responses. cytokine production triggered by activating fcgr signaling events promotes antiviral responses and activation of dcs, leading to increased antigen uptake and processing. in addition, these cytokines allow recruitment of monocytes and dcs to the site of infection so as to aid in virus clearance [ ] . activating fcgr signaling has been shown to phosphorylate spleen tyrosine kinase (syk) that results in up-regulation and activation of stat- , leading to up-regulation of cytokines and isgs critical for antiviral responses. while the intermediate signaling molecules have yet to be completely elucidated, we and others have shown that the activation of stat- can occur independently of interferon signaling [ , ] . the significance of stat- in immunity has been shown in stat knockout studies, where stat knockout results in significant defects in isgs production and dysregulation of tcell, macrophage and dc differentiation, resulting in t h -biased immune responses that delay viral clearance [ ] . besides stat- , protein kinase c expression and activation have been shown to be important in mhc class ii stimulation and presentation [ ] , a process pivotal for optimal cd + t-cell activation. the strength and nature of immune responses triggered by activating fcgrs, however, are determined by pairing of activating and inhibitory responses. activating fcgrs signal through itam to recruit and activate kinases to trigger immune effector functions. by contrast, inhibitory receptors signal through immunoreceptor tyrosine-based inhibitory motif (itim) to activate phosphatases that can inhibit itam function. for example, immune aggregates can coligate fcgriib, resulting in recruitment of phosphatases such as ship- and shp- that inhibit fcgrmediated phagocytosis [ , ] . as described previously, some viruses can also coligate inhibitory receptors such as lilrb to activate shp- to reduce cytokine and isg production. inhibitory receptors hence control a range of cellular responses mediated by activating fcgr-signaling, which include fc-effector functions such as fcgr-mediated phagocytosis, cytokine production, adcc and cdc. while inhibitory receptor coligation can potentially reduce the therapeutic efficacy of mabs, not all of these effects are undesirable. for instance, intravenous immunoglobulin can signal through fcgriib to induce antiinflammatory responses that can potentially reduce the risk of cytokine release syndrome in mab therapy [ ] [ ] [ ] . given the importance of kinases and phosphatases in mediating innate and adaptive immune responses, determining kinase and phosphatase profiles in fcgr-bearing cells mediated by therapeutic antibodies could hence predict the efficacy of these antibodies in controlling virus infection. although kinases triggered by igg and immune complexes are important for fc-mediated effector functions, overproduction of cytokines (e.g., il- b, tnfa, il- and il- ) due to immune complexes can result in immunogenicity-related adverse events [ ] . for instance, the phase i trial of the anti-cd mab tgn resulted in immune-mediated cytokine storm and multiorgan failure in healthy volunteers, highlighting the importance of assessing the likelihood of such adverse events [ ] . the need to assess the risk of such adverse events is even more crucial for engineered antibodies that have modified fc regions and increased binding to activating fcgrs. while improved fc-fcgr interactions can increase therapeutic efficacy of antibodies, this can also lead to increased risk of adverse events. hence, a cytokine signature encompassing the relevant proinflammatory and anti-inflammatory genes can potentially adcc is critical for clearance of antibody-opsonized cell surface receptor targets. when surface antigens are opsonized with antibodies, fc-receptor interaction with the host immune cells such as nk cells, monocytes, macrophages, neutrophils and mast cells can result in direct lysis of the infected cells, eventually leading to virus clearance. as many of these cell types express both activating and inhibitory fcgrs, fcgriib may hence affect activating fcgr-dependent adcc. among the activating fcgrs, fcgriiia is the most abundant fcgr expressed on nk cells, and adcc signaling is better characterized with fcgriiia relative to other activating fcgrs [ ] . the importance of fcgriiia in mediating adcc has been highlighted by studies showing a lack in clinical efficacy for several antibodies (e.g., infliximab and rituximab) in individuals with different fcgriiia polymorphisms [ ] . these observations were correlated with in vitro adcc analysis where peripheral blood mononuclear cells obtained from fcgriia h/h and/or fcgriiia v/v genotype showed higher adcc activity compared with those of other genotypes with lesser affinity to igg [ ] . previous research has also demonstrated the importance of adcc in viral defense and clearance. for instance, studies have shown both in vivo and in vitro the importance of adcc in controlling influenza, rsv, and hiv- infections in vivo and in human studies [ ] [ ] [ ] [ ] [ ] . complement-mediated activation by antibodies, on the other hand, can cause direct cell lysis by formation of the membrane attack complex (mac). this process, also termed as cdc, is initiated by binding of c q to antibody complexes. this results in the formation of membrane-bound c and c convertases that cleave and activate c and c , respectively. c cleavage by c convertase then forms c a and c b, where c b mediates the formation of membrane attack complex (mac) that can directly cause cdc by forming membrane pores on targeted cells. simultaneously, the cleavage of c results in the formation of c b that can be rapidly degraded into fragments ic b and c dg. deposition of these fragments on the target cells can then bind to cr expressed on leukocytes, leading to phagocytosis and adcc (figure ) [ ] , resulting in the neutralization of viruses. several clinically approved mabs have been shown to mediate their functions by activating complement. for instance, antibodies such as edrecolomab (antiepithelial cell adhesion molecule mouse mab) and alemtuzumab (anti-cd mab) can activate complement and mediate adcc [ , ] . several modifications to igg can directly affect binding to activating fcgrs and subsequent cellular activation. for instance, the igg triple mutant (s a/e a/l a) has been shown to increase binding to fcgriiia and adcc activity [ ] . fc variants (s d/i e and s d/i e/a l mutations) designed computationally possessed increased binding to fcgriiia and reduced binding to fcgriib, resulting in increased effector functions [ ] . the s d/i e/a l mutation, however, led to significant reduction in cdc [ ] . an anti-her antibody engineered to bear multiple substitutions (l v, f l, r p, y l and p l) also showed increased binding to fcgriiia and enhanced killing of her -expressing cancer cells [ ] . finally, given the critical role of fc-linked glycans in fcgr-binding, differences in glycan structure can affect binding and function of therapeutic antibodies. for example, afucosylated antibodies bind fcgriiia with better affinity and have enhanced ability to mediate adcc [ ] . while these modified antibodies can have improved efficacy, it is essential to ensure that they do not cause immunogenicity or adverse events when administered in patients. as such, assays to assess antidrug antibody responses are now a mainstay at most drug regulatory authorities for evaluating therapeutic antibodies during preclinical development. by improving binding to activating fcgrs and enhancing effector functions, fc modifications could benefit patients with fcgr polymorphisms that render them less responsive to therapeutic antibody treatment. enhanced adcc and cdc effector functions could also engage the immune system in a synergistic manner for more potent virus neutralization. the treatment of viral infections using therapeutic antibodies remains a challenging task. besides generating high-affinity antibodies against viruses, therapeutic antibodies should also trigger fc-mediated effector functions that facilitate virus neutralization. these effector functions include increased uptake via fcgr-mediated phagocytosis, increased cytokine production leading to enhanced antigen presentation and antiviral responses, increased adcc and cdc. combinatorial therapeutic strategies that trigger fc-mediated effector functions while occluding the interaction with immune inhibitory receptors could also enhance the clinical efficacy of therapeutic antibodies. finally, for viruses that infect and replicate within fcgr-bearing cells, therapeutic antibodies should mediate virus neutralization even in the presence of fcgr-mediated uptake, thus mitigating the risks of ade. with the increased focus on antibody-based therapeutics, there is a burgeoning need to develop robust methods that can rapidly and accurately assess antibody binding and fc-receptormediated effector functions. routine testing and measurement of fc-mediated effects should hence be considered for characterization of therapeutic antibodies, as this would allow selection and development of therapeutic antibodies with improved efficacy and reduced side effects. in addition, as the antiviral antibody responses are polyclonal and comprise different antibody isotypes, the effects of therapeutic antibodies in the presence of these antibodies should also be tested. given the importance of fc-fcgr interaction in antibodymediated effector functions, fc modification could lead to the development of therapeutic antibodies with improved interaction to activating fcgrs. this could enhance fcgr-mediated uptake, cytokine production, antigen presentation, adcc and cdc. future optimization to improve interaction with fcrn may also increase half-life of therapeutic antibodies, thereby reducing the risk of ade associated with the use of therapeutic antibodies against viruses that infect fcgr-bearing cells. with a growing number of methods to assess fc-mediated effector functions and technologies in antibody modification, we believe that more therapeutic antibodies against viral diseases will be developed in the near future. this will be useful for disease management, especially during epidemics. given the absence of licensed vaccines for many viruses, we believe that therapeutic antibodies will likely be important for treatment of viral infections, particularly during epidemics. we speculate that future advancement in antibody engineering resulting in improved fc-fcgr interaction will continue to aid the development of therapeutic antibodies that possess improved clinical efficacy. better and more robust methods that can predict fc functionality and fc-associated effects clinically will also continue to be essential to select for therapeutic antibodies with good efficacy but with minimal side effects. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. . the lack of effective vaccines against multiple infectious diseases necessitates the development of new therapeutics. . therapeutic antibodies are increasingly used for the treatment of infectious diseases as they are well-established and well-tolerated by humans. . therapeutic antibodies targeted against similar antigenic targets may differ in clinical profile, depending on the type of fcgrs that are engaged. . interaction between antibody fc region and activating fcgrs can trigger fc-mediated effector functions that are essential for virus neutralization. . for viruses that can infect fcgr-bearing cells, therapeutic antibodies should be able neutralize viruses intracellularly to minimize the risk of ade. . cytokine production from activating fcgr signaling is essential for enhanced antigen presentation and antiviral responses. . antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity mediated by therapeutic antibodies can directly kill virus infected cells. . a better understanding of how therapeutic antibodies neutralize virus infections can allow development of modified therapeutic antibodies with improved therapeutic efficacy and reduced side 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antibody tgn human cd as a lysis receptor mediating direct natural killer cell cytotoxicity antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells passively acquired antibody-dependent cellular cytotoxicity (adcc) activity in hiv-infected infants is associated with reduced mortality broadly neutralizing human anti-hiv antibody g is effective in protection against mucosal shiv challenge even at low serum neutralizing titers fc receptor but not complement binding is important in antibody protection against hiv heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in hiv- elite controllers complement function in mab-mediated cancer immunotherapy monoclonal antibodies as therapeutics in oncology new chimeric anti-pancarcinoma monoclonal antibody with superior cytotoxicity-mediating potency high resolution mapping of the binding site on human igg for fc gamma ri, fc gamma rii, fc gamma riii, and fcrn and design of igg variants with improved binding to the fc gamma r engineered antibody fc variants with enhanced effector function anti-tumor activity and toxicokinetics analysis of mgah , an anti-her monoclonal antibody with enhanced fcgamma receptor binding properties lack of fucose on human igg n-linked oligosaccharide improves binding to human fcgamma riii and antibody-dependent cellular toxicity papers of special note have been highlighted as: . of interest .. of considerable interest palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. key: cord- -mn r x authors: hodgins, douglas c.; chattha, kuldeep; vlasova, anastasia; parreño, viviana; corbeil, lynette b.; renukaradhya, gourapura j.; saif, linda j. title: mucosal veterinary vaccines: comparative vaccinology date: - - journal: mucosal immunology doi: . /b - - - - . - sha: doc_id: cord_uid: mn r x infections of mucosal surfaces are major causes of morbidity, mortality, and economic loss in species of veterinary interest, and a concern for animal welfare. vaccines are used extensively in veterinary medicine, and innovative vaccine technologies such as recombinant dna-vectored and distinguishing infected from vaccinated animals (diva) vaccines and automated in ovo vaccination (of embryonated chicken eggs) have been rapidly adopted commercially. immunological research using outbred, nonrodent animal models has contributed to a broader understanding of mucosal defenses, and has provided the initial impetus for investigation of the common mucosal immune system. studies of the potential of novel adjuvants to improve vaccine efficacy against genetically unstable, immune-subverting rna viruses, such as porcine reproductive and respiratory syndrome virus in pigs, should assist in the control of pathogens with similar characteristics in other species. successful development of vaccines to prevent and treat ascending infections of the reproductive tract of cattle set a precedent for applications in other species including humans. studies of mucosal adjuvants and delivery systems continue at the interface between passive and active immunity, with the goal of inducing the earliest possible protection against enteric and respiratory pathogens of neonates. despite advances in nutrition, genetics, housing, and therapeutics, diseases of the respiratory, reproductive, and gastrointestinal tracts of domestic animals and poultry continue to be major causes of morbidity and mortality. although vaccines have been developed and licensed for prevention of many of these diseases, there is a need for improvement in vaccine efficacy and for new vaccines. reasons for low vaccine efficacy include inappropriate, unstable, or outdated antigens in vaccine preparations (e.g., in vitro expressed antigens instead of in vivo expressed antigens), inappropriate immune responses (e.g., systemic instead of mucosal, or th type instead of th type or vise versa), and inappropriate use of otherwise efficacious vaccines (e.g., inappropriate timing of vaccination) (tizard, ). an overview of some of the vaccines currently licensed for vaccination of domesticated animals and wildlife by mucosal routes is provided in table . many of these vaccines consist of pathogens attenuated by traditional methods, but engineered virus-vectored vaccines are now used extensively (by both mucosal and parenteral routes) in veterinary medicine. the majority of current poultry vaccines are attenuated agents delivered in ovo, orally, intranasally (in) or by other mucosal routes, for reasons of ease of administration, economy, and protective efficacy. in comparison, fewer vaccines for domestic mammals are delivered by mucosal routes. management practices for mammals differ from those for poultry; mass vaccination techniques for mucosal delivery have been developed for poultry, but have not been pursued as zealously for mammals. recently however, a number of attenuated live vaccines for in administration have been developed for respiratory tract infections, using traditional methodologies. improved protective efficacy and rapid onset of immunity compared to killed vaccine products have led to widespread acceptance. attenuated live oral vaccines for enteric diseases have also been marketed, but in many cases efficacy has been disappointing due to lack of potency in adults or interference by maternal antibodies in suckling animals. better strategies for induction of immunity in the gastrointestinal tract are needed, especially for neonates in the presence of maternal antibodies. in contrast, effective parenteral vaccines for the most common diseases of the reproductive tract in veterinary species have been available for years, and there has been little motivation to develop mucosal vaccines. many of the diseases of the respiratory and gastrointestinal tracts are most devastating in the neonatal period. for these diseases active immunization may not provide protection before natural exposure to the pathogen. maternal vaccination to enhance passive immunity has been widely used in veterinary medicine, especially for control of enteric diseases. practical difficulties arise, however, with diseases such as parvovirus enteritis in puppies in which a smooth transition must be made from protection by passive maternal antibodies to protection by active immunity, without permitting a window in between of disease susceptibility. this transition is difficult to achieve because induction of active immunity is commonly inhibited by maternal antibodies. various strategies are used to address this problem, but improved vaccines, adjuvants, and antigen delivery systems would improve the reliability of neonatal immunization. although progress is being made in disease prevention in veterinary species, ever changing management practices (e.g., earlier weaning of piglets, larger animal operations) generate new patterns of disease, requiring new control strategies. the emergence of new pathogens (e.g., porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus- ) provides new challenges for vaccine research before some of the older challenges have been met. in this chapter, we focus on mucosal veterinary vaccines and vaccine concepts related to selected pathogens of economic importance. our intent is to highlight progress, to review existing and future vaccination strategies, and to acknowledge the unique contributions of this research to our understanding of mucosal vaccines and immunology. respiratory tract infections are a major cause of morbidity and mortality in farm animals, poultry, and pets. disease conditions are intensified by current management practices such as mixing of recently weaned, stressed beef calves from multiple sources in auction barns. certain disease conditions, such as atrophic rhinitis of pigs, result from the interplay of several pathogens, and multiple agents must be represented in vaccines. some respiratory pathogens such as mycoplasma hyopneumoniae in young pigs are causing new patterns of disease as management practices change (e.g., weaning at an earlier age), requiring changes in vaccine strategies. other pathogens such as prrsv have only recently emerged and improved vaccines await advances in understanding of the agent and of disease pathogenesis. a discussion of respiratory vaccines for even the major pathogens of veterinary species is beyond the scope of the present review. this section will focus on vaccines for prrsv infections in pigs and influenza in horses to illustrate general principles. porcine reproductive and respiratory syndrome (prrs) is a chronic reproductive and respiratory disease of pigs caused by prrsv, a member of the family arteriviridae (benfield et al., ) . signs associated with prrs are anorexia, fever, lethargy, pneumonia, red/blue discoloration of the ears and vulva, delayed return of sows to estrus after weaning, abortion, fetal mummification, stillborn or weak born piglets, and high preweaning mortality (mengeling et al., ) . prrsv is prevalent in swine-producing countries worldwide. annual economic losses to the pork industry in the united states due to prrs have been estimated at $ million (holtkamp and kliebenstein, ) . according to the american animal and plant health inspection service (aphis) report of january , . % of unvaccinated pigs in the united states are seropositive to prrsv. infected pigs excrete prrsv in saliva, nasal secretions, urine, milk, colostrum, and feces at low levels or intermittently, and also in semen of infected boars (rossow et al., ) . in the absence of control measures, prrsv is spread by aerosols, fomites, and personnel. prrsv is divided into two distinct genotypes, type i (european) and type ii (north american). each genotype contains subtypes and strains, which are genetically diverse and vary in virulence and pathogenicity (kim et al., ) . immunity to one genotype of prrsv may provide partial or no protection against reinfection, reflecting the complexity of prrsv genetics and immunological variation among strains (botner et al., ) . swine are the only known species susceptible to prrsv. alveolar macrophages (mΦs) are the primary permissive cells to prrsv; mΦs present in pulmonary intravascular spaces, lymph nodes, thymus, heart, spleen, placenta, and umbilical cord may also be infected by the virus (halbur et al., ) . the most efficient and rapid host response against viruses consists of production of type i interferons (ifns) (ifnα and ifnβ). in prrsv-infected pigs, innate ifnα secretion is significantly suppressed (albina et al., ) and the virus dampens innate nk cell-mediated cytotoxicity as early as day postinfection (renukaradhya et al., ; dwivedi et al., ) . in pigs, a significant correlation has been observed between the prrsv infection and an increased expression of il- , associated with reduced expression of ifnα, ifnγ, il- , and tnfα (gómez-laguna et al., ) . induction of il- is mediated by interaction between prrsv proteins and mΦs/dendritic cells (dcs). the prrsv nucleocapsid protein induces il- production by peripheral blood mononuclear cells (pbmcs) and alveolar mΦs (wongyanin et al., ) , delaying the onset of protective cell-mediated immunity (cmi) to prrsv (dwivedi et al., a . dysregulated immune responses in infected pigs affect viral pathogenesis, disease severity, and susceptibility to secondary microbial infections (thanawongnuwech et al., ; renukaradhya et al., ) . immune responses against prrsv are inadequate to completely clear the virus. viremia disappears in - weeks, but the virus persists in the tonsils and lymphoid tissues for several months (moyron-quiroz et al., ) . prrsv infection in both germ-free and conventionally raised pigs is associated with polyclonal b cell activation, hypergammaglobulinemia, lymphoid adenopathy, renal lesions, and lymphoid hyperplasia (cooper et al., ) . polyclonal lymphoplasia also occurs in mice infected with lactate dehydrogenase-elevating virus, a related virus (grossmann et al., ) . primary antibody responses occur promptly after prrsv infection (loemba et al., ) , but the majority of secreted antibodies are autoantibodies or prrsv nonneutralizing antibodies (lemke et al., ) . the virusneutralizing antibody (na) response is weak, and delayed (mateu et al., ) . both killed and modified live virus (mlv) prrsv vaccines are available commercially for intramuscular or intradermal administration (charerntantanakul, ) . mlv vaccines confer protection against genetically homologous prrsv, but incomplete protection against heterologous viruses (mengeling et al., ) . in both prrsv-infected and mlv-prrsv-vaccinated pigs, virus-specific cell-mediated immune responses are either delayed or dampened . reversion to virulence is a major concern with modified live vaccines. there are numerous reports of reversion of prrsv vaccine virus to virulence and of the presence of reverted vaccine strain prrsv in unvaccinated sows and pigs . vaccine-derived virus has been isolated from fetuses, stillborn, and dead piglets, indicating the spread of disease from vaccinated pigs. identification of mutations in multiple vaccine-derived isolates at identical positions of the viral genome suggests a strong selective pressure on critical viral proteins in field situations. available killed prrsv vaccines fail to protect even against homologous virus, do not elicit na, and induce weak cell-mediated immune responses (bassaganya-riera et al., ) . pregnant sows and gilts and breeding boars are not protected from prrsv due to a lack of safe and protective vaccines. control of prrsv in breeding stock is critical in preventing vertical and horizontal transmission of the virus. early attempts to develop killed prrsv vaccines using recombinant prrsv proteins, plasmids expressing viral genes, and inactivated prrsv administered with or without adjuvants have been unsuccessful ( charerntantanakul, ) . however, recent studies show promise. killed prrsv vaccine coadministered in with cpg oligodeoxynucleotide adjuvant (a tlr- ligand) induced antibodies, virus-specific t cells, and secretion of ifnγ and il- (zhang et al., ) . in another study, prrsv inactivated by uv light or binary ethylenimine induced na and protected pigs against homologous viral challenge. the suppressive effect of live prrsv on ifnα production was lost when the virus was inactivated by uv irradiation (vanhee et al., ). an oral immunization strategy using prrsv nucleocapsid protein genetically fused to cholera toxin (ct) stimulated prrsv antibody responses in the intestines, but no detectable response in vaginal secretions (hyland et al., ) . lack of success in developing protective killed prrsv vaccines may reflect an inability to present killed prrsv antigens effectively to the pig immune system, or (like live prrsv) killed virus may induce immunosuppressive effects. in addition, the antigenic mass used in killed vaccines may be insufficient, suggesting the need for potent adjuvants or novel delivery systems. attempts have been made to improve mlv-prrsv vaccines using adjuvants. in one study, pigs were injected with recombinant porcine il- , il- , il- , or ct within week of intramuscular administration of mlv-prrsv. il- and ct enhanced ifnγ and gp antibody responses, respectively (foss et al., ) . however, use of these adjuvants did not reduce the severity of viremia. unfortunately, prrsv (na) responses were not assayed, and protection against heterologous challenge was not assessed in this study. prrsv gains entry through respiratory and reproductive mucosal surfaces and causes disease primarily at mucosal sites. therefore, a mucosal vaccine against prrsv may be an effective strategy for controlling the disease. until recently, attempts to elicit protective mucosal immunity against prrsv have been unsuccessful, probably due to a lack of appropriate adjuvants. because prrsv infection rapidly subverts the host immune responses, an effective adjuvant must overcome immunosuppression caused by vaccine virus and simultaneously potentiate virus-specific adaptive immunity. a panel of bacterial preparations derived from mycobacterium, vibrio, and streptococcus species, which were potent adjuvants in rodent models, were tested in with mlv-prrsv (bonavida et al., ; barral and brenner, ) . based on mucosal and systemic immune responses, three of the preparations, mycobacterium tuberculosis whole cell lysate (m. tb wcl), ct b subunit, and ok- (a product of streptococcus pyogenes), were selected for viral challenge trials. only m. tb wcl significantly dampened prrsv immunosuppressive effects, and enhanced virus-specific adaptive responses (renukaradhya et al., ; dwivedi et al., b) . historically, heat-killed mycobacteria are recognized to have as potent adjuvant effects as components of freund's complete adjuvant (fca) and have been used extensively in experimental animals. the use of fca in humans and food animals is unacceptable due to severe granulomatous inflammatory reactions to toxic cell wall components ( bekierkunst, ). however, a nontoxic water-soluble purified fraction of m. tb wcl (werner et al., ) has adjuvant properties in rodents, guinea pigs, and rabbits. pigs inoculated in with m. tb wcl have no detectable signs of toxicity locally or systemically (dwivedi et al., a,b) . a recombinant vaccine (prrsv gp and m expressed in bacillus calmette-guerin (bcg)) is reported to reduce clinical signs of prrs with decreased viremia and viral load in the tissues (bastos et al., ) . cross-protective immunity against prrsv has been evaluated in pigs receiving mlv-prrsv and m. tb wcl in, by challenging with virulent heterologous prrsv virus (kim et al., ) . reduced clinical disease, viremia, and virus-mediated immunosuppression were noted. in addition, secretion of ifnγ and il- by lung and blood lymphocytes in response to prrsv m and nucleocapsid proteins was enhanced (dwivedi et al., a) . guillonneau et al. ( ) reported similar findings in mice vaccinated in with adjuvanted influenza vaccines. enhanced virus-specific cytotoxic t cell and memory responses to internal viral proteins were noted, as well as cross-protective immunity. studies of passive protection of pigs by prrsv na have indicated that modest na titers (≤ / ) protect pregnant sows against reproductive failure, block placental transmission of infection, prevent viremia in piglets, and provide sterilizing immunity (osorio et al., ) . pigs immunized in with mlv-prrsv alone or with m. tb wcl developed na titers < / and / , respectively, postimmunization (dwivedi et al., b) . in pigs immunized (mlv-prrsv + m. tb wcl) and challenged with prrsv mn , an na titer > / persisted until pid (dwivedi et al., a) . relatively low numbers of circulating virus-specific ifnγ secreting cells have been reported for pigs vaccinated in with mlv-prrsv without adjuvant, ( - cells per million pbmcs) (dwivedi et al., a) . in contrast, meier et al. ( ) have reported - ifnγ secreting cells per million pbmcs in pigs vaccinated against aujeszky's disease virus. pigs immunized in with mlv-prrsv with m. tb wcl and challenged with prrsv had greater than ifnγ secreting cells per million pbmcs, and more than a twofold higher frequency of cd + cd + t cells (dwivedi et al., a) . the literature concerning mucosal immune responses in the pig respiratory tract is limited compared to studies of rodents and humans. recent research highlights the advantages of activating the mucosal immune system using vaccines delivered with potent adjuvants. induction of adequate immune responses in the respiratory and reproductive tract will be essential for control of prrs in swine. in-delivered, potent mucosal vaccines generate better cross-protective immunity against genetically variable prrsv field viruses. efforts to control prrs outbreaks using conventional parenterally delivered vaccines have had limited success; development of an alternative approach of generating immunity in the respiratory tract should be a priority. in particular, mucosal adjuvants based on components of mycobacterial species show promise. respiratory tract disease affects virtually every aspect of equine husbandry, including working, pleasure, and race horses. considerable efforts are expended to prevent epizootics of respiratory disease in stables, fairs, shows, and race tracks. equine influenza virus will be discussed in detail as an example of past, current, and future vaccine approaches. equine influenza virus causes epizootics of upper and lower respiratory tract disease almost worldwide. until equine influenza was excluded from the continent of australia through import restrictions and quarantine. in august of that year, importation of an infected horse led to an explosive epizootic of equine influenza with an estimated , horses infected over a -month period (callinan, ) , demonstrating the potential of the virus to spread in a naïve, unvaccinated population. infection can occur in horses of all ages, but epizootics often involve younger animals (van maanen and cullinane, ) . clinical signs include high fever, a persistent dry cough, nasal discharge, anorexia, and depression. secondary bacterial pneumonia may complicate the clinical picture. equine influenza viruses are classified as type a influenza. antigenic differences in the hemagglutinin (h) and neuraminidase (n) glycoproteins define the two recognized subtypes, a/equine/ (h n ) and a/equine/ (h n ) (wilson, ) . two lineages of a/equine/ , american and european, have been identified; multiple virus strains are included in vaccines since protection against heterologous strains is incomplete (yates and mumford, ) . antigenic drift is sufficient to require regular reappraisal of strains included in vaccines (mumford and wood, ) . natural infection induces iga antibodies in nasal secretions, igga and iggb antibodies in serum nelson et al., ) , and circulating cytotoxic t lymphocytes . protection against reinfection persists for at least a year (hannant et al., ) . vaccination with inactivated virus vaccines induces serum igg (t) antibodies without detectable iga in nasal secretions (nelson et al., ) and without cytotoxic t cell activity (van maanen and cullinane, ) . two or three doses of vaccine are typically administered in the primary series, with booster doses at least once a year thereafter. more frequent vaccination is advised for horses at high risk of infection (wilson, ) . protection is typically incomplete and of limited duration. improved adjuvants can enhance the level and duration of antibody responses to inactivated virus vaccines (mumford et al., c) . suppressive effects of maternal antibodies on responses to inactivated vaccines have led to recommendations not to vaccinate foals before months of age (van oirschot et al., ) . a subunit equine influenza vaccine based on the immune stimulating complex (iscom) adjuvant technology has been licensed and marketed in europe since (newmark, ) . antibody responses to iscom-based vaccines typically are of higher titer and are more persistent than for conventional inactivated vaccines (mumford et al., a) . protection has been demonstrated against experimental challenge, months after a three dose vaccination series (mumford et al., b) . protection may be due in part to the ability of iscom-adjuvanted vaccines to induce cytotoxic t lymphocytes (morein et al., ) . although iscom-based vaccines can induce iga antibody responses following in administration (hu et al., ) , the commercial influenza iscom vaccine is administered parenterally. in administration of inactivated equine influenza virus with ct b has been reported to induce local iga antibodies, and protection against experimental challenge (hannant et al., ) . a cold-adapted, temperature-sensitive live vaccine for in administration is available commercially . protection against experimental challenge has been demonstrated months after a single vaccination . this is a notable improvement in efficacy and practicality over conventional killed vaccines. experimental plasmid vaccines encoding the hemagglutinin gene of equine influenza have been examined in horses. three doses of plasmid administered to the skin and mucosal sites (tongue, conjunctiva, and third eyelid) induced protection against clinical disease and partial protection against viral shedding. protection against clinical disease was reduced if plasmid was administered only to the skin (lunn et al., ) . a canarypox-vectored equine influenza vaccine, expressing hemagglutinins from two strains of h n equine influenza, has been available commercially (intramuscular administration) since (toulemonde et al., ) . in the australian epizootic of , this vaccine was used extensively during the government program to eradicate equine influenza. the vectored vaccine was chosen for this program because of its diva (distinguishing infected from vaccinated animals) characteristics ( kirkland and delbridge, ) . infected horses mount antibody responses to the viral nucleoprotein in addition to the viral hemagglutinin, whereas vaccinated horses respond only to the hemagglutinins expressed by the vaccine vector. a recent report indicates that adequate antibody responses are generated with as little as days between primary and secondary vaccination (el-hage et al., ) . protection against experimental challenge has also been noted weeks after a single dose of vaccine in ponies ( toulemonde et al., ) . for some respiratory pathogens (e.g., m. hyopneumoniae in pigs) the critical antigens associated with protective immune responses have not been identified. for other pathogens (e.g., prrsv) there is also a need to identify the appropriate type of immune response (th or th ) needed for protection. for some complex disease conditions (e.g., bovine respiratory disease complex) there is continuing uncertainty whether all of the relevant contributing pathogens have been identified. although many parenteral vaccines are efficacious in reducing lower respiratory tract disease, there is a need to investigate whether induction of mucosal immunity in the upper airways, in combination with systemic immunity, can further reduce infection rates, transmission of pathogens, and economic losses. finally, there is a need to devise and implement changes in management procedures to reduce disease exposure (by nonimmunological methods), and to optimize immune interventions by improved timing of vaccinations. vaccines to prevent reproductive tract disease have received much emphasis in veterinary medicine. this is especially true of food-producing animals because reproductive failure is a major economic problem. although vaccines to prevent reproduction are of interest for abandoned pets or deer in areas of overpopulation, this section will deal only with vaccines designed to prevent infectious disease of the reproductive mucosa. infections causing adverse pregnancy outcome can be classified by route of infection: hematogenous or ascending. several hematogenous infections have a predilection for the gravid uterus resulting in early to late abortions . these include leptospirosis, chlamydial infection, and brucellosis in several animal species, histophilus somni infection in cattle and sheep and neospora caninum in cattle. although vaccines are available for several of these infections, the vaccine for brucellosis has been available since the s and is the most well known. several brucella species cause abortion or epididymitis/orchitis in the primary hosts ( brucella abortus in cattle, brucella suis in swine, brucella melitensis in goats, brucella ovis in sheep, brucella canis in dogs, and brucella marinum (or brucella delphini) in marine mammals). infection may be acquired via the gut mucosa or the conjunctiva/upper respiratory mucosa and localizes in the reticuloendothelial system and endometrium/placenta by systemic spread. thus, systemic vaccines are effective. a modified live b. abortus vaccine, along with a "test and slaughter" eradication program, has been successful in controlling bovine brucellosis in north america. the modified live vaccine (b. abortus strain ) is very effective in stimulating cmi that is critical for protection against this facultative intracellular pathogen. strain now has been largely replaced by a new attenuated strain, rb , which does not stimulate an antibody response known to interfere with diagnostic serologic assays. there is considerable information on mechanisms of systemic immunity to brucellosis. neospora caninum also causes abortion by a hematogenous route and a vaccine is available (weston et al., ) . histophilus somni can infect either by the hematogenous route or by an ascending genital route to cause abortion or infertility and vaccines are available. since the focus of this volume is mucosal immunity, no more will be said concerning protection against hematogenous infections of the genital tract. ascending local infections of the reproductive tract are usually transmitted sexually. the two best examples of vaccines for sexually transmitted infections of animals are campylobacter fetus subsp. venerealis (formerly vibrio fetus subsp. venerealis) and tritrichomonas foetus. both are host-specific bovine sexually transmitted diseases (stds) that only infect the reproductive mucosa. both are extracellular pathogens that do not invade the mucosa of the reproductive tract but may be found in the placenta and fetus. the localized nature of these infections and transmission limited to coitus suggest that mucosal immunity must be important. because a vaccine has been available for c. fetus subsp. venerealis for several decades and its use has controlled the disease in developed countries, that vaccine will be discussed first. vibriosis (or campylobacteriosis) is a chronic bacterial genital infection with no overt clinical signs other than reproductive failure (corbeil et al., ) . after months of infection, the uterus is cleared first, followed by the vagina. convalescent immunity is partially protective for a limited time. antibody is effective in protection against this extracellular pathogen as demonstrated by systemic passive immunization (berg et al., ) . the antibody response to infection is primarily iga in the vagina and igg in the uterus (corbeil et al., ) . systemic immunization with a whole cell vaccine results in both igg and igg antibody responses to surface antigen in serum, and uterine and vaginal secretions (corbeil et al., ) . this response prevents infection and can rapidly clear infected cows (corbeil and winter, ) . that is, the vaccine can be used prophylactically and even therapeutically. immunization is efficacious even though surface antigenic variation occurs in the face of a local immune response (corbeil and winter, ) . presumably, immune clearance occurs when the dynamic interaction between protection and evasion is shifted in the favor of the host. this appears to occur earlier when the response is primarily igg than when iga predominates (corbeil et al., ) . this may be related to the ability of the igg antibodies to mediate opsonization and intracellular killing of the bacterium, an ability which iga antibodies lack (corbeil and winter, ) . although this work was done many years ago, it sets a precedent for systemic immunization for prophylaxis and therapy of reproductive mucosal infections. trichomoniasis is a similar chronic genital mucosal infection of cattle. it is caused by the protozoan, t. foetus, and results in pregnancy loss. trichomonas vaginalis causes a human std also associated with adverse pregnancy outcome. thus, bovine trichomoniasis serves as a model for immune prevention of both bovine and human reproductive mucosal infections. because of the economic significance of bovine trichomoniasis and because no chemotherapy is approved, investigations have focused on immunoprophylaxis and immunotherapy. t. foetus colonizes the vaginal and uterine or preputial surfaces for months, like c. fetus subsp. venerealis infection. in fact, mature bulls are often infected for life whereas young bulls may clear the infection with time (cobo et al., ) . this is probably related to innate immunity. trichomonads are anaerobic parasites and are found deep in uterine glands and epithelial crypts of the penis and prepuce (rhyan et al., ) where the oxygen tension is probably lowest. older bulls have deeper epithelial crypts, which may have lower oxygen tension. in order to understand protective acquired immune responses, monoclonal antibodies (mabs) with putative protective functions were chosen for immunoaffinity purification of a highly glycosylated surface antigen (bondurant et al., ; corbeil et al., ) . analysis of many isolates of t. foetus indicated that two mabs recognized different epitopes of the same antigen, which was conserved in all isolates tested. this glycosylated surface antigen was later shown to be a lipophosphoglycan (lpg)/protein complex. systemic immunization with the immunoaffinity-purified lpg-containing surface antigen, followed by intravaginal challenge with t. foetus, resulted in significantly earlier clearance of the parasite from vaccinated animals than from controls (bondurant et al., ; corbeil et al., ) . more importantly, clearance of immunized animals most often occurred before weeks of infection. parsonson et al. ( ) showed that significant inflammation accompanied by reproductive failure did not occur until after weeks of infection, so the vaccine should protect against fetal loss. analysis of vaccine-induced antibody responses demonstrated predominantly igg responses in the serum and iga plus igg antibodies in secretions (corbeil et al., , . ige antibodies were also increased during infection. as ige antibodies increased, mast cells degranulated and clearance of t. foetus occurred. these studies raised several questions. first, why is the systemic response to the lpg/protein complex skewed toward igg (a th response in cattle) and not igg (a th response)? this question is under investigation. second, are igg or iga antibodies to the lpg-containing antigen most protective and how can that ig isotype be enriched to enhance protection? to address the latter questions, preliminary studies were done in mice to determine the best routes and adjuvants to enrich for igg or iga in genital secretions . subcutaneous priming with the immunoaffinity-purified lpg-containing surface antigen (called tf . antigen) in quil a adjuvant and subcutaneous boosting with whole cells enriched for igg anti-tf . antibodies in genital secretions whereas subcutaneous priming and intravaginal boosting greatly enriched for iga antibodies in genital secretions. when cattle immunized by these two methods were challenged intravaginally with t. foetus, those with predominantly iga or predominantly igg anti-tf . antibodies in genital secretions were equally protected. later studies with similar in immunizations showed that stimulation of the common mucosal immune system gave results similar to those of intravaginal immunization . this raised the question of inductive sites for local immune responses in the genital tract. others have suggested that the genital tract is not an inductive site because m cells and mucosa-associated lymphoid tissue (malt) are not present. this is true of cattle as well as mice and people. however, even though control cows did not have histologically demonstrable malt in the uterus and vagina, cows experimentally infected with t. foetus did . similar lymphoid nodules and follicles under a modified epithelium were detected in preputial and penial surfaces of bulls infected with t. foetus (rhyan et al., ) . immunostaining of parallel sections with mab to tf . antigen indicated uptake of antigen by epithelial cells and large mΦ or dc under the basement membrane near the lymphoid follicles (rhyan et al., ) . similar antigen uptake has been detected in the infected female uterine and vaginal mucosa (corbeil et al., ) . thus, even though the parasite is noninvasive, released tf . antigen appears to be taken up by epithelial cells. rat uterine epithelial cells can present antigen to t helper cells (wira and rossol, ) . also, mΦ/ dc positive for antigen should be antigen-presenting cells (apcs). detection of igg and iga antibodies in genital secretions of infected bulls (cobo et al., ; rhyan et al., ) and cows and the histologic demonstration of follicles and putative apcs suggests that inductive sites in the genital tract are formed in response to antigenic stimulation. like c. fetus subsp. venerealis, t. foetus has mechanisms for evasion of immune responses. these include coating of the surface with ig nonspecifically , epitope variation (ikeda et al., ) , and cleavage of igg , igg , and complement component by extracellular cysteine proteinase (talbot et al., ; kania et al., ) . studies with cysteine proteinase inhibitors demonstrated decreased cytotoxicity of t. foetus for bovine trophoblast cells and decreased infectivity in a mouse model, confirming the likely role of cysteine proteinases in pathogenesis (cobo et al., ) . even with these mechanisms for immune evasion, as with c. fetus, it is clear that the dynamic interaction between host and parasite can be made to favor the host by systemic or local immunization. the usefulness of whole cell vaccines in preventing t. foetus infection and reproductive failure in cows has been demonstrated in clinical trials (kvasnicka et al., ) . first-generation whole cell vaccines are now commercially available for prevention of trichomoniasis in cows. earlier, clark et al. ( ) demonstrated efficacious immunization of bulls with whole t. foetus cells or crude membrane glycoproteins that probably contained tf . antigen. this study indicated that vaccination of bulls could both prevent infection and clear already established infection. so vaccines for bovine trichomoniasis are both immunoprophylactic and immunotherapeutic. recent studies showed that vaccination of bulls with a commercially available whole cell killed t. foetus vaccine in oil adjuvant resulted in protection against challenge and high levels of igg antibodies in serum and preputial secretions (cobo et al., ) . igg antibodies to whole cell t. foetus antigens predominated but fairly high levels of igg antibodies were also detected. the above studies with c. fetus subsp. venerealis and t. foetus show that: . stds can be prevented or even cured by systemic vaccination of both males and females. . at least for one std, igg and iga of the same antigenic specificity are equally protective at the mucosal surface. either systemic immunization or mucosal (in or intravaginal) immunization with systemic boosting is protective. . inductive sites are formed in the mucosa of infected male and female genital tracts even with noninvasive pathogens. . strong and appropriate immune responses will clear microbial infection from the genital tract even when the microbe has multiple immune evasive mechanisms. . the fact that protection against two stds has been demonstrated in the natural outbred host (cattle) has advantages over murine models of std vaccines. in the latter, the human pathogen is usually inoculated into the unnatural murine host and the disease does not mimic the human infection. furthermore, although inbred mice provide a homogeneous experimental animal, they do not represent the variation in immune responses seen in the outbred human population. the work on bovine vibriosis and trichomoniasis demonstrates protection under field conditions for two stds that cause adverse pregnancy outcome in an outbred host. this is an encouraging precedent for control of human stds and related adverse pregnancy outcomes. future needs include identification of the protective antigens for most stds. for antibody-mediated protection of the genital mucosa, several questions have not yet been addressed. as far as we know, the role of ige in the genital tract is largely unstudied even though it does seem to be involved in clearance of trichomonads from the bovine genital tract. lastly, manipulating genital immune responses to enhance th or th type responses is an unexplored research area. enteric disease is a major cause of mortality and morbidity in animals. agents causing diarrhea in animals include viruses (e.g., adenoviruses, pestiviruses, caliciviruses, coronaviruses, parvoviruses, rotaviruses, toroviruses), bacteria (e.g., campylobacter spp., clostridium spp., diarrheagenic escherichia coli, salmonella spp., yersinia spp.), and parasites (e.g., coccidia spp., cryptosporidium parvum). these infections occur most commonly in suckling animals or in poultry under weeks of age, but may also be common postweaning and in susceptible seronegative or stressed adult animals (saif and jackwood, ) . induction of local secretory iga (s-iga) antibodies (prevent attachment, invasion, and neutralize toxins or infectious agent) and mucosal cellular immune responses (against intracellular bacteria and viruses) by vaccines are essential for protection from enteric pathogens. peyer's patches (pp) and mesenteric lymph nodes are two important organized gut-associated lymphoid tissues (galt) in domestic animals and serve as the induction site for gut immune responses. ileal pp in some domestic animals (sheep, cattle, pigs, dogs) differ from jejunal pp, serving as a primary lymphoid organ similar to the bursa of fabricius in chickens, unlike in humans and rodents where pp are secondary lymphoid organs (chu and liu, ; yasuda et al., ) . cryptopatches or clusters of lymphoid cells in the basal lamina propria occur in mice, but are absent in pigs (pabst et al., ) . mucosal lymphoid tissues and lymphoid cells in domestic animals and humans differ in toll-like receptor (tlr) expression and function (after binding their ligand) when compared to mice (iwasaki and medzhitov, ; tohno et al., ; guzylack-piriou et al., ) . these differences in immune components suggest that vaccine studies carried out in mice may fail to translate to domestic animals or humans. enteric pathogens have different characteristics related to their intestinal tropism and replication, requiring different vaccination strategies. enteric viruses have predilections for replication in distinct vertical and longitudinal regions of the small or large intestines. cytolytic infection of enterocytes leads to varying degrees of villous loss and fusion, reduced small intestinal absorptive capacity, and a malabsorptive, maldigestive diarrhea (saif, a) . secretory diarrhea is also induced by some viruses such as rotavirus (rv), which involves the viral enterotoxin, nonstructural protein (nsp ), and/or stimulation of the enteric nervous system (ball et al., ; lundgren et al., ) . thus, viral diarrheas can be of variable severity and act via multiple mechanisms that differ from those of enteric bacteria, most of which cause secretory diarrhea mediated by enterotoxins (fairbrother and gyles, ) . enteropathogenic viruses can be divided into three types (types i, ii, and iii) according to their preferred site of replication in the intestine (reviewed by saif ( a) ). type i (transmissible gastroenteritis virus (tgev) and rv) and ii virus infections can be prevented by inducing local intestinal immunity, whereas type iii viruses (parvovirus), which infect crypt enterocytes basolaterally, can be prevented by inducing systemic immunity. the enteropathogenicity of bacteria is determined by their virulence factors including adhesion factors (fimbriae or pili) and enterotoxins; therefore, bacterial vaccines generally need to prevent attachment and toxin action within the intestine (fairbrother and gyles, ) . in the following sections, we will review vaccine strategies for these different types of enteric infections. tgev and rv vaccines in pigs will be reviewed to illustrate findings using domestic outbred animals instead of inbred laboratory rodent models. neonates can be protected from enteric infections by providing passive lactogenic immunity, which can be achieved by immunizing mothers preparturition. pioneering work done by bohl and saif in the early s with live oral virulent tgev in pigs was the foundation for the gut-mammary gland-s-iga immunologic axis, which later became the basis for the concept of the common mucosal immune system. their studies showed that in tgev seronegative sows, only oral immunization with virulent tgev induced high rates of protection in suckling neonates, which was associated with high titers of s-iga antibodies in colostrum and milk, whereas systemic immunization induced mainly igg antibodies saif et al., ) . rotavirus is a major cause of dehydrating diarrhea in young livestock, infants, and poultry (saif and fernandez, ) . multiple rv serogroups, based on common inner capsid vp antigens (a-h), and multiple g (vp , glycoprotein) and p (vp , protease-sensitive) serotypes (based on neutralizing epitopes) or genotypes (based on sequence analysis) for group a rvs have been detected in humans, sheep, swine, cattle, horses, dogs, cats, poultry, and wildlife (estes and kapikian, ; martella et al., ) . among the distinct rv serogroups and g/p serotypes/genotypes, cross-protection is limited. the antigenic divergence among different sero/genotypes of rvs presents a challenge for the design of vaccines that are capable of inducing heterotypic protection. commercial modified live and killed rv vaccines for rv diarrhea in livestock and poultry are limited to group a rvs (saif and fernandez, ) . mebus et al. ( ) developed the first oral rv vaccine for calves in ( year prior to the discovery of human rv) using a cell cultureadapted neonatal calf diarrhea rv strain. although a significant reduction in morbidity and mortality was observed in a field trial among vaccinated calves, in the majority of herds (compared to previous years), subsequent field studies revealed variable efficacy. experimental studies suggested that maternal antibodies interfered with live oral vaccine replication and suppressed development of active immunity (saif and fernandez, ) . the neonatal gnotobiotic pig model has been used to investigate immune responses to rv vaccines and infection for nearly three decades (saif et al., , yuan and saif, ; gonzalez et al., ) . gnotobiotic pigs are free of maternal antibodies (placental transfer of igs does not occur in swine), but they are immunocompetent at birth. they are maintained aseptically and are free of exposure to extraneous wildtype rvs, assuring that exposure to a single pathogen can be analyzed. initial studies were conducted to mimic natural rv infection (bohl et al., ) and to study immune correlates of protection gonzalez et al., ; yuan et al., ) . understanding the sequence and kinetics of immune responses stimulated by virulent rvs allows for the determination of markers of protective immunity and pathogenicity, which can then be used to design vaccines that will stimulate protective immunity without inducing pathology. gnotobiotic pigs orally inoculated with virulent porcine or human rvs were completely protected from homotypic but not heterotypic (distinct g/p types) rv challenge (hoshino et al., ; saif et al., ) . significant correlations were observed between the protection to rv-induced diarrhea and shedding and the following immune parameters: the number of intestinal iga rv antibody-secreting cells (ascs), serum and intestinal iga rv antibody titers, and the frequency of intestinal rv-specific ifnγ producing cd + and cd + t cells yuan et al., ; to et al., ; yuan and saif, ; yuan et al., ) . these immune parameters were significantly higher in virulent rv inoculated pigs than in those inoculated with attenuated or inactivated rv (ward et al., b) . pigs inoculated with two or three doses of attenuated rv were moderately protected against diarrhea ( %- doses, %- doses) and virus shedding ( %- doses and %- doses) after homotypic challenge, suggesting a need for multiple doses and suitable mucosal adjuvants to enhance the efficacy of rv vaccines yuan et al., yuan et al., , yuan and saif, ) . gnotobiotic pigs inoculated with virulent rv developed an acute proinflammatory serum cytokine profile (il- , tnfα) coinciding with peak diarrhea and viremia, followed immediately by increased th (il- , ifnγ) cytokines and convalescent th (il ) and tr (il- ) cytokine responses. gnotobiotic pigs inoculated with one dose of attenuated rv showed lower early ifnγ and proinflammatory cytokine responses compared to virulent rv inoculated pigs. both attenuated and virulent rv inoculated pigs developed th /th /tr cytokine-secreting cell (csc) responses at - weeks postinoculation; however, attenuated rv inoculated pigs developed lower intestinal ifnγ and higher intestinal and splenic il- cscs compared to virulent hrv inoculated pigs (azevedo et al., ) . virulent rv inoculated pigs had significantly higher protection rates against rv challenge ( % and % against diarrhea and shedding, respectively) compared to one dose of attenuated rv inoculated pigs ( % and % against diarrhea and shedding, respectively) (azevedo et al., ) . these findings suggest that higher protection rates are associated with early proinflammatory and th cytokine responses, which promote cytotoxic t cell activity and viral clearance, and later th induced cytokines, which are important for protective s-iga antibody responses. thus, protection against rv in pigs requires balanced th /th /treg responses (azevedo et al., ; gonzalez et al., ) . infection of gnotobiotic pigs with virulent rv causes early recruitment of innate apcs (monocytes/mΦ and dcs) and γδ t cells into the ileum, and enhanced tlr , tlr , and tlr expression among apcs in spleen (zhang et al., c; wen et al., wen et al., , . in virulent rv-infected pigs, plasmacytoid dcs are major producers of serum ifnα and, along with other innate immune cells (γδ t cells and apcs) and cytokines (tnfα, ifnγ and il- ), are important for controlling early rv viral replication and subsequent development of protective adaptive immune responses . development of attenuated rv vaccines with mucosal adjuvants that mimic immune responses to virulent rv may improve existing vaccines. immunogenicity and protective efficacy of rv vaccine formulations (attenuated replicating virus, inactivated virus, and recombinant baculovirus-expressed virus-like particles (vlp)), administration routes, and adjuvants have also been evaluated using the gnotobiotic pig model (bohl et al., ; yuan and saif, ; gonzalez et al., ) . inactivated oral or intramuscular rv vaccines failed to protect against virulent rv challenge, despite high igg antibody responses induced in serum and systemic lymphoid tissues. serum igg antibodies did not correlate with protection. however, a recent study showed that an inactivated reassortant rv strain (cdc- strain) formulated with aluminum phosphate and administered systemically in gnotobiotic pigs resulted in induction of serum igg antibody titers, coinciding with partial protection against shedding and diarrhea, suggesting that adjuvant may have stimulated local specific (gut iga antibodies) or nonspecific immune responses, which were not assessed in this study (wang et al., ) . rotavirus subunit vaccines consisting of double-layered vlp composed of rv inner capsid proteins vp and vp ( / -vlps) administered in or orally with mutant heat-labile toxin of e. coli (mlt) or iscoms as adjuvants (yuan and saif, ) induced igg asc responses in systemic lymphoid tissues and low or no iga asc responses in intestinal lymphoid tissues and also failed to mediate protection, contrary to results in adult mouse studies (yuan and saif, ) . the failure of in or oral / -vlp vaccines suggests that protective immunity to rv diarrhea in neonatal pigs requires mainly the presence of systemic or intestinal iga antibodies to the outer capsid rv proteins, vp and vp , each of which induces na. however, when / -vlps adjuvanted with mlt or iscom were used as in or oral booster doses in pigs orally primed with attenuated rv, the protective efficacy increased significantly. an oral attenuated rv prime and in / -vlp-iscom boost regimen (attrv/ / -vlp) induced the highest numbers of intestinal iga ascs and serum and intestinal iga antibody titers, and protection rates were similar to or higher than those induced by three oral doses of attenuated rv (gonzalez et al., azevedo et al., ) . interestingly, priming with two doses of / -vlp (in or oral) followed by live attenuated rv was ineffective for inducing iga antibodies or protection. thus the use of a replicating vaccine to prime at one inductive site (galt) followed by boosting with a nonreplicating vaccine at a second mucosal inductive site (nasal-associated lymphoid tissue, nalt) is an effective strategy for stimulating protective mucosal immune responses, which can be applied to other enteric viral vaccines. furthermore, efficacy of the prime/boost strategy (replicating/nonreplicating vaccine, attrv/ / -vlp) was examined in the presence of high and low titers of passive antibodies to mimic neonatal pigs receiving maternal antibodies (nguyen et al., a,b) . similar to attrv/ / -vlp prime/boost vaccine studies, plasmid dna containing vp was used as a booster subsequent to oral attenuated rv vaccine priming. this regimen showed high protection against shedding, but poor efficacy against rv diarrhea (yuan et al., ) . collectively these findings suggest that mucosal boosters are effective in enhancing iga antibody titers to rvs in orally primed animals . studies have shown that immunogenicity and efficacy of mucosal vaccines can be improved by the use of appropriate strains of probiotics that modulate mucosal and systemic immune responses, by interaction with epithelial cells and the underlying intestinal immune cells (fukushima et al., ; sanz and de palma, ) . certain probiotic strains are reported to enhance immune responses to rv vaccines in children; others reduce rv diarrhea severity, but the mechanisms are not well defined (fang et al., ; holscher et al., ) . supplementation with lactobacillus acidophilus in attenuated rv vaccinated pigs is reported to enhance intestinal ifnγ-producing cd + t cells, intestinal iga and igg rv ascs, and serum iga and igg rv antibody titers (zhang et al., b) . these findings suggest that probiotics are an alternative, cheap, and safe adjuvant for enhancing efficacy of oral attenuated rv vaccines in animals and potentially in humans. colonization of pigs by two different strains of lactic acid bacteria (lab, l. acidophilus and lactobacillus reuteri) and subsequent virulent rv infection resulted in higher and balanced th /th /treg cytokine responses (il- , ifnγ, il- , il- , tgfβ), higher total intestinal iga-secreting cells, total serum igm, and intestinal igm and igg titers, although the numbers of iga rv ascs and serum and intestinal rv antibody titers did not differ compared to virulent rv-only infected pigs. no overall difference in protection rates was noted when compared to virulent rv-only inoculated pigs, which was likely because of the short interval (only days) between lab colonization and virulent rv challenge (zhang et al., a; azevedo et al., ) . dual colonization of the aforementioned lab strains also modulated innate immune components in virulent rv inoculated pigs as follows: ( ) enhanced frequency of γδ t cells in the intestine and their distribution; ( ) enhanced tlr and tlr -expressing cd + apc, and tlr -expressing cd − apcs in the blood, but reduced tlr -and tlr expressing cd − apcs in the spleen, and ( ) reduced frequency of mΦ and cdcs in the spleen. collectively, these findings suggest that probiotics may reduce systemic inflammatory responses induced by virulent rv. effects on tlr expression on apcs in the ileum were not determined (zhang et al., c; wen et al., wen et al., , . probiotics may not only modulate immune responses to rvs or other enteric vaccines, but may also directly ameliorate diarrhea/infection by enhancing gut barrier integrity and maintaining intestinal permeability, by stimulating nonspecific immune responses, by changing gut microbial populations, and by aiding in the regulation and prevention of apoptosis ( madsen et al., ; yan and polk, ; preidis et al., ) . using the tgev model for evaluation of active protection against diarrhea in pigs, researchers also delineated compartmentalization in the common mucosal immune system and its impact on mucosal vaccine strategies and protection (vancott et al., (vancott et al., , . the natural occurrence of a deletion mutant of tgev with exclusive respiratory tropism, referred to as porcine respiratory coronavirus (prcv), provided a unique opportunity to study asc responses and protective immunity to two antigenically related porcine coronaviruses with enteric (tgev) versus respiratory (prcv) tropism. oral immunization of pigs with tgev induced high numbers of iga asc in the intestine and provided complete protection against tgev challenge, whereas in immunization of pigs with prcv induced mainly systemic responses (igg asc) and provided only partial protection against tgev challenge. thus, the in prcv alone failed to elicit sufficient intestinal iga asc to provide full protection against the enteric pathogen, tgev. findings from this study and rv vaccine studies suggest that the use of multiple mucosal inductive sites in a prime/boost vaccination regimen may be an effective approach for overcoming compartmentalization in the common mucosal immune system. canine parvovirus (cpv) infects crypt enterocytes causing hemorrhagic gastroenteritis in pups (saif and jackwood, ) . because cpv is likely disseminated to the basolateral surface of crypts by the hematogenous route, serum na (derived maternally or actively produced) is protective against the disease. pollock and carmichael ( ) demonstrated that pups with hemagglutination inhibition (hi) serum antibody titers of > : were immune to oronasal cpv type challenge. cpv is highly stable in the environment and pups were susceptible to infection as soon as maternal antibodies declined to hi titers of : - : . a maternal hi antibody titer as low as : severely affected the efficacy of a live low titer cpv vaccine ( - th passage in culture) in generating active immune responses (carmichael et al., ) , which resulted in a window of susceptibility for pups. others have shown that an hi titer of less than : in cpv- (low passage, high titer) vaccinated pups did not severely affect active antibody responses (burtonboy et al., ; hoare et al., ) , suggesting that increasing the dose or reducing the attenuation of the virus may help to overcome inhibitory effects of maternal antibodies. studies with modified live variant cpv- b strain (low titer) have shown that these vaccines, when given either parenterally ( th passage) or in ( th tissue culture passage), elicited almost % protection in pups with maternal antibody titers of : to : and even % protection in pups with antibody titers of : . higher efficacy of these vaccines can be attributed to either strong inherent immunogenicity of these new vaccines or antigenic differences among cpv- and cpv- a and cpv- b (pratelli et al., ) . overall, during the past four decades of cpv vaccine development, modified live viruses have proven superior to inactivated intramuscular (im) vaccines (appel, ) . in brief, various strategies such as the use of less attenuated virus (low serial passage), high titer vaccines, multiple doses, or the use of the in route of immunization have been attempted to overcome inhibitory influences of maternal antibodies and to reduce the window of susceptibility in pups. recently new variant cpv- c has emerged, which is highly pathogenic and causes more severe diarrhea in dogs. currently used vaccines (cpv- or cpv- b strains) are effective in protecting dogs against challenge with cpv- c virus under experimental conditions (spibey et al., ) , although efficacy in the field is unknown (reviewed by decaro and buonavoglia ( ) ). antigenic differences between original cpv- and its variants may reduce the efficacy of current cpv vaccines, which is supported by in vitro virus-neutralization tests conducted on vaccinated animals that showed low cross-reactivity between heterologous cpv variants (cavalli et al., ) . however, this may not represent actual cross-protection in clinical cases. there is a need not only to make current vaccines effective in the presence of maternal antibodies, but also to update them based on continuous epidemiological surveillance studies. dna plasmids expressing vp (jiang et al., ) , replicon-based cpv dna vaccine expressing vp (dahiya et al., ) , b cell epitope ( l peptide of vp ) fused to ct b subunits expressed in transgenic tobacco chloroplasts (molina et al., ) , chimeric virus particles expressing cpv peptide (different prime/boost strategies) (nicholas et al., ) , and recombinant vlps formed by baculovirusexpressed vp (casal, ) have been evaluated in dogs or rodents without maternal antibodies and have demonstrated good immunogenicity and/or protective efficacy. further efficacy tests in pups with maternal antibodies are needed to assess their commercial potential. oral vaccines for the induction of active immunity against bacterial diarrhea are not commonly used in livestock, although e. coli diarrhea is an important problem in neonatal and postweaning calves and pigs. f (k ) and f are the major fimbrial adhesins present on swine enterotoxigenic e. coli (etec) and are major targets for e. coli vaccines (fairbrother et al., ) . whole bacteria vaccines are routinely administered parenterally to pregnant cattle, sheep, and swine to protect their suckling neonates against etec infections (moon and bunn, ) . such vaccines are practical and effective because: ( ) fimbriae are required for the adhesion-colonization of bacteria early in the pathogenesis of the disease; ( ) most fatal etec infections in farm animals occur in the neonatal period; ( ) more than % of the etec in farm animals belong to a small family of fimbrial antigen types, ( ) and mothers are seropositive to etec so booster responses are elicited. recent vaccine studies have focused on administration of purified bacterial subunits (transgenically expressed adhesin of f fimbriae in plants) and mucosal adjuvants (verdonck et al., ; joensuu et al., ) and have shown promising results. overall, the vaccine strategy used, parenteral vaccination of field-exposed seropositive mothers, to induce lactogenic immunity is the same as that shown to be effective for parenteral application of rv vaccines in rv seropositive mothers (bohl et al., ; saif and jackwood, ; saif and fernandez, ) . studies of live oral enteric vaccines in animals have clarified the mechanisms of induction of protective immunity against enteric disease and contributed to our understanding of the common mucosal immune system. however, commercial live oral vaccines often have shown inadequate or inconsistent efficacy under field conditions (saif and jackwood, ; saif and fernandez, ) . major obstacles to improved efficacy of oral vaccines include: ( ) maternal antibodies in the intestine of neonates (mainly colostrum and milk antibodies), which interfere with live vaccine replication; ( ) qualitative and quantitative limitations in the neonatal immune system, although neonates are immunocompetent; ( ) the inability of attenuated vaccine strains to adequately infect (too high attenuation) or stimulate s-iga antibodies in the intestine; ( ) the use of inappropriate (or unstable) antigens or route for subunit vaccines; ( ) the lack of oral delivery vehicles or mucosal adjuvants for subunit vaccines; and ( ) infection by pathogens prior to vaccination. studies of neonatal pigs indicate that protection rates against rv diarrhea upon challenge correlate with the magnitude of iga asc and memory b cell responses in intestinal lymphoid tissues gonzalez et al., ; yuan and saif, ) . studies conducted in immunodeficient or specific gene knockout adult mice have shown that neither cd + or cd + t cells nor antibodies were essential for induction of protective immunity to rv infection, but usually one of these effectors (t or b cells) was necessary for elimination of primary rv infection (mcneal et al., (mcneal et al., , . in pigs, recent studies have shown that cd + and cd + ifnγ producing t cells play a role in protection against rv diarrhea and infection (yuan et al., ) , but it is difficult to create genetically modified pigs similar to knockout mice, to assess the contribution of b cells and t cells. the redundant nature of immune responses to rv in mice, the multiple immunologic and possibly nonimmunologic pathways to resolve rv infections (franco and greenberg, ; ward, ) , the age factor and host differences in rv pathogenesis in mice and pigs (saif et al., , ward, ) , and the use of inbred mouse strains contribute to the discrepancies seen between the adult mouse and the neonatal pig models. the majority of pathogens enter and initiate infection at mucosal surfaces, making mucosal sites relevant targets for vaccines to prevent infection. to develop effective mucosal vaccines, it is important to determine correlates of protection against enteric pathogens. generally for localized gut infections, s-iga antibodies and intestinal t cells play an important role. improved vaccines that induce high levels of intestinal iga antibodies against the appropriate microbial antigens can be achieved by choosing the proper vaccine formulation and delivery method. vaccines should also induce: ( ) heterotypic protection; ( ) active immunity in the presence of maternal antibodies; and, ( ) long-lasting immunological memory. novel vaccines (e.g., vlps, transgenic plants), adjuvants (e.g., mlt, iscoms, tlr ligands (e.g., cpg), mycobacterial extracts, α -dihydroxyvitamin d , retinoic acid, probiotics, and cytokines), and vaccine delivery systems (e.g., recombinant plant or animal viruses or bacterial vectors, genetically engineered probiotics, iscoms, liposomes, and nanoparticles) should be explored and evaluated in relevant animal models to further enhance the efficacy of current or new vaccines. recent studies have shown that the targeting of antigens directly to apcs (via surface receptors) is an effective way to tailor immune responses to optimize protection and should be explored further in large animals (trumpfheller et al., ) . the passive transfer of maternal immunity provides essential protection in newborns. although most neonatal immune systems are competent to mount primary immune responses against pathogens, primary responses often do not develop quickly enough to prevent disease. maternal immunologic transfer provides a critical (though temporary) aid to survival for neonates. the enhancement of passive immunity through vaccination of the mother has been a successful disease prevention strategy in domesticated animals. vaccinated mothers develop higher levels of specific antibodies in colostrum and milk and increased levels of immunity in their offspring (glezen, ; saif and fernandez, ) . passive immunity can also be enhanced by oral administration of immune milk or heterologous antibody preparations (e.g., chicken egg yolk igy (ikemori et al., ; kuroki et al., ) or monoclonal antibodies) or by parenteral administration of hyperimmune plasma (becu et al., ) . recent studies using monoclonal, single-chain antibodies (variable heavy domain (vhh) nanoantibodies) of llama origin open new possibilities for providing passive immunity to humans and animals (garaicoechea et al., ) . immunoglobulins of the igg and igg isotypes of camelids consist of heavy chains without associated light chains (hamers-casterman et al., ) . the distinctive biochemical characteristics and binding qualities of vhh antibodies overcome some key limitations of conventional antibodies (see below). unfortunately, passive antibodies often interfere with active immunization of young animals and birds. various vaccination strategies have been developed to minimize the suppressive effects of maternal antibodies, but improved adjuvants and antigen-delivery systems are needed to facilitate efficient induction of active immunity in the presence of maternal antibodies. this section will address past, current, and future approaches for enhancing passive immunity in veterinary species. the transfer of systemic passive immunity from mother to offspring can occur prenatally, via the placenta or yolk sac, or postnasally via ingestion of colostrum and milk, depending on the species. the main ig isotype transferred in most mammalian species is igg. in avian species igy, the functional equivalent of mammalian igg, is transferred to the yolk to passively protect the developing chick (kovacs-nolan and mine, ) . in primates and rabbits, maternal igg is transferred across the placenta to the fetus. in rodents, transplacental transfer occurs in combination with prolonged ( - days) postnatal transfer by means of colostrum and milk (husband, ) . in dogs and cats transfer of igg occurs by a combination of prenatal and postnatal mechanisms, with % to % of total transfer occurring before birth (tizard, ) . in ruminants, horses, and pigs, offspring are born virtually agammaglobulinemic and transmission of ig occurs only via colostrum for a limited time after birth (tizard, ) . after the transition from production of colostrum to milk, ig are no longer absorbed from the intestines and only act locally in the gastrointestinal tract. immunoglobulin absorption in neonates of large domestic species is facilitated by the presence of protease inhibitors in colostrum and its efficiency declines rapidly after birth, with maximal absorption occurring in the first h. the cessation of absorption of intact macromolecules is termed "gut closure," and occurs at different ages in different species. in calves and pigs closure normally occurs by - h after birth. absorption of colostrum ig can be highly effective, supplying the newborn with serum ig at concentrations similar to those of the dam. failure of passive transfer (fpt) is a common problem, however, in newborn calves and foals (besser and gay, ; tyler-mcgowan et al., ) . fpt may occur because of the production of low quantities of colostrum, because of production of colostrum with low concentrations of maternal antibodies, because of ingestion of low quantities of colostrum, or because of inefficient absorption (quigley and drewry, ) . colostrum supplements, colostrum replacers, and plasma products have been developed commercially to address this problem, with variable success. vaccination of the dam in late pregnancy can enhance antibody titers in colostrum and after suckling in the serum of the offspring (saif and fernandez, ) . the benefits of vaccination of the dam for enhancing passive immunity can be lost if colostrum is of low quality or if absorption of colostral ig is inefficient. the half-life of ig varies considerably among species of domestic animals and with the ig isotype. neonatal receptor for fc of igg (fcrn) is involved in homeostasis of serum levels of igg in general, but distinct mechanisms may function in neonates. the main route of clearance of passively acquired maternal igg in calves is transfer from the serum to the intestine (besser et al., ) . approximately % of passively acquired igg is eliminated by this route. if titers of passive circulating antibodies are high enough, the transfer of antibodies from the circulation to the intestinal lumen can mediate short-term protection against rotavirus diarrhea (besser et al., ) . the same mechanism may be functional in piglets (saif and wesley, ; parreño et al., ; ward et al., a) . the persistence of titers of circulating maternal antibodies is generally considered in designing vaccination strategies for young animals because of suppressive effects of maternal antibodies on active immune responses. induction of immune memory can occur in the absence of a detectable serum antibody response (boersema et al., ; parreño et al., ) . the presence of passive antibodies in the intestines can interfere with mucosal immune responses to natural infection as well as to vaccination parreño et al., ; nguyen et al., a,b) . experiments in colostrum-deprived lambs (jones et al., ) and calves (mosier et al., ) have demonstrated the ability of parenterally administered immune antisera to mediate protection following experimental challenge with mannheimia haemolytica. parenteral administration of hyperimmune plasma raised against rhodococcus equi has been shown to protect against pneumonia in young foals in experimental (hooper-mcgrevy et al., ) and field studies (becu et al., ) . hyperimmune plasma is available commercially for use in foals. prepartum vaccination of beef (van donkersgoed et al., ) and dairy (hodgins and shewen, ) cows has been demonstrated to increase antibody titers to m. haemolytica in their colostrum and in the serum of their calves. vaccination of broiler breeder chickens can be used to provide passive protection against respiratory/septicemic disease caused by avian pathogenic e. coli (kariyawasam et al., ) . rodents have been a popular model for the study of passive protection by milk antibodies. however, rats and mice actively transport igg from the gut into the circulation during the first - weeks of life. thus, antibodies in ingested milk contribute to both local and systemic immunity in rodents, in contrast to the strictly local effects occurring in humans and most domestic animals. in pigs, horses, dogs, and cats, igg is the most abundant ig in colostrum but iga predominates in milk. parenteral vaccination, by enhancing serum igg antibody titers, contributes to igg antibodies in colostrum but has limited effects on iga antibodies in milk. milk antibodies provide passive protection to the neonatal intestinal tract by immune exclusion preventing the attachment of viruses, bacteria, and parasites and by neutralizing viruses or enterotoxins. s-iga antibodies, because of their resistance to cleavage by digestive enzymes, and higher levels in milk are more efficient in mediating protection in the gut of pigs and other monogastrics (saif and jackwood, ) , but high persisting levels of passive igg antibodies are also protective. in ruminants igg antibodies, relatively resistant to proteolytic enzymes (brock et al., ) and predominant in milk, have functions similar to those of s-iga. numerous vaccines are marketed for vaccination of cows and sows to provide lactogenic immunity to rotavirus, coronavirus, and e. coli in suckling offspring. vaccine efficacy has been variable. ideally, suckling animals become subclinically infected with enteric pathogens while receiving adequate passive antibodies to prevent disease, and develop active immunity to prevent subsequent diarrhea. this balance between passive immunity and disease can be disrupted in intensive animal production systems by exposing animals to pathogens in confined, contaminated environments. earlier weaning practices reduce intake of milk antibodies. maternal enteric vaccines are commonly used in two populations of pregnant animals. to control epidemic infections, they are used in naïve, seronegative animals to induce primary immune responses. to control endemic infections (such as rotavirus and e. coli), booster vaccines are used in seropositive, field-exposed animals to stimulate anamnestic memory responses. virulent tgev given to pregnant sows stimulates high levels of iga antibodies in milk and passive protection (saif and jackwood, ) . oral attenuated tgev vaccines, which replicate poorly in sows, induce lower iga milk antibody titers and low or variable efficacy in the field (moxley and olson, ) . parenteral killed tgev vaccines induce only low igg milk antibody titers and have the lowest protection rates. attempts to develop maternal tgev recombinant subunit vaccines based on the surface tgev spike (s) protein that induces na, or live vector vaccines expressing the s protein, have also been of limited success in tgev seronegative swine (saif and wesley, ) . however, prime/boost strategies such as im administration of tgev s protein following oral/in priming with attenuated tgev have shown promise as a means of enhancing iga milk antibody titers (park et al., ) . booster vaccination strategies are required to enhance lactogenic immunity to endemic enteric pathogens, such as rotavirus and e. coli, because antibody titers in milk decline dramatically during lactation. breast milk iga antibodies are increased in women endemically exposed to cholera following parenteral boosting with a cholera vaccine (svennerholm et al., ) . in pigs infected with rotavirus, iga memory b cells initially reside in the ileal pps but are subsequently present in substantial numbers in spleen (yuan et al., ) . systemic stimulation of such iga memory b cells by parenteral booster vaccines can yield dimeric iga antibodies in serum for transport to mucosal secretions via the polymeric ig receptor. under field conditions, antibodies to endemic intestinal pathogens are also common in bovine colostrum and milk, but without the boosting effect of highly immunogenic vaccines, antibody titers are often too low to protect calves (besser and gay, ; saif and fernandez, ) . thus vaccines are marketed for prepartum vaccination of cows against rotavirus, coronavirus, and e. coli to enhance passive immunity in their calves, but the field efficacy of these vaccines has been questioned (waltner-toews et al., ) . vaccination of pregnant dairy cows with modified live or binary ethyleneamine inactivated rotavirus or recombinant / / / vlps has been shown to increase igg and virus na titers to rotavirus in colostrum and milk and mediate passive protection in calves against oral rotavirus challenge (saif and fernandez, ; kim et al., ) . prepartum vaccination of cows and sows with bacterins prepared from enteropathogenic (epec) e. coli for prevention of diarrhea in their offspring is also commonly practiced. under modern farming practices, dairy and veal calves rarely are fed whole milk from their dams for more than or days. thus vaccine efficacy is based on antibodies absorbed from colostrum or retained temporarily in the gut, rather than on a continuing supply of immune milk. piglets, in contrast, continue to receive immune milk until weaning at - weeks of age. the importance of a continuous supply of passive antibodies for protection against tgev has been demonstrated (saif and wesley, ) . numerous commercial ig preparations with antibody activity against specific enteric pathogens have been marketed. products intended for prevention of e. coli enteritis in calves include dried bovine colostrum and whey, hyperimmune sera raised in horses, and mouse monoclonal antibodies to the k (f ) antigen of e. coli. these products are administered orally in the first h of life to prevent adhesion of epec e. coli. orally administered bovine colostral whey containing rotavirus antibodies also passively protects piglets against rotavirus (schaller et al., ) . immunization of chickens shows promise as an efficient method for producing polyclonal antibodies for passive protection. specific antibodies of the igy isotype are induced by vaccination and are concentrated in egg yolk. laying hens can produce about g of igy per year. yolk antibodies with virus na provide calves with partial protection against diarrhea caused by rotavirus (kuroki et al., ; vega et al., ) , and etec e. coli (ikemori et al., ) . in a recent study, supplementation of the milk ration of neonatal calves with egg yolk containing igy antibodies to rotavirus for days provided % protection against rotavirus diarrhea after challenge, and also enhanced mucosal asc numbers in the duodenum (vega et al., ) . egg yolk lacking rotavirus specific igy did not provide clinical protection, but did enhance asc responses, suggesting the presence of immune modulators in egg yolk. protective effects of yolk antibodies are dependent on antibody titers in oral preparations (marquardt, ) . development of better means to protect yolk antibodies from digestive processes will improve both the efficacy and the economic viability of yolk antibodies for clinical applications (kovacs-nolan and mine, ). for many diseases of newborns and neonates, passive immunity is the only practical means of providing timely protection. unfortunately, it is well documented that maternal antibodies can suppress active immune responses following vaccination. this effect has been observed with both live and nonreplicating vaccines, and for both systemic and mucosal responses (siegrist et al., ; hodgins et al., ; parreño et al., ; nguyen et al., a,b) . antibody responses especially are affected; t-lymphocyte responses may not be suppressed (siegrist et al., ) . titers of maternal antibodies are maximal for most species of interest in the first week of life and then decline gradually over the next few months, but variability of titers among individuals is high. with many vaccines, a "window of disease susceptibility" of variable duration occurs when titers of maternal antibodies are too low to mediate protection, but are too high to permit effective vaccination. a number of strategies are used to cope with this problem. some veterinary vaccines for cattle are sold with the disclaimer that "animals vaccinated before months of age should receive a booster dose of vaccine at months of age." this provides little solace for the many diseases of cattle occurring in the first weeks or months of life. a common strategy for vaccines of dogs and cats is to administer a series of doses of vaccine from an early age (at which time only a few individuals will be responsive) and to continue vaccinating until an age at which virtually all can respond to vaccination. this strategy has economic disadvantages for the pet owner. some manufacturers produce low passage, high virus titer vaccines especially for use in situations where high titers of maternal antibodies and high pathogen exposure are anticipated. this is similar to a strategy once (but no longer) approved by the world health organization for vaccination of children in developing countries against measles (gellin and katz, ) . preliminary evidence suggests that incorporation of vaccine antigens in highly structured iscoms or in application of vaccines can enhance immune responses in the presence of maternal antibodies (van binnendijk et al., ; brockmeier et al., ) . immunoglobulin g in most mammalian species is composed of two heavy chains, covalently linked by disulfide bonds, and two light chains. "conventional" heavy chains consist of one variable domain and three constant domains (ch , ch , and ch ); light chains are composed of a variable and constant domain. in contrast camelid species produce "heavy chain immunoglobulins" that lack light chains and the first constant heavy domain (hamers-casterman et al., ) . the antigen-binding site of these unusual heavy chain antibodies is formed by a single domain, designated vhh in camelids. vhh are easily produced as recombinant proteins, designated single domain nanoantibodies or nanobodies ® and represent the smallest molecule in nature capable of binding a specific antigen. the cdr region of these nanobodies has the capacity to form long loops that can extend into cavities on antigens, e.g., the active site crevice of enzymes. other advantageous features of nanobodies include their high solubility, thermal stability (resist pasteurization), refolding capacity, and optimal tissue penetration in vivo (reviewed by vanlandschoot et al. ( ) ). nanobodies have demonstrated efficacy as agents of passive immunity for infectious diseases. vhh specific for rotavirus inner capsid protein vp are able to broadly neutralize rotaviruses independently of serotype, and in mouse experiments provide passive protection against challenge with human rotavirus (pant et al., ; garaicoechea et al., ) . nanobodies against other viral diseases with veterinary impact have been developed (foot-and-mouth disease, influenza, rabies (vanlandschoot et al., ) ) and represent a promising next-generation biologic platform for passive immunity. maternal vaccination to enhance passive immunity is already widely used in veterinary medicine. some of these vaccines, especially vaccines against enteric viruses, have limited efficacy. new approaches to enhance immunogenicity are promising, but await commercial development. a clearer understanding of protective mechanisms and immune modulation mediated by passive antibodies would contribute to more effective interventions. there is an urgent need for adjuvants and delivery systems capable of reliably inducing active immunity in neonates despite the presence of maternal antibodies. the ability to provide continuity of immune protection from birth, by combining passive immunity with active immunization, would have a major impact on neonatal morbidity and mortality. the rapid expansion of commercial fish farming (aquaculture) in many countries in recent decades has been accompanied by an urgent need to develop vaccines to prevent (infectious) fish diseases that were previously unknown or obscure. added to the difficulties involved in identifying the pathogens responsible, there has been the challenge of delivering vaccine efficiently with minimal stress to very large numbers of fish. some commercial vaccines for fish are administered by intraperitoneal or im injection, but mucosal vaccines are also widely used (reviewed by brudeseth et al. ( ) ). fish are routinely vaccinated by immersion in tanks of diluted bacterin, modified live bacteria or viruses, with exposure times ranging from s up to min, depending on the vaccine and age of the fish. it is unclear whether the main route of antigen uptake is oral or via the mucosal surface of the gills. several commercial vaccines are now available that consist of recombinant viral proteins for mixing into the feed (evensen and leong, ) ; days of feeding is recommended by the manufacturer. research on mucosal veterinary vaccines has contributed new concepts to the field of mucosal immunity. investigations of pathogen-host interactions in outbred animals have illustrated the complexity of these interactions. early studies of an enteric coronavirus infection of swine (tgev) led to the concept of the gut-mammary gland s-iga immunologic axis and provided part of the basic tenet for a common mucosal immune system. later studies of tgev and a deletion mutant of tgev with respiratory tropism (prcv) revealed functional compartmentalization within the common mucosal immune system whereby in inoculation of pigs with prcv failed to elicit sufficient intestinal iga antibody to fully protect against the enteric pathogen tgev (vancott et al., (vancott et al., , . subsequent studies have explored new prime/boost mucosal immunization strategies to elicit intestinal immunity to rotavirus in naïve pigs (saif, b; yuan and saif, ; gonzalez et al., ) . in these studies only oral priming with attenuated virus led to successful in booster responses using nonreplicating (vlp) vaccines combined with mucosal adjuvants such as iscom or mlt. thus use of a replicating vaccine to prime lymphocytes at a major mucosal inductive site (galt) followed by boosting with a nonreplicating vaccine at a second inductive site (nalt) effectively stimulated intestinal iga antibodies and induced active protection against rotavirus diarrhea. although there is progress in developing safe and effective nonreplicating vaccines to boost mucosal immune responses (saif and fernandez, ) , the dilemma remains to develop effective vaccines to prime for mucosal immunity. mucosal adjuvants (mlt, iscom, cpg, cytokines) and new delivery systems (replicating vectors, microparticles) have shown promise in animal studies reviewed in this chapter. however, their economical production and final evaluation under field conditions, including in the presence of maternal antibodies as relevant, are needed. considerable research effort has been devoted to development of vaccines for respiratory diseases of domestic animals. in some instances attenuated organisms delivered by mucosal routes have demonstrated improved efficacy over nonreplicating antigens given by systemic routes. for many respiratory diseases, however, further progress in the development of mucosal vaccines will have to await advances in understanding of disease pathogenesis and identification of protective antigens. in contrast, studies of ascending infections of the reproductive tract in cattle have demonstrated the efficacy of systemic vaccination to clear established infections, and highlight the possibility of therapeutic vaccines. finally it is important to realize that there are considerable species differences in mucosal immunity. for example, the primary ig in mammary secretions of ruminants is igg which is actively transported to the mammary gland from serum and provides effective passive immunity to the nursing offspring against enteric pathogens. thus parenteral immunization of the dam stimulates passive immunity in ruminants against enteric pathogens. in contrast, in monogastrics, iga predominates in milk and iga lymphoblasts that traffic to the mammary gland originate in the intestine. therefore oral vaccines in monogastrics may provide a more effective vaccine strategy to induce iga antibodies in milk (saif and fernandez, ) . by applying the aforementioned vaccine concepts with new and effective mucosal 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adjuvant and immunostimulating activities of water-soluble substances extracted from mycobacterium tuberculosis (var. hominis) efficacy of a neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle comparative field evaluations of in ovo applied technology equine influenza antigen-presenting cells in the female reproductive tract: influence of estrous cycle on antigen presentation by uterine epithelial and stromal cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin- and regulatory t-lymphocytes (treg) probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells the sheep and cattle peyer's patch as a site of b-cell development equine influenza vaccine efficacy: the significance of antigenic variation induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model mucosal and systemic 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acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs key: cord- -s nbr y authors: nan title: section virology date: - - journal: zentralblatt für bakteriologie doi: . /s - ( ) - sha: doc_id: cord_uid: s nbr y nan s contains a human endogenous retroviral element related to simian sarcoma virus (ssv) and its associated virus ssa v. by sequence analysis and comparison with the corresponding ssvlssav sequences the genomic organization of s element was determined to be ' gag-s-nrs-pol-ltr '. s-nrs represents a region of bp in s that consists of nonretrovira!'sequences. a probe containing the complete s element was used to screen two human cdna libraries under low-stingency conditions. clones were isolated which had yielded strong hybridisation signals with the s probe. hybridisation of these clones with different fragments of the s genome revealed that of these clones contain s-nrs related sequences. five of the isolated clones were sequenced and compared with s . one of them contains a homologous to the u -region of the s l tr. the outer four clones sequences with - % identity to s s-nrs on nucleotide level. the deduced amino acid sequences of the four s-nrs clones are only - % homologous to s s-nrs. none of the sequences represent a contiguous open reading frame. these results indicate that s-nrss probably do not have protein encoding function. hybridisation of human genomic dna shows that s-nrs belongs to a multicopy family of related sequences. human immunodeficiency viruses, human t lymphotropic viruses, and the human spumaretrovirus (hsrv) constitute the natural occurring human retroviruses. molecular cloning and sequencing of hsrv revealed four open reading frames (orfs) additional to gag, pol, and env. three additional orfs, bel - , are located berween the env gene and the 'ltr, one additional orf, sl, is loacted in the central region of the genome. functional analysis of the bel and s genes requires an infectious molecular clone of hsrv, which was not available so far. we have constructed such a clone (phsrv). the biological activity of the clone was confirmed by a variety of criteria: induction of characteristic cpe in susceptible cells infected with cell-free supernatant from cultures transfected with phsrv; indirect immunofluorescence; radioimmunoprecipitation of viral proteins and electron microscopy. furthermore, it is shown that phsrv is able to transactivate the hsrv-ltr. glycoprotein iv of bovine herpesvirus (bhv-l) is one of the major immunogenic glycoproteins of the viral envelope and is involved in absorption and/or penetration of the virus. it is esential for the production of infectious virus and cannot be deleted from the viral genome. in contrast to the analogous proteins of herpes simplex virus (gd) and pseudorabies virus (gp ) the giv of bhv- shows a size heterogeneity among different strains. this indicates that in at least one part of the polypeptide backbone the aminoacid composition is variable. comparison of the nucleotide sequences of four bhv- strains revealed that the size heterogeneity in these strains is due to different repetitions of a bp sequence, which is located upstream the sequence coding the membrane spanning domaine of giv. we are currently testing the possibility to introduce heterologous sequences into this part of giv to analyse whether the giv can contain antigenic epitopes of different viruses without affecting the functional activity of the giv. the molluscum contagiosum virus (mcv), a member of the family poxviridae, induces epidermal proliferation in man. the genome of mcv type (mcv-l; kbp) had been characterized by physical mapping using a defined gene library of the viral genome and by dna-dna hybridization. the physical maps of the viral genome were constructed for the restriction endonucleases bamhi, clai, ecori, and hindiii. detailed hybridization experiments revealed the presence of repetitive dna sequences located within the terminal regions of the viral genome, e. g. bamhi dna fragment b ( kbp; to . mu) and e ( . kbp; . to mu). the fine mapping of these particular regions indicates that the repetitive dna sequences are located within the hindiii dna fragments jl ( . kbp; . to . mu), k ( . kbp; . to . mu), pi ( . kbp; to . mu), and p ( . kbp; . to mu). nucleotide sequence analysis was carried out. it was found that the hindiii fragments jl and k each contained an inverted repeat of bp and bp, respectively. the homology between the both repetitive dna elements was found to be %. the analysis of the coding capacity of the determined dna sequences revealed the presence of and open reading frames in the hindiii dna fragment k and the corresponding region in the hindiii dna fragment jl, respectively. positive in-patients (n = ) and outpatients (n = ) showed an increasing prevalence of antibodies to herpes viruses. the difference was particularly evident for cmv-igg ( % prevalence in hospitalized, . % in out-patients and . % in hiv negative patients). anti-hbc, with a low prevalence in controls ( . %) was markedly associated with progression of hiv infection ( . % prevalence in out-patients, . % in in-patients). the antibody pattern to opportunistic viral infections may be regarded as a marker of pathogenicity in hiv infected patients. because of the strong crossreactivity of the two serotypes of herpes simplex virus (hsv) it is very difficult to distinguish between hsv-l and hsv- -typespecific antibodies in patients' sera. although genital herpes can be caused by hsv-l in % of the cases, hsv- is the cause of most recurrent genital herpes ( %). the serological differentiation of past hsv- from past hsv-l infections is necessary for identifying pregnancies likely to be complicated by recurrent maternal hsv- infection. by improving the enzyme-linked immunosorbent assay (elisa) for hsv- -antibodies and additional testing of sera by western blot, we were able to specifically identify hsv-l-and hsv- -antibodies in serum samples. -for the elisa, hsv- -antigen was immobilized on -well micro titer plates (nunc), and patients' sera were added. antibodies were detected by biotin/streptavidin peroxidase. -for the western blot the electrophoretically separated hsv- -antigen was used. the antigen was blotted to a polyvinylidenedifluoride (pvdf) membrane (immobi- n™, pharmacia) and incubated with the test sera. antibodies against typespecific glycoproteins of hsv type (gg- ), forming a sharp band, were visualized by the application of biotinlstreptavidin peroxidase. our results showed that the elisa and the western blot correlated in . %. serum samples with high antibody titers against hsv-l showed false positive reaction in the hsv- -elisa in . % of the cases. the optical density of these sera ranged ± , around the cut-off value of the elisa. these samples could easily be identified by western blot. -serological studies showed, that % of the prostitutes (n = ) have antibodies against hsv-l and % against hsv- . in % both types of antibodies could be found. furthermore % of female patients in gynecological treatment (n = ) had antibodies to hsv type and in % of the cases against both serotypes. % showed no antibody reaction. roughly the same prevalence of hsv-l and hsv- -typespecific antibodies was found in patients from the dermatological department. a. m. eis-hubinger, k. kurowski, and k. e. schneweis inst. f. med. mikrobiologie u. immunologie, univ., d- bonn a nonneutralizing monoclonal antibody (mab), directed against glycoprotein gc of herpes simplex virus type (hsv-l) and neutralizing mab to glycoprotein gb were evaluated for their ability to protect mice from genital hsv-infection. the nonneutralizing mab had only little effect on virus replication in the mucous membranes, but completely protected the mice from peripheral skin lesions, neurological illness and death. progression of the virus to the central nervous system was obviously inhibited by a modified course of the ganglionic infection: the number of ganglia presenting infectious virus and final latency were reduced, although early latency was not induced. in addition to the effects of the nonneutralizing mab, the neutralizing mab effectively shortened viral shedding from the vagina and converted proliferative ganglionic infection into latency. the different modes of action of the monoclonal antibodies are discussed in context to the possible defense mechanisms of the humoral immunity against virus proliferation in mucous membranes and in ganglia. the thymidine kinase (tk) gene of bovine herpesvirus (bhv-l) can serve as an integration locus for foreign dna into the viral genome, because expression of a functional active tk is not essential for viral replication. the tk -gene of bhv - was located by conversion of tk--negative cells to the tk+ -phenotype and marker rescue experiments. it contains an open reading frame of bp. sequence comparison with other herpesviral tk-proteinsequences revealed, that only certain parts of the amino acid sequence as the nucleotide binding loop are highly conserved. hybridization of rna from infected cells with tk-dna showed, that tk-mrna has a size of . kb, whereas the size of tk-mrnas of other herpesviruses measures about kb. '-and '-end of the tk transcription unit were characterized using nuclease -analysis. an open reading frame, when translated into protein, is homologous to the glycoprotein h (gh) of herpes simplex virus type and lies downstream the open reading frame of the tk-gene. we conclude, that the expression of,bhv-l-gh correlates with the expression of thymidine kinase. preliminary results suggest, that transcription of both genes is initiated from one common promoter. in foot-and-mouth disease virus (fmdv) infected cells disappearance of the nuclear protein histone h and the simultaneous appearance of a new chromatin associated protein termed pi can be observed. we have sequenced the amino terminus of pi and clearly showed that protein pi derives from histone h by proteolytic cleavage. the n-terminal amino acid residues are specifically cleaved off early during infection. in addition using an in vitro transcription/translation assay with different fmdv clones we showed that the histone h -pi transition is fmdv c protease dependent. this protease until now has only been found to be responsible for the processing of the viral polyprotein. the c protease (i) is the only fmdv protein required to induce this histone h -pi transition, (ii) no other viral protein can perform this specific cleavage, and (iii) no viral precursor fusion protein is necessary for this specific cleavage, as it is reported for the processing of the poliovirus p precursorpolyprotein. the c mediated histone h cleavage is not restricted to chromatin derived from natural host cells. as the deleted part of the histone h corresponds to the domain presumed to be involved in the regulation of transcriptional active chromatin in eucaryotes, it is postulated that this specific cleavage of h is a mechanism which fmdv utilizes to switch off host cell rna synthesis, as is reported for picornaviruses. in combination with the reported mechanism of host cell translation shut off by cleavage of the cap-binding protein complex, this specific histone h cleavage could contribute to the almost complete breakdown of host cell functions during infection. the tk of hsv - contains three regions of homology to highly conserved sequences of other nucleotide binding enzymes. these are the residues to (nucleotide binding loop), residues to and residues to . comparison of the viral and cellular tk protein sequences and -d structure analysis led to the hypothesis that the asp might be involved in binding of acyclovir which is used as a therapeutic for hsv infection. site specific mutagenesis which replaced asp by asn led to a polypeptide with no tk activity. the same was found when residues to , which are not present in cellular tks, were deleted. both mutant genes were integrated into vaccinia virus to study the biochemical properties of the resulting polypeptides and to analyze the importance of the mutated sequences for the functional activity of the enzymes. . in contrast the human glioma cell line u and the adenovirus transformed kidney cell line revealed replication of both organspecific variants as early as hours p.t. nevertheless the amount of replicated dna of the kidney variant was clearly reduced in glioma cells and replicated dna was detected later than with the cns variant. from these data we conclude, thatjcv replication is regulated not only host type specific, but also in a cell type specific manner. young human volunteers were vaccinated with a killed hepatitis a vaccine produced in human embryo fibroblast cells. different amounts of vaccine were administered by the intramuscular route. weeks after the first vaccination with a . !-lg vaccine . % of the volunteers seroconverted. weeks after the second injection the seroconversion rate was . %. anti-hav was determined quantitatively and results showed anti-hav titers to times higher than those after gamma globulin administration. one year after the vaccination / volunteers still had anti-hav titers in their sera to times higher than those after gamma globulin administration. human papillomaviruses (hpv) associated with the epidermodysplasia verruciformis syndrome (ev) represent a group of closely related viruses, clearly distinct from other hpvs. the most interesting features of ev-viruses are: (i) high oncogenic potential of some specific virus types, (ii) extremely narrow host range. to get some insight into the molecular basis of processes controlling the viral expression we studied the sequence-specific dna-protein interactions within the genomic regulatory regions. using the nuclear extracts of hela cells and a combination of exonuclease iii-and dnase i-footprinting techniques the protein binding maps have been constructed for hpv and hpv , the prototypes of viruses with high versus low oncogenic potential. the sequences in question showed a complex array of protein binding domains, covering almost the entire length of the regulatory regions. a cluster of prominent binding sites, which overlapped with the sequences of the motif (m ), a highly conserved element in most of the ev-viruses, was investigated in more detail. in transient cat assays the m motif of hpv as dimer or trimer was found to act as a strong expression activator. the analysis of m sequences in band-shift tests revealed partially different sets of dna binding proteins, interacting with the elements from hpv and . one of these proteins, at least in case of hpv , was shown to be the regulatory factor api. a part of m sequences in the vicinity of the ap binding site display a homology to enhancer elements in other small dna viruses. a. gerritzen, j.-p. kleim, and a. friedrich inst. for med. microbiology, univ., d- bonn semiquantitative detection of hepatitis b virus (hbv) dna in sera of infected individuals has become an important means of modern serological hepatitis diagnostics. it enables investigators to draw conclusions on infectivity, prognosis and therapeutical success. molecular hybridization using radioactively (i. e. phosphorus) labeled dna probes with subsequent autoradiography is characterized by both high sensitivity and specificity. alternatively digoxigenin-labeled probes which are detected by enzyme immunoassay have been employed. using this method - pg of hbv dna in aqueous solution have been detected with high specificity on membranes made of cellulose nitrate. testing sera of hepatitis patients pg of hbv dna has been detected on nylon membranes of . m pore size e p: . pg). unfortunately specificity remained disappointing even after centrifugation, digestion by proteinase k and treatment of the sera with phenole/chloroform. objective. to assess the risk for laboratory personnel resulting from patients with unknown hiv infection, sera sent in to the institute for medical virology and immunology (imvi essen) for immunologic and/or virologic testing were anonymously screened for hiv antibodies. methods. between july and december, , a total of sera from different departments of the university clinics of essen were selected for screening under code on the basis that no hiv antibody test was requested for diagnostic purposes at the same time, and sera from patients found to be anti-hiv positive during the previous years were disregarded (n = ). sera reactive in elisa (enzygnost-anti-hiv, behring) were further tested in a second (dupont) and third elisa (pasteur) and in a confirmatory test (western blot -wb-, dupont). results. in of sera from patients not suspected for hiv infection, hiv antibodies were confirmed by wb ( . %) (table) . an additional sera showed less than specific we bands and were considered indeterminate. sera from unknowingly infected patients were sent in most often from the internal (n = ) and dermatology departments (n = ), whereas sera with indeterminate hiv antibody results mostly originated from patients who received tumor treatment (n = ). conclusions. the prevalence of . % unknown hiv infections reflects a higher rate than generally assumed but possibly results from a selection in a university of patients with immunological disorders and underlying hiv infection. an overall prevalence of % anti-hiv positive sera suggests the strict adherence to infection control measures in medical laboratories. bovine viral diarrhea virus (bvdv) is a member of the pestivirus group. in culture of bovine cells, a non cytopathic (ncp) and a cytopathic (cp) biotype can be distinguished. using radioimmunoprecipitation a monoclonal antibody (mab) detected a kd nonstructural (ns) polypeptide in cells infected with ncpbvdv. in cells infected with cpbvdv an additional kd protein was precipitated. a second set of mabs was directed against the kd minor glycoprotein. the distribution of both proteins in cells infected with the two biotypes was analyzed by immunofluorescence analysis (ifa). none of the two proteins was detected on the surface of live, infected cells. the ns protein was homogenously distributed in the cytoplasm of cells infected with both biotypes. the second set of mabs displayed different staining patterns in cells infected with each of the biotypes. in cells infected with cpbvdv, a homogenous cytoplasmic fluorescence was visible. in cells infected with ncpbvdv, the stain was largely restricted to the perinuclear zone, giving a distinct staining with each of the mabs. the possible significance of these results for cytopathogenicity are discussed. in sera of patients with a variety of inflammatory rheumatic diseases autoantibodies to selfantigens (autoantigens) are found. one of the hypotheses on the molecular basis of autoimmune diseases which is discussed intensively since decades, assumes that structure-or sequence-related epitopes of virus and host proteins might be involved in initiation of autoimmune processes (molecular mimicry). -to search for crossreactive or sequenceidentical epitopes of cell and virus proteins, we started to map in detail antigenic regions and individual epitopes on autoantigens. a most detailed study has been performed for the ulsnrnp specific p protein which is the major target of autoantibodies in systemic rheumatic diseases such as mixed connective tissue disease and systemic lupus erythematosus. -by immunoblotting and elisa assays performed with recombinant p fusion proteins and peptides four antigenic regions and many patient specific epitopes could be mapped. in one of the antigenic regions an epitope amino acids long could be identified which is also present on the matrix protein m of influenza b viruses. with affinity purified p specific autoantibodies the reaction with the m influenza b virus protein and vice versa could be experimentally verified. -these results demonstrate for the first time that autoantibodies from patients with rheumatic diseases recognize an epitope of identical sequence on a highly prevalent and pathogenic virus and a major auto antigenic target. whether the existence of this common epitope is by chance or causually related is currently investigated. detection of antibodies against cytomegalovirus (hcmv) induced "early" -antigens by immunoblotting contradictory informations about the diagnostic assessment of antibodies against the "early" -antigens of human cytomegalovirus (hcmv) have been numerously published. we investigated the correlation between the incidence of antibodies against hcmv "early"proteins and the state of infection. ninety-six hcmv-igg-positive sera (elisa) were tested for specific igg-antibodies against hcmv "early"-antigens by immunoblotting. three groups were examined: (a) renal transplantation patients, (b) aids-patients and (c) randomly selected healthy individuals. each group yielded approximately the same percentage of positive immunoblots ( %, %, %). sera belonging to group (a) or (b) reacted stronger and recognized a greater number of polypeptides ( ± ; ± ) when compared to healthy persons ( ± ). all immunoblot-positive sera reacted at least with the kd protein (major "immediate early"-protein). fourteen out of hcmv-igm-positive sera (elisa) belonging to group (a) or (b) recognized "early"-antigens ( %). twenty-two out of hcmv-igm-negative sera ( %) reacted as well. we conclude that an acute hcmv infection does not cause the formation of antibodies against "early" -antigens in all individuals and furthermore these antibodies can persist during a subclinical/latent infection. berne virus (bev), isolated from a horse, is the prototype of the toroviridae, a proposed new family of positive-stranded rna viruses. bev virions consist of a peplomer-bearing membrane which envelops a tubular nucleocapsid of helical symmetry; this structure can be straight (resulting in a tubular particle) or bent into an open torus (conferring the shape of a kidney or biconcave disc to the virion). the nucleocapsid contains a single polyadenylated rna molecule of > kb and the most abundant polypeptide, an . kd basic phosphoprotein with nucleic acid binding properties. upon infection, a set of '-coterminal sub genomic mrnas is synthesized by leader-independent transcription; only the unique ' sequences of each mrna are translated. -the combination of virion structure, nucleocapsid protein size and leader-independent transcription is unique in virology and justifies a family status for bev and related viruses. however, coronaviruses have a similar genome organization, a '-coterminal nested set of mrnas and sequence similarities in the second orf of the polymerase gene of bev. since the latter suggest common ancestry, toro-and corona viruses together may be considered as a third evolutionary cluster of positivestranded rna viruses, in addition to the alpha-and picornavirus superfamilies. w. ]ilg, h. mairhofer, c. markert, and h. wolf max v. pettenkofer-inst. for hygiene and med. microbiology, univ., d- munich in order to characterize viral and nonviral structures responsible for the recognition of ebv-infected lymphocytes by the immune system, we studied the reactivity of ebv-specific cytotoxic t cells towards autologous ebv-positive lymphoblastoid cell lines (lcls). lcls were established from different ebv-positive donors by either spontaneous outgrowth of cells transformed by the donors own virus or by infection with a laboratory strain of ebv (b - ). these cell lines were used for the generation of ebv-specific cytotoxic t cells by weekly stimulation and addition of interleukin ; cells were cloned by limited dilution. in chromium release assays these cd positive t cell lines and clones were able to discriminate between two autologous lymphoblastoid cell lines infected by either the "own" virus strain or the b - strain, respectively. further experiments using various concentrations of effector cells showed that structures recognized by different t cell clones were expressed only in a certain percentage of cells ( - %) of each lcl, and that nonviral components differently expressed on different lcls also seem to playa role ctlllcl interactions. the large-scale production and purification of recombinant gp leads to a native and biologically active protein, which bounds specifically to the cd receptor. anti-hbs response of hbs vaccine recipients within an ongoing immunization course was studied in vitro. antigen specific antibody production and proliferative response as well as the expression of cd and cd and the secretion of scd were detected. it was the aim of the study to analyze the effect of various cell stimuli (mitogens, lymphokines and antibodies against cell surface antigens) on the antigen-specific immune response in vitro. pwm driven enhancement in hbs specific antibody production was shown to be time dependent. in contrast to iii and il , incubation with il led to a significant increase in anti-hbs antibody synthesis. low doses of led to a significant increase in hbs induced cd expression. moreover, induced scd secretion is enhanced by hbs antigen. il receptor expression of hbs vaccine non-responder pbmc is reduced as compared to the responder group. cd receptor expression of responder pbmc is influenced by antigen as well as iii whereas no modulation can be seen with non-responder pbmc. the capacity of nonresponder cells to respond to hbs antigen is reduced whereas the capacity of these cells to respond to iii is markedly enhanced. in summary, our data show that the hbs specific immune response in vitro is influenced by lymphokines. . % of sera from women (n = ) and . % of sera from men (n = ) were found to contain antibodies directed against the l gene products of the hpv types mentioned above. . % of female and . % of male sera exhibited antibodies directed against the hpv- e and/or e gene products. on the other hand in an individual female serum antibodies directed against hpv- el and e could be found, and another one harboured antibodies directed against hpv- e . all of the antibodies were of igg type. in order to specify whether antibodies against papillomaviruses are associated with sexual activity, we used sera from female individuals of age years old. we tested also the sera from the same individuals which were subsequently taken at time intervals of and years. it could be shown that l antibodies were present in the sera over the whole period of time. however, also an increase in the frequency of antibodies directed against hpv - ll with growing age of the women was observed. these data indicate that beside the distribution of human papillomaviruses by sexual intercourse other routes of papillomavirus infection may exist, for instance perinatal infection. further experiments revealed differences in salt stability and ph dependence between gd, gb, and gc with respect to their binding ability. thus, binding of gd could be disrupted at mm nacl and had a ph optimum between ph and ph , whereas binding of gc and gb showed a ph optimum near ph and was stable even at salt concentrations above mm nacl. the results given above provide direct evidence for a functional role of gc, gb, and gd in binding of hsv- to the cell surface. to h. as demonstrated by electron microscopy, the mechanism of this effect is due to irreversible binding of the metal ions to the viral membrane, presumably to mercaptan groups of the viral glycoproteins. adsorption of zinc inactivated virus is reduced to some extent only whereas a heavy impact has to be presumed on virus penetration. only in a small concentration range ( to i!m zn in culture medium) zns is inhibitory to the virus replication in infected cells without serious toxicity to the cells themselves. -therefore the mechanism of the in vivo efficacy of zinc preparations against hsv lesions of the mucosa is mainly based upon inactivation of free virus and only partly due to virustasis or inhibition of hsv adsorption. experiments done either with synthetic peptides or with cells transfected with the gene encoding the ebv latent membrane protein bnlfi suggested that this protein is a target for ebv specific cytotoxic t-cells (ctls). as such experiments do not closely resemble the natural situation we wanted to expand on this topic and established a system which should better reflect in vivo conditions. we constructed two recombinant vaccinia viruses, one carrying the complete bnlf l-ma reading frame, the other encodes a truncated form of bnlf i-ma containing the third exon only which was found on ebv infected burkitt lymphoma in addition to the downregulated full length protein. the continuous coding sequence of the complete gene which is necessary for expressing foreign proteins in recombinant vaccinia was cloned from genomic fragments and synthetic oligonucleotides. after infection of several cell types including freshly isolated pbls the expression of both forms of lmp was clearly demonstrated in immunoblots and by immunofluorescence. -ebv -specific cytotoxic t-iymphocytes (ctl) generated by repeated stimulation with autologous ebv infected lymphoblastoid cells in the presence of il- were reacted with autologous pha stimulated blasts infected with the two vaccinia viruses and the wild type virus as a control. ctls of two persons recognized the full length bnlfi protein but not the shortened protein whereas ctls of two other persons did not recognize any of the two bnlfi proteins. from these experiments we conclude that ., the n-terminus of the bnlfi protein contains a determinant which is recognized by some ebv-specific ctls and ., that there are additional proteins which are targets for other ebv-specific ctls. the expression of the epstein-barr virus regulation gene bmlfi is regulated by a promoter/enhancer complex located upstream from the short orf bslfi (transcribed as a spliced unit). the region '-proximal to the tata box contains consensus sequences for binding transcription factors (e.g. nf-l, ap-l). we analyzed the activity of this control region in different cell systems using appropriate reporter assays (chloramphenicol acetyltransferase and hepatitis b virus surface antigen as reporter genes). -a bp aluilbsteii fragment including the tata box was shown to be sufficient to promote the transcription in raji cells induced to lytic ebv expression by different procedures. responsiveness to phorbolester was shown in ebv negative cells ( t ). the upstream region of the bmlfi promoter ( bp bsteiiissti fragment) was identified as regulatory segment in transfection assays showing both positive and negative effects depending on the presence and state of ebv. -in ebv negative cells (hela), the upstream element clearly responded to the ebv transactivator brlfl. this specific trans-activation of the distal control region by brlfi (expression vector pksvr kindly provided by a. sergeant, lyon) was down-regulated in latently infected cells (raji) while in ebv producer cells (p hr-l) trans-activity by brlfi was detected. following insertion into a heterologous expression system the bmlf upstream control region enhanced transcription of the sv early promoter independent of its orientation. we have previously shown that mutants of prv that do not express the nonessential glycoprotein gill adsorb less readily to cells than does wildtype virus. we show here that the first interactions of prv with target cells occur via binding of the virus to a heparin-like component on the cell surface and that the viral protein mediating this binding is glycoprotein gill. this conclusion is based on the following findings: ) heparin inhibits adsorption of wildtype prv effectively as measured by plaque formation as well as by attachment of radioactively labelled virus to cells. however, it affects adsorption of gill-mutants only slightly. ) while wild-type prv binds well to matrix-bound heparin binding of a giiimutant is dramatically reduced. ) pre-treatment of cells with heparinase reduces plaque formation and adsorption of wildtype pry by % but does not affect gill mutants. ) of the pry membrane proteins glycoprotein glii binds most abundantly and specifically to heparin sepharose beads. our results indicate that binding of pry via glycoprotein glii to a heparin-like cellular component promotes efficient adsorption. in the absence of glii or of the heparin-like cellular receptor the virus adsorbs by an alternative less efficient mode. a. e. metzler and r. wyler inst. of virology, univ., ch- ziirich, switzerland bovine herpesvirus (bhv ) causes two well established entities, namely infectious bovine rhinotracheitis (ibr) and infectious pustular vulvovaginitis (ipv). encephalitis, a third entity, is less well understood. whereas ibr usually follows horizontal virus spread by aerosols, ipv is acquired by venereal contact. it remains controversial if the conditions were associated with distinct viruses. restriction endonuclease analysis of viral dna or evaluation of viral proteins following separation in sds-polyacrylamide gels may be used to distinguish between bhv . , bhv . and bhv . . the proteins of european field isolates, recovered from distinct disease episodes within the time period to , were compared with the proteins of established laboratory strains. of field isolates were classified as bhv . , as bhv . and none as bhv . . bhv . was most regularly recovered from cattle with genital afflictions. however, some bhv . strains originated from animals with ibr and others from aborted fetuses as well. the first recognition of bhv . among the field isolates in coincided with sequential waves of severe ibr outbreaks throughout europe. results obtained with laboratory strains indicated that bhv . , obviously of limited pathogenic potential, must have existed at earlier times. together with published data the results suggest that recognition of a strain as bhv . or bhv . does not necessarily reflect a specific pathogenic potential nor a distinct organ tropism. nevertheless, the severe ibr incidences were clearly associated with a virulent bhv . it has been demonstrated that the late gene products ll and l of certain papillomavirus types exhibit dna-binding activity. hepatitis b virus core proteins are also an example for the interaction of viral capsid proteins with nucleic acids. in this case dna binding is mediated by carboxy terminally located clusters of basic aa residues. similarly, the ll gene product of hpv- contains also groups of basic amino acids at its carboxy terminus. to examine whether this region is in fact the dna binding site of ll, we expressed different parts of this gene in e. coli as ~-gal fusion proteins. the vector system which we used allows the cleavage of the viral protein from ~-gal by collagenase digestion. out of the different expression products only the whole l protein and its carboxy terminal part bound dna. to specify the dna binding site, the coding sequence for the last amino acids of l was fused to ~-gal. by this measure dna binding activity could be transferred to ~-gal, which did not formerly exhibit such property. collagenase digestion and use of l specific polyclonal antibodies ensured that dna binding was a genuine attribute hpv- l gene products. the latter enzyme differentiates between a , -and a , -linkages, cleaving preferentially sialic acid attached to galactose via an a , -linkage. na-treated cells were resialylated using specific sialyl-transferases and cmp-sialic acid. only a transferase attaching sialic acid in an a , -linkage to gal was able to restore receptors for bfdv. accordingly, cultured chicken embryo cells were resistant to bfdv infection following destruction of this receptor by na treatment. detection of cytomegalovirus (antigen) in body fluids has become increasingly important.-for that purpose, in our study blood and urine specimens from immunosuppressed patients ( renal transplant recipients, heart and liver transplant recipients, patients with aids) were tested for hcmv. indicator cell cultures (foreskin fibroblasts) were inoculated with urine and leucocytes, respectively, and resp. , , and days after inoculation, subjected to an immunoperoxidase staining (ips) using a monoclonal antibody (dupont, f.r.g.) directed against hcmv early antigen. as controls, cell cultures inoculated with leucocytes were examined for hcmv-specific cytopathic effects (cpe) for days. additionally, leucocytes from patients were subjected to in-situ-hybridization with biotin-labeled hcmv ad -dna (eco ri j-fragment) probes. serum samples were tested for the presence of hcmv-specific antibodies of ig classes g, m., and a by elisa (behring, f.r.g.). -hcmv was detected in blood and urine samples from patients. blood samples were positive by both ips and cpe, by ips only, and by cpe only. hybridization assays were all negative. in virologically positive cases, titres of hcmvspecific antibodies amounted to (reciprocal titres): igg ;::: ( cases, with a significant rise of titre in case), igm ;::: ( cases), and iga ;::: ( cases). -our results indicate that, for laboratory diagnosis of active hcmv infections, the detection of hcmv early antigen in urine is, with regard to practicability and rapidity, superior to the test for viremia and is to be considered a helpful extension of serological testing. moreover, with urine samples being easily obtainable, the detection of hcmv early antigen in urine is especially appropriate, too, for controlling the course of active hcmv infections. kai-olaf netzer, axel rethwilm, and volker ter meulen inst. f. virologie u. immunbiologie, univ., d- wiirzburg the human spumaretrovirus (hsrv) is a distinct member of the foamy virus subfamily of retroviridae. its genome of about kilo bases has been sequenced. it comprises the typical retroviral genes gag, pol, and env, and at least three more open reading frames possibly coding for regulatory proteins. thus far, not much is known about the gene products of hsrv. by means of radioimmunoprecipitation, we have identified and partly characterized the major immunogenic antigens of hsrv. radiolabeled viral proteins precipitated by hsrv-positive sera (but not by hsrv-negative control sera) were in the range of to kilodalton (kda) apparent molecular weight. labelling with c-glucosamine, or with s_ methionine in the presence of tunicamycin led to the identification of three viral glycoproteins of , and kda apparent molecular weight, respectively. these glycoproteins most likely represent env gene products. a phosphorylated protein of kda may be related to the gag gene of hsrv. the results of this study show that radio-immunoprecipiation provides a powerful diagnostic and research tool to identify hsrv-positive sera. thomas nowak! and gerd wengler inst. f. virologie, univ., d- gief~en, !present address: behringwerke ag, d- marburg the primary structure of the nonstructural proteins ns , ns a, ns b, ns , ns b and ns of the wnv has been determined. the nonstructural proteins were isolated from nuclear membrane fraction of wnv infected bhk cells. aminoterminal sequence data of these purified proteins were determined. together with the published amino acid sequence of the nonstructural coding genom region (castle et ai., , virology , - ) we obtained the sequences of the nonstructural proteins ns ( kd), ns a ( kd), ns b ( kd), ns ( kd), ns b ( kd) and ns ( kd). the gene order, the sizes of the virus coded proteins and the processing of the nonstructural proteins appears to be identical between the flaviviruses. it is well accepted that the functional impairment and killing of infected cells, hypersensitivity and autoimmunity contribute to the symptoms and pathology of viral diseases. we will discuss evidence for an additional mechanism can be defined as the uncontrolled activation of host effector functions not focused on viral antigens. "autotoxicity" is evident in the following situations: (i), certain paramyxoviruses and influenza viruses are capable of activating the generation of reactive oxygen species in phagocytes in the absence of antiviral antibody. this reaction is triggered by the binding of viral surface glycoproteins to the plasma membrane of the phagocytes. when injected into the blood stream, these viruses exert toxic effects independent of viral replication. -(ii), liver damage is observed in a model of murine influenza despite the apparent lack of viral replication in this organ. this effect may be mediated by cytokines. -(iii), in canine distemper, demyelination occurs despite the apparent lack of viral replication in oligodendrocytes, the cells which form myelin. degeneration of uninfected oligodendrocytes is also observed in dissociated brain cell cultures. as measured by luminol-dependent chemiluminescence, antiviral antibody stimulates the generation of reactive oxygen in microglial cells by linking fe receptors with viral antigen expressed on the surface of infected cells, e. g. astrocytes. in these cell cultures, oligodendrocytes can readily be destroyed by reactive oxygen species generated by xanthine/ xanthine oxidase. about , sera (january, to august, ) were analyzed retrospectively for hepatitis a or hepatitis b markers; , of these were evaluable. age distribution and seasonal distribution of acute hepatitis a virus infections in groups of patients with german or foreign names were determined. most cases of acute hepatitis a in the foreign population occurred between september and december, and most patients were below years of age. in the german population no seasonal peaks could be found. however, there was one peak between and , and another between and years of age. -we did not see seasonal peaks of acute infections with hepatitis b virus. no age was preferentially affected, but infections occurred earlier in foreign population than in german people. -in both groups cases of acute infections with both hepatitis a and hepatitis b viruses were only rarely found. more foreign people than german had markers for hepatitis a or hepatitis b ( % vs. %). the glycoprotein complex gil belongs to the essential membrane proteins of pseudorabies virus (prv). therefore mutational analyses of viral gil require growth of the respective mutant in a complementing, gil-expressing cell line. a cell line capable of supplying gil in trans was isolated after co-transfection of a genomic pry-dna fragment encompassing the complete viral gil-expression unit and the plasmid psv neo conferring resistance against the antibiotic g . the viral expression unit is usually silent but can be transactivated after superinfection by herpes simplex virus (hsv-l). transient expression of the pry "immediate early" protein is not sufficient to induce transactivation. by immunofluorescence and radioimmunoprecipitation using gil-specific monoclonal antibodies expression of authentic gil could be demonstrated. attempts to isolate a constitutively gil-expressing cell line failed, presumably due to the toxicity of the expressed glycoprotein. results regarding processing of gil without the context of a pry-infection will be presented. furthermore, availability of this cell line enables us to specifically mutate the gil-gene in the pry genome and characterize the resulting mutants biologically. cellular and humoral immune reactions are important for the pathogenesis of viral infections. in our animal model of mv encephalitis, lewis rats develop a subacute cns disease process (same) in the presence of only low levels of neutralizing antiviral antibodies. the contribution of the mv-specific cellular immunity to this disease is yet unknown. therefore, the in vitro reactivity of polyclonallymphocyte cultures to mv structural components was determined. additionally, the possibility to replace purified viral antigens by bacterially expressed fusion proteins was studied. t cells were primed by immunization with antigens in emulsified in freund's complete adjuvant. t cells prepared from regionallymphnodes were taken into culture - days later and lymphoproliferation was measured by the incorporation of tritiated thymidine in the presence of specific and irrelevant antigens with a panel of antigen-specific t cell lines. with all polyclonal t cell populations except those primed with recombinant haemagglutinin (pbd-h) a specific proliferation was obtained when either whole inactivated mv or the immunizing antigen was used for restimulation. while the nucleocapsid (n) or matrix (m)-specific cell lines recognize equally well pbd-n and virion purified n (nv), or pbd-m and mv respectively, such substitution for pbd-h and hv was not found. our observations indicate the induction of a differential t cell dependent immune response against the procaryotically expressed h compared to the h glycoprotein purified from virion. k. rittert, b. nellent, h. eiffertl, h. kratzin , and r. thomssen in the course of acute epstein-barr virus (ebv) infection igm antibodies always occur against two cellular antigens that were characterized as proteins with a molecular weight of kd (p ) and kd (p ), respectively. purified p was identified as a monomer of human triosephosphate isomerase (htpi). p is a so far unknown protein that possesses a high homology with triosephosphate isomerase of rabbits (rtpi). -the two autoantibodies are produced only as igm class antibodies, there is no switch to igg. presumably these antibodies are monoclonal in percent of the patients. -in percent of the cases with acute hepatitis a virus (hav) infection anti-htpiirtpi antibodies were found, too. acute ebv infection as well as acute ha v infection may be complicated by hemolysis of different extent. igm anti-htpiirtpi antibodies purified by affinity chromatography caused an increased tcr release from human erythrocytes (rh negatice, group ). the contribution of these autoantibodies to hemolysis in acute viral infections is likely and will be further investigated. in first attempts we tried to express hbv pre-s( ) epitopes as aminoterminal fusions with hbc, all attempts resulted in no detectable expression due to instability or toxicity. we then inserted oligonucleotides coding for two overlapping pre-s( ) epitopes (dprvrglyfpa) o. immunol. : immunol. : , ) at the site where in duck hepatitis virus core antigens an insertion of amino acid is found compared to the mammalian hepatitis viruses and predicted to be at the surface of particles. the insertion results in the expression of stable fusion proteins with pre-s antigenicity in western-blots. the chromatographic behaviour of hbdpre-s( ) chimaera on sepharose b is similar to that of authentic recombinant core particles, suggesting that they assemble to particles. a rabbit anti-pres( ) - antiserum (kindly provided by r. neurath) recognizes the hbdpre-s( ) particles in a non-denaturing elisa type assay, a monoclonal antibody to the second epitope does not, which shows that a part of the inserted sequence is accessible on the particles. since we can now express hbc particles carrying pre-s epitopes in e.eoli the immunologic properties (induction of t-cell and b-cell responses) of purified particles can be investigated. w. the characterization of an enzyme-linked immunosorbent assay, ®enzygnost-hsv (ag), for the identification and typing of hsv is discussed. -differentiation of hsv was evaluated against laboratory hsv strains. it was shown that the typing indices were valid over a broad dilution range, which is essential for samples of both low and high antigen content. -in a preliminary clinical study (n = specimens) direct testing revealed an identification sensitivity of . %, a typing sensitivity of . % and overall specificity of . %. in a confirmatory test, a % agreement for both identification and typing was obtained. -on the basis of these results, it was concluded that ®enzygnost-hsv (ag) is a suitable alternative to the cell culture method, since it overcomes the failure of virus isolation due to possible inactivation resulting from improper transport and/or storage of the specimens. -in comparison with conventional hsv detection and typing test systems, ®enzygnost-hsv (ag) offers the following advantages: -ease of performance (within h), -less expensive, -subjective reading of test results using elisa photometers, -computer-controlled automation using microtitre plates is possible. sera of persons in the age between -to -years were tested parallel with the biotin! avidin western blot (b/awb), with the biotin!avidin enzyme-linked immunosorbent assay (b/aelisa) and with the complement fixation technique (cft). antigens used in all tests of the study were preparations of the human adenovirus type . specific igg-antibodies directed against two immunoblotted group-specific major adenoviral polypeptides, the hexon epitope a-antigen and the penton base ~-antigen, were found in more cases ( %) than antibodies found with the b/aelisa ( %) and with the cft ( %). the prevalences of igg-and iga-antibodies to human adenovirus were studied by b/aelisa in serum samples obtained from different age-groups of frankfurt/main, west germany. the lowest igg-antibody prevalence ( %) was measured in the six-to -month age-group, increasing to % in the six-to seven-years age-group. a lot of adenovirus positive sera with specific iga-antibodies were measured in the one-to two-( % and two-to three-( %) years age-groups. a second peak of iga-antibody prevalence ( %) appeared in the six-to seven-years and seven-to eight-years age-groups, where the highest prevalence of anti-adenovirus igg was seen. reverse transcriptase is necessary for viral replication, therefore it is an attractive candidate for antiviral therapy. detailed functional and structural analysis like e. g. cristallographic studies or neutron solution scattering are predisposition for the development of new inhibitors affecting reverse transcriptase activity. -for this purpose we have constructed a plasmid which allows high level expression of the active reverse transcriptase after introduction into e. coli. by partially adopting the e. coli codon usage and adding the original amino-and carboxy terminal sequence by synthetic oligonucleotides we were able to produce the authentic enzyme in e. coli in considerable amounts (up to % of the total e. coli protein) and in a very stable form, as shown by coomassie staining and by western blot analysis. mutants with additional amino acids fused at the aminoterminal sequence show a lower expression and an increased accessibility to the bacterial proteases. -the activity of the authentic and of the amino terminally altered enzyme was compared by standard methods and by an activity gel procedure. enzyme activity could be detected only at kd and kd, wereas no activity could be detected at kd. the recombinant produced enzyme can be used for purification and crystallographic studies. the genome of hog cholera virus (hcv) consists of an rna of kb in length. the rna contains one large open reading frame(l) which is probably translated into a polyprotein and then processed proteolytically. -metabolic labeling and radioimmunoprecipitation with a polyspecific antiserum led to the identification of four different glycoproteins -gp , gp , gp and gp -in hcv infected cells ( ) . inhibition of n-linked glycosylation through treatment of infected cells with tunicamycin resulted in distinct changes in migration behavior of these proteins in sds-page. -various cdna fragments located in the region coding for hcv encoded glycoproteins were expressed as fusion proteins in bacteria. the purified fusion proteins were used to prepare antibodies specific for the respective hcv glycoproteins. these serological reagents were used in radioimmunoprecipitation assays and western blots. the following conclusions can be drawn: .) gp and gp represent differently processed forms of a single protein. in patients with epidermodysplasia verruciformis (ev) hpv induces lesions with a higher risk of malignant conversion. there is evidence that the virus occurs also in the normal population. as hpv can not be propagated in tissue culture capsid antigens are not readily available for seroepidemiologic studies. we therefore expressed the major structural protein l of hpv and fragments thereof as ~-gal fusion proteins in bacteria. the expression vector pros encodes a fxa-cleavage site, which allows the separation of viral and bacterial moiety by proteolytic digest. purified viral antigens were used to test sera by western blot analysis. % of the sera revealed antibodies against hpv l at a dilution of : . in some cases the titers exceeded : . the antibody prevalence was similar in all age groups. western blots with l fragments showed that the humoral immunoresponse of different persons is not always directed against the same epitopes. the molecular basis for the lack of hbeag in viremic sera of some acute and chronic infected patients is not clear. in this study it was investigated whether this group of patients is infected with hbv variants which cannot synthesize hbeag. -viral dna isolated from sera of hbeag negative chronic carriers was amplified (per) and sequenced directly or after cloning. in all patient's sera hbv variants were found which had in common a stop codon in the precore region. -since the precore protein is the precursor for synthesis of the classical hbeag, one can conclude that the precore mutation of the hbv variants are responsible for the lack of hbeag in the serum of these patients. these results also implicate that the expression of hbeag is not essential for the viability of hbv. at least under immunosuppressive conditions the precore mutation seems not to drastically effect viral replication since a high hbv-dna titer was observed in the serum of one patient after liver transplantation. -whether the lack of hbeag expression is also responsible for the frequent severe and progredient course of infection and the lacking response in interferon therapy is currently investigated with a test specific for precore variant's infection and with animal models. inst. of virology, univ., d- giessen a highly purified nucleoprotein (np) preparation from influenza virus infected cells yielded in addition to the commonly known kd protein a kd component which could not be detected in virus particles. among a series of np-specific monoclonal antibodies some reacted with both proteins and others were only bound by the kd protein. among both types of np-specific monoclonal antibodies only a limited number were bound at the surface of murine cells infected with any type a virus. another category of antibodies bound to cells infected with a given subtype, but failed to react with the surface of cells infected with a different subtype. the results indicate that only restricted antigenic domains of the native np and perhaps np fragments are exposed at the surface of infected murine cells. additionally, the protective capacity of cell-associated np was determined by immunization of mice with the purified np preparation. in parallel, and in order to determine the immunogenic potency of newly synthesized np, mice were immunized with a vaccinia virus recombinant containing the gene for np prior to challenge with infectious virus. although immunized mice produced monospecific antibodies and a cytotoxic t cell response to the employed forms of np, they were not protected from influenza virus infection. the s segment rna of nephropathia epidemica virus (nev) strain hiillniis b was isolated by molecular cloning of the corresponding cdna. therna is nucleotides long with the ' and ' termini being complementary for bases. the viral messengersense rna contains one major open reading frame (orf) with a coding capacity of amino acids encoding a kda polypeptide. compared to the hantaan s segment cdna sequencing there is a nucleotide homology of % and % homology at the amino acid level. many of the amino acid differences are conservative exchanges. the c-termini of the nev and hantaan nucleocapsid proteins are nearly identical. the hydrophilicity profiles are very similar and most of the potential kinase dependent phosphorylation sites have been conserved. in contrast, the following differences are significant: the calculated isoelectric points of the nev and hantaan nucleocapsid proteins are . and . , respectively. the most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the c-terminus of nev and close to the n-terminus of hantaan virus nucleocapsid polypeptides. -bmft project a. foot-and-mouth diesease virus-infected cells suffer from cytopathic effects. one causative agent is the virus protease c, as shown by transient expression of respective cdna in baby hamster kidney cells. in contrast to other edna-encoded virus proteins c is not detectable by indirect immunofluorescence. it is, however, detectable as de novo synthesized protein hours after transfection by radioimmunoprecipitation. the enzyme is then indistinguishable in size from that found in virus-infected cells, indicating similar autocatalytical release from fused protein. transcription of protease c-encoding edna fragments is inhibited, as well as that of co-transfected fragments which do not encode protease c, as analysed by northern blot hybridizations. the shut off of transcription which is one of the cytopathic effects observed in infected cells correlates thus to the production of active protease c. the inhibitory molecular mechanisms may involve truncation of the nuclear protein histone h at its n-terminus as found by western blotting. this protein is found similarly truncated in virus-infected cells. virusprotein-specific antigenicity and immunogenicity of tissue culture rabies vaccines o. thraenhart, . marcus, and k. ramakrishnan inst. f. med. virologie u. immunologie, univ., d- essen pre-and postexposure treatment with inactivated rabies vaccines prevent rabies virus infection without complications in contrast to vaccines of nervous tissue origin. a considerable diversity concerning virus strains (pm, flury lep, era), cell strains (wi- , mrc- , chick fibroblast, bhk) and concentration procedures (ultrafiltration, ultracentrifugation) exists between vaccines of different producers. the influence of the various criteria on the antigenicity and immunogenicity were tested in in vitro experiments using mono-and polyclonal antibodies against rv proteins with antigen-elisa, western blot and immuneelectronmicroscopy in unfractionated and fractionated vaccines after rate zonal ultracentrifugation. the immunogenicity was tested in vivo by protection induction (pi) in mice, the results were correlated with the virusspecific antigenicity (vps-ag) in tissue culture supernatants. vps-, immunglobulin-specific and functional antibody-(ab) and interferon-(ifn) induction was tested in human vaccinees. -rv glycoprotein (gp) and nucleocapsid (np) concentration in vaccines were correlated with pi in mice. the gp : np ratio was > in tissue culture vaccines in contrast to brain vaccines. the harvest of gp and np in tc depends mainly on the cell strain. the virion-associated : soluble gp ratio is productionspecific. the concentration virion tc vaccines contain gp, np, m, l, which induce in vaccinees an early, high and long lasting vps-, igg specific ab-response; anti-gp and -np is induced as early as - days. ifn-induction is correlated with the applied dosis. no nonresponder was observed and all vaccinees had protecting levels of neutralizing ab from - days onwards and still after year. the frequency and specificity of antibodies to p-gene encoded proteins of human hepatitis b virus was tested in sera of acute and chronically infected patients with and without hepatocellular carcinoma (hcc). for antibody detection an immunoprecipitation gel assay was performed with radioactively labeled polypeptides produced by in vitro translation of rna of different p-gene regions. thus, five antigenic regions were identified. all anti-p antibody positive sera reacted with carboxy terminal p-polypeptides, a subset with polypeptides of the aminoterminal and middle region, and none reacted with p-protein derived from the most sequence variable region. anti-p antibodies were detected at very high frequency in sera of acute ( %) and chronically infected patients without hcc ( %), but less often in hcc patients ( %). anti-p antibodies appear early in infection and decline prior to hbsag/anti-hbs seroconversion. -these data indirectly demonstrate the expression of most hbv p-gene sequences and the high immunogenicity of p-proteins in vivo. moreover, they establish anti-p antibodies as a frequent serological marker of infection and identify the carboxy terminal region of the p-proteins (s) as immunodominant. the human polyoma virus jc produces brain tumors in hamsters. in these tumors jcv tantigen, the major product of the early genes is generally expressed. the protein plays a central role in the regulation of polyomavirus replication. since tissue culture as a major source for t-antigen is limited to human primary fetal glial cells we characterized jcv tantigen in the hamster cell line hjc derived from a jcv induced medulloblastoma. -southern blot analysis revealed the presence of jcv dna in hjc cells. the physical state was predominantly integrated. the transcription of early genes was demonstrated by presence of jcv specific mrna in northern blots. the nuclear localisation of t-antigen could be established by immunohistochemical staining and by immunoprecipitation with a crossreacting monoclonal sv antibody (pab ) a molecular weight of to kd was determined. furthermore from western blot analysis it could be assumed that high molecular forms of jcv t-antigen are present in the hamster cell line. taken together these data demonstrate that the molecular weight and nuclear localization were as expected from sv transformed cell lines. therefore jcv t-antigen in hjc cells shall be used to characterize its dna binding capacity to the origin region of jc virus dna. current laboratory diagnosis of a coxsackie-b-virus (cbv) infection is mainly based on virus isolation, supported by the detection of rising or high virus-specific neutralizing antibody titers. since such high titres have also been found in apparently healthy peopleprobably originated from a subclinical infection and persisting for a year or more -cbv-igm detection may be a more reliable criteria for serological diagnosis. attempts to identify these antibodies in routine context using conventional techniques (nt, immunodiffusion) or solid phase immunoassays (eia, ria, reverse elisa, immunoblot) have failed, despite high sensitivity and specificity, due to high costs or technical disadvantages. in the present study we developed a modified western blot technique (modi-western blot), which enables a rapid and reliable identification of cbv-igm antibodies. in this assay the antigen was electrophoretically separated using an integrated electrophoresis system (phastsystemunit) and transferred to a solid matrix by diffusion blotting. subsequent immunodetection was performed using a biotin-avidin amplification and monoclonal antibodies. to appraise the efficiency of the method human serum samples were analysed for virus-specific antibodies (igm, iga and igg subclasses) to coxsackie-b-viruses. -group specific igm responses could be found in of ( . %) of igm seropositive cases. type-predominant igm antibodies ( / ) were detected in cases against cbv , in cases against cbv and in case against cbv . moreover igg/lga assay in patients seropositive for igg and igm revealed in all cases igg and iga antibodies, which supports the evidence of an acute cbvinfection. improvement of diagnosis of cytomegalovirus (hcmv) infections in immunosuppressed patients by detection of hcmv using a monoclonal antibody directed against hcmv-early d- frankfurt primary or recurrent hcmv infections can cause severe clinical problems in immunocompromised patients. as far as active hcmv infections in these patients are concerned, the diagnostic significance of serological data can be restricted tiel: virology, in press an unsatisfactory reproducibility and comparibility was observed when six available enzyme immune test kits for anti-hbc were evaluated by four german red cross bloodtransfusion centers. only of blood donors were consistently positive, but samples produced discrepant results, although five of the assays used the same inhibition procedure of labeled anti-hbc (caspari et ai., j. clin. microbiol., in press). we reexamined the clearly and discrepantly positive samples of this study (besides some negative controls) by an assay which measures the binding of igg to hbc antigen by peroxidase labeled anti-igg. practically all consistently positive samples were confirmed by the different test principle while practically all discrepant samples were negative. this shows that consistent results by different inhibition assays are indeed reliable while divergent results can be neglected. anti-hbc is used in some countries as surrogate test for hcv carriers. using an experimental test kit from ortho diagnostics, of confirmed anti-hbc positiive and of confirmed anti-hbc negative donors reacted repeatedly as anti-hcv positive. these data do not provide evidence that anti-hbc is useful for elimination of hcv carriers. in this study we compared directly the detection level, sensitivity, and specificity of the most sensitive radioactive and the most sensitive non-radioactive method for detecting hepatitis b virus (hbv) dna in patient serum by dot blot hybridization, based on our previous experience with different assay systems. the former employed the p-labeled hbv rna probe included in the hepprobe kit (gibco-brl), detected autoradiographically. an advantage of this kit is the low level of radioactivity of the pre-labeled probe « f,tc), thereby permitting its use even in those laboratories lacking a license for radioisotopes. the non-radioactive method involved the use of an hbv dna probe sulfonated with sodium bisulfite (chemiprobe kit, orgenics), followed by immundetection and an enzymatic color reaction. the detection level of the p probe was found to be . pg hbv dna, corresponding to x genomes in f,tl serum, compared with only pg with the sulfonated probe. subsequently, sera from patients with various constellations of hbsag, and anti-hbe were tested with both methods. the concordance rate was % (r = . ). compared with p results, sulfonation showed a sensitivity of % and a specificity of %. radiolabeling, therefore, still allows the most sensitive and reliable detection of hbv dna in serum. sulfonation could eventually provide a feasible alternative to radioactivity if the viral dna in serum could be sufficiently amplified in vitro, for example with the polymerase chain reaction. such studies are being planned in our institute. key: cord- -g jiaenw authors: stoevesandt, oda; taussig, michael j; he, mingyue title: protein microarrays: high-throughput tools for proteomics date: - - journal: expert rev proteomics doi: . /epr. . sha: doc_id: cord_uid: g jiaenw protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. they are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. while conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding dna arrays. this article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics. protein microarray technology has rapidly developed into one of the most active areas in biotechnology today, and is being increasingly applied for the high-throughput discovery of molecular interactions, profiling of protein expression and monitoring of protein modifications. while genome sequencing and annotations provide comprehensive information on protein-encoding genes, and dna arrays analyze gene expression at the mrna level, these technologies need to be complemented by functional proteomic studies in order to better understand protein activities, expression levels and interactions at the system level. owing to alternative mrna splicing and a multitude of post-translational modifications (ptms), the number of cellular protein species far exceeds that of the genes. in addition, expression levels of individual proteins can range over as much as six orders of magnitude when cells are exposed to different environments [ ] . it is clear that, for large-scale analyses, proteomics studies require sensitive, high-throughput tools with a wide dynamic range of detection. one such tool is the protein microarray, consisting of several thousands of protein species immobilized on a solid surface in miniaturized features, enabling sensitive, high-throughput screening with economical use of samples and reagents. in contrast to screening systems such as yeast two-hybrid, the in vitro nature of protein arrays allows for the control of the interaction conditions, protein concentrations, ptms and specific cofactor requirements [ ] . the generation of a large diversity of proteins, such as the array elements, presents a major challenge for protein microarray systems, despite the develop ment of high-throughput proteinproduction methods. for many applications, the proteins also need to remain stable and functional on the array surfaces during long-term storage, which can pose an additional difficulty. a recent advance to overcome these problems is to produce protein microarrays through in situ cell-free synthesis directly from arrayed dna templates [ ] [ ] [ ] . this article reviews the conventional methods, as well as more recent developments, in protein microarray techno logies, together with novel applications of these technologies in medical and proteomics studies. review stoevesandt, taussig & he or antigens (that may be nonfolded) are arrayed for profiling the expression of proteins [ , ] or for the quantification of antibodies [ ] in complex samples such as serum. applications of antibody arrays include biomarker discovery and monitoring of protein quantities and activity states in signaling pathways. antigen arrays are applied for profiling antibody repertoires in auto immunity, cancer, infection or following vaccination. moreover, antigen arrays are tools for controlling the specificity of antibodies and related affinity reagents. reverse-phase arrays comprise cell lysates [ , ] or serum samples [ ] . analysis is multiplexed by probing replicates of the arrays with different antibodies. reverse-phase arrays are particularly useful for studying the changes in the expression of specific proteins and protein modifications during disease progression and, thus, are primarily applied for biomarker discovery. figure shows the two general strategies for making protein micro arays. in conventional approaches, individual proteins are expressed by recombinant technologies and are then purified and spotted onto a solid support, often glass slides. by contrast, in the newer in situ arrays, proteins are directly produced onto the array surface through cell-free protein synthesis. the most commonly used strategies for the production of proteins for arrays are heterologous expression in escherichia coli [ ] or by cell-free systems [ ] . the choice of protein production method depends on the downstream applications, in which protein yields, folding and modifications must be taken into account. clearly, high-throughput formats are desirable for generating proteins. the advantages of using e. coli are rapidity, low cost and ease of adaptation to high-throughput production. however, some eukaryotic proteins are poorly expressed in e. coli or are produced, but at low solubility, and eukaryotic ptms are lacking in prokaryotic cells. screening assays have been developed for the rapid assessment of protein solubility, including using reporter proteins to directly monitor protein expression and correct folding [ ] [ ] [ ] . cell-free protein synthesis provides an effective and rapid alternative by using pcr dna fragments as templates, circumventing the need for e. coli cloning [ , ] . cell-free methods are capable of making proteins that are difficult to express in in vivo systems. cell-free lysates can be prepared from different species, allowing protein synthesis to be carried out in prokaryotic or eukaryotic systems and at defined temperatures. these open systems can also be adjusted through the addition of further components, so as to provide environments suitable for protein folding or modifications [ ] . in conventional methods, proteins expressed by e. coli or cell-free systems are purified prior to spotting onto the array surface. purification is usually performed using affinity tags attached at either the n-or c-terminus, of which the most popular tags are hexahistidine (his ) and glutathione-s-transferase (gst) [ , ] . by contrast, by spotting unpurified tagged proteins onto capture surfaces, the immobilization and purification steps can be combined into a single step [ ] . proteins are often arrayed onto functionalized glass slides, using four main physicochemical principles, which may be combined: adsorption, covalent binding, affinity interaction by specific tags and physical entrapment into gels [ ] . surface adsorption is generally mediated by electrostatic charges (e.g., on polylysine-coated slides) or hydrophobic interactions. despite its simplicity, the main drawback of this method is the possibility of denaturating proteins on the surface. nevertheless, studies have suggested that surface-adsorbed proteins may still retain their functionality [ ] . surface adsorption is convenient where native protein structure and function are not essential; for example, antigen arrays. covalent binding of proteins to derivatized surfaces is a more efficient and robust approach [ ] . the surfaces usually carry reactive groups, such as epoxides, aldehydes, succinimidyl esters or isothiocyanates, which react with nucleophilic groups (e.g., amino, thiol or hydroxyl groups) of amino acid residues. covalent immobilization via random attachment also tends to denature arrayed proteins. affinity interaction by specific tags provides a means of immobilizing proteins in a defined orientation on a tag-capture surface, often retaining full protein activity. examples include the use of biotin, his -and gst-tagged proteins binding to streptavidin, ni-nta and glutathione-coated slides, respectively [ , ] . proteins fused with a carbohydrate-binding module have been successfully immobilized on a cellulose-coated surface [ ] . affinity-capture surfaces are also capable of immobilizing tagged proteins directly from complex mixtures without prior purification [ ] . recently, an enzymatic method has been described for covalent immobilization of tagged proteins directly from cell lysates [ ] . the use of d matrices (hydrogels) to entrap proteins into a structured environment maintains the function of arrayed proteins. usually, a layer of a polymer, such as polyacrylamide, agarose or gelatine, covers the slide and provides a porous structure filled with an aqueous buffer. the matrix can be additionally modified with functional groups for covalent or affinity immobilization of proteins. gel matrices permit the arraying of enzymes, while conserving their activities [ ] . a systematic study comparing eight different commercially available array surfaces, combined with detection by different fluorescent dyes, has revealed critical parameters affecting the reproducibility and reliability of protein microarrays [ ] . robotic spotting of proteins is generally carried out by either contact printing with solid or split metal pins, or via non contact delivery in an ink-jet printer-like fashion. unlike arraying of dna samples, procedures for spotting functional proteins must ensure that they are hydrated, stable and folded during the process. however, the distributed volumes of solutions are typically in the subnanoliter range and the spots are therefore prone to drying rapidly by evaporation. buffers for protein arraying therefore need to combine physiological conditions and compatibility with both the surface-immobilzation mechanism protein microarrays: high-throughput tools for proteomics and the robotic-arraying process in terms of viscosity and surface tension. typical buffer additives for protein stabilization against drying are hydroxylated cryoprotectants (e.g., glycerol, polyethylene glycol, trehalose and sucrose), but their effectiveness must be evaluated with the surface of choice [ , ] . several new nonnucleophilic substances have recently been identified as buffer additives that improve spot morphology without interfering with immobilization [ ] . an innovative method for preventing droplet evaporation was demonstrated by spotting under a layer of water-immiscible liquid fluorocarbon, coupled with a liquid-bridge, noncontact spotting technology [ ] . depending on the types of slides and the viscosity of the buffer used, printing density can generally be up to , individual features on a standard microscope slide, corresponding to a center-to-center spacing of spots in the range of µm [ ] . to avoid separate expression and purification of individual proteins by cell-based methods, cell-free expression has been exploited to produce protein microarrays by synthesising proteins directly onto the surface from arrayed dnas (figure ) . a number of different methods have been developed [ ] . in the protein in situ array (pisa) method, pcr dna constructs and the cell-free expression system are co distri buted onto protein-capture surfaces, so that newly synthesized proteins are captured in situ and unbound lysate material can be washed away (figure a ) [ ] . pisa first demonstrated the advantages of 'on-chip' synthesis of proteins, notably the ability to convert dna directly into functional proteins in an array format, without separate e. coli cloning, expression and purification. pisa also avoids the problem of maintaining protein stability on the array surface in long-term storage, since the proteins are only expressed as and when the protein array is needed. the pisa concept was first exemplified using an engineered double-his tag, which permits single-step immobilization of the tagged proteins onto ni-nta-coated surfaces from the cellfree lysate [ , ] . a highly miniaturized version, based on a multiple spotting technique (mist), has been developed, allowing in situ synthesis of proteins on the surface in subnanoliter volumes ( pl). this makes it possible to produce protein microarrays with a potential density of up to , spots per slide [ ] . instead of codistributing soluble dna and cell-free lysate as in pisa, the nucleic acid programmable protein array (nappa) method creates a protein array from immobilized dna templates [ , ] . plasmid dna is immobilized together with a proteincapturing antibody onto a glass slide, which is then covered with a rabbit reticulocyte lysate cell-free system to express the proteins. the newly synthesized proteins become trapped by the antibody colocalized at each spot ( figure b ). nappa has been applied to produce protein arrays (up to spots per slide) identifying immune-response signatures of breast cancer autoantibodies in patient sera [ , ] and to isolate target proteins with agonist activities from a cdna library [ ] . as the proteins immobilized by nappa remain on the same surface as the dna array, the technology does not generate a 'pure' protein array, but rather one in which individual proteins are colocalized with both their encoding dna and the general capture reagent. in situ synthesis of protein array (see figure recently, a novel system has been introduced that converts a reusable dna array into multiple protein array copies ( figure c ) [ protein microarrays: high-throughput tools for proteomics dna array (but with a mirror-image layout). fully consistent with the concept that diffusion is the mechanism of protein transport to the capture slide, the protein-density profiles of dapa protein spots are well-defined gaussian distributions. dapa is capable of producing at least copies of the same protein array by the repeated use of a single dna array template [ ] . this technique may find particular use for the repeated printing of protein arrays in laboratories that do not have access to microarray spotters. dapa has been validated by arraying a number of different proteins, including antibody fragments, green fluorescent protein and transcription factors [ ] . in situ puromycin capture from mrna arrays a puromycin-capture strategy has been described to fabricate protein arrays by capturing nascent polypeptides in situ [ ] . it requires extra steps to make mrna-ssdna hybrid molecules, and protein yields are limited by the amount of mrna spotted. readout technologies to detect interactions of probes on protein microarrays can be grouped into label-based and label-free methods. to date, approaches based on fluorescent labeling are the most widely used, with fluorophores either directly attached to the interactors or introduced on secondary reagents, such as in sandwich-assay formats. label-free approaches, by contrast, measure an inherent property of the interactors themselves, such as mass or dielectric properties. fluorescence-based methods use widely available reagents and instrumentation, but the covalent introduction of fluorophores may affect the protein function or epitope being monitored. label-free technologies circumvent this problem and may allow real-time monitoring, but they require more sophisticated equipment. to date, the sensitivity of the routinely used label-based methods is higher than that of label-free approaches [ ] . current microarray scanners are typically equipped for fluorophore excitation at and nm, allowing dual color fluorescence detection in the cy and cy ranges. scanners with a wider range of excitation wavelengths, including nm suitable for gfp-fusion proteins, are also becoming increasingly available. direct labeling of proteins for interaction screens is usually by covalent conjugation of n-hydroxysuccinimide ester-linked fluorescent dyes to primary amines of the protein n-terminus or ε-nh of lysine residues. alternatively, thiol-reactive dyes are used to couple to cysteine residues. these methods can be applied to purified proteins as well as to complex mixtures (e.g., sera). for sample comparison, ratiometric two-color labeling strategies are used, analogous to those used for expression profiling on dna microarrays [ , ] . random chemical conjugation of dyes may interfere with protein function. cell-free expression systems provide alternatives for site-directed introduction of fluorophores at the protein n-or c-terminus. using a modified initiator trna, the n-terminal methionine can be replaced by a fluorophore-amino acid conjugate, with labeling efficiencies of % in an e. coli cell-free system [ ] . c-terminal labeling proceeds through the incorporation of fluorophorecoupled puromycin derivatives, with labeling efficiencies of up to % [ , ] . besides direct coupling of fluorophores, primary and secondary immunofluorescent staining protocols are often applied [ , ] . in antibody arrays, such sandwich-detection strategies provide a second level of target recognition, significantly increasing the specificity and sensitivity (down to picogram per milliliter) of target detection. enhanced sensitivity can also be achieved with a number of amplification systems. tyramide signal amplification uses peroxidase-coupled reagents to catalyze the coupling of reactive fluorophore derivatives to tyrosine residues in the vicinity [ , ] and can detect femtogram amounts of protein. dna-based technologies, such as rolling circle amplification (rca) [ , [ ] [ ] [ ] , as well as proximity ligation of oligonucleotides coupled to antibodies recognizing different epitopes [ , ] , provide high levels of amplification, essentially converting from protein to dna detection. quantum dots are emerging as the new-generation fluorophores for ultrasensitive readout of binding events [ , ] . recently, single-walled carbon nanotubes coupled to antibodies were used as multicolor raman labels for the highly sensitive detection of proteins arrayed on raman-scattering gold surfaces [ ] . it is likely that novel combinations of existing tools will continue to enhance the detection sensitivity of protein microarrays. current label-free methods applicable to array formats include mass spectrometry (ms), optical biosensors on metal surfaces (e.g., surface plasmon resonance [spr] and nanohole arrays) and conductance biosensors on carbon nanotube and nanowire surfaces [ ] . mass spectrometry offers intriguing possibilities through its ability to directly identify proteins, and is 'hypothesis-free' compared with identification via the use of specific antibodies. the direct interfacing of d polymer-based protein arrays to maldi-ms for the detection of antigen-antibody interactions and enzymatic activities has been demonstrated [ ] . porous silicon surfaces structured with arrays of nanovials have been applied for rapid tryptic digestion of biomarker proteins, subsequently identified by maldi [ ] . biosensor-based approaches detect the binding of a protein to a sensor surface by virtue of the change of the local refractive index or conductivity on the surface. they typically enable the detection of real-time interactions to determine association and dissociation kinetics [ ] . based on the principle of spr, gratingcoupled spr [ ] allows higher degrees of multiplexing, with up to arrayed elements [ ] . a recently developed interferometric biosensor, based on optical-phase differences due to molecular associations, allows for real-time kinetics with picogram per square millimeter sensitivity [ ] . nanohole-array sensors utilize the optical properties of light transmission through - -nm diameter holes in metal surfaces, which are susceptible to changes in the local refractive index brought about by molecular interactions [ ] . biosensors of carbon nanowires and nanotubes, which review stoevesandt, taussig & he detect changes in conductivity on the surface upon binding of molecules, can also be used to detect molecular interactions with proteins, small molecules and nucleic acids [ , ] . interactions of proteins, both with other proteins and with nonprotein partners (e.g., nucleic acids, small molecules and lipids), form the basis of many cellular pathways. global identification of these interactions greatly contributes to our understanding of signal transduction and its aberration in disease, which, in turn, facilitates target discovery and drug design. with regard to identifying protein interactions and networks, protein microarrays will not generally be the sole definitive technique, but are likely to be complementary and confirmatory to a range of other methods, from isolation and characterization of protein complexes by pulldown and ms, to yeast two-hybrid screening and similar in vivo systems. nevertheless, protein arrays provide the benefit of studying defined individual protein components under controllable conditions. functional proteome arrays provide a high-throughput system for exploring protein interaction networks, as first exemplified by the landmark application of a yeast proteome array [ ] . one-onone screening of interactions by functional protein micro arrays often identifies novel interactions. for example, screening of arrayed coiled-coil domains from human leucine zipper transcription factors against each of the domains in their soluble form led to the discovery of a number of previously unknown pairwise interactions. their relative binding strengths, as determined on the array surface, were also in good agreement with those from solution-based studies [ ] . in another study based on in situ arraying by nappa, a protein functional array containing proteins involved in dna replication initiation was probed with each of these proteins in turn. a total of pairwise interactions were identified, of which were previously unknown [ ] . novel interactions with calmodulin and calmodulin-related proteins in the presence of calcium have been identified through the use of high-density functional protein microarrays prepared from open reading frames of arabidopsis spp. [ ] . the regulatory functions of the proteins for a wide range of targets and cellular activities were revealed. unknown interactions were also identified directly from cell lysates using arrays of functional proteininteraction domains, such as sh , sh , pdz, ww, ff, ph, fha and . . [ ] . in an approach based on parallel capture of native protein complexes from lysates of differentially stimulated t cells, peptide microarrays were used for the ana lysis of signaling-dependent protein-interaction networks [ ] . functional arrays of nuclear hormone receptors and coactivators were used for the systematic detection of receptor dimerization, functional classification of recruited coactivators and pharmaco logical characterization of receptor ligands [ ] . recently, an interesting example has demon strated the use of human protein microarrays for identifying polyanion-binding proteins [ ] . arrayed proteins, such as transcription factors, can be probed with dna or rna, enabling simultaneous identification of both the nucleic acid-binding proteins and their target sequences [ ] . the first proteomic array employed for the ana lysis of dna-binding events contained yeast proteins. after screening with fluorescently labeled genomic ssdna or dsdna, proteins that bound at least one of the dna forms were identified [ ] . in another assay, genomic dna from saccharomyces cerevisiae was used to probe proteome chips, identifying dna-binding proteins, including known transcription factors. this report also identified a novel dna-binding protein with a metabolic enzyme activity directly regulating eukaryotic gene expression [ ] . recently, commercial protein microarrays have been used for the large-scale search of protein-dna interactions [ ] . a microarray composed of known and predicted yeast transcription factors detected numerous pairings of transcription factors and their binding dna sequences. target genes for one previously uncharacterized dna-binding protein were identified and shown to be involved in the stress response and oxidative phosphorylation [ ] . the sequence-specific binding to dna of two bacterial transcription factors (argr repressor protein and camp-binding protein) and the dna-replication protein (dnaa) have also been demonstrated on protein microarrays [ ] . using p-labeled dna as the probe, p dna-binding variants were identified on functional arrays, and their relative affinities were simultaneously measured [ ] . a screen for protein-lipid interactions was performed on functional yeast proteome arrays [ ] . probing with several phospho inositides pointed to six new proteins associating with signaling lipids. the f irst example of using a protein array to analyze protein-small molecule interactions was between fkbp and three fluorophore-coupled small molecules, showing differences in the affinity of their interactions [ ] . binding of gtp to gtp-binding protein on functional arrays has also been demonstrated [ ] . a significant finding was that three different g-protein-coupled receptors arrayed together with their associated membrane lipids retained high-affinity, selective interactions with small-molecule ligands, demonstrating the functionality of arrayed membrane proteins [ ] . screening of yeast protein microarrays with small molecules led to the discovery of a target protein required for the suppression of rapamycin growth arrest [ ] . it is anticipated that, in the future, functional protein arrays will be further explored for screening a variety of biological and synthetic small molecules for their potential as drug candidates [ ] . antibody and antigen arrays are ideal 'capture' tools for profiling protein expression or immune responses, with applications in diagnostics and biomarker discovery. protein microarrays: high-throughput tools for proteomics arrays comprising large numbers of immobilized anti bodies of different specificities are a sensitive means for qualitative and quantitative determination of protein expression [ , [ ] [ ] [ ] . antibody arrays can be used to capture specific proteins from a variety of biological samples (e.g., lysates of cultured cells, tissue lysates and body fluids), enabling the identification of possible diagnostic signatures of disease-associated proteins [ , , ] , and have been applied with success to cancer studies. a recombinant antibody (scfv) microarray allowed profiling of proteins from the sera of pancreatic adenocarcinoma patients, producing a protein signature of nonredundant proteins that could discriminate cancer patients from healthy individuals [ ] . in a further example of antibody array application to biomarker discovery, out of proteins were identified as having significant changes in their expression profiles in biopsies of human squamous cell lung cancer carcinoma compared with normal tissues [ ] . the recent development of antibody microarrays in suspension using color-coded beads offers a fast, flexible, sensitive and multiplexed means for screening large numbers (> ) of clinical serum samples [ ] . the content of antibody arrays, in terms of their range and quality, is a critical issue with regard to their applications. antibody arrays used in protein profiling must recognize the native conformations. antibodies have different affinities for their specific targets, so the ana lysis of ligand binding in parallel and on the same surface may require the optimization of antibody concentrations prior to spotting. the concentration range of the target proteins and the conditions under which different ligands bind may also require optimization of buffers. in addition, cross-reactivity of antibodies can cause a variable background. protein mutations, modifications and epitope changes may result in the failure of antibody arrays to detect these proteins. to help reduce these problems, the 'antibodypedia' database has been established, providing details of individual antibody validation from publicly available resources [ ] . antigen arrays are used to profile antibody responses in autoimmunity, cancer, infection or following vaccination. proteomewide antigen arrays generated through cell-free synthesis of proteins from vaccinia virus [ , ] or proteins from francisella tularensis have been used to study humoral immune responses to infection with these agents [ ] . they produced specific antibody profiles in sera from vaccinated or infected humans and animals that, in turn, facilitated the rational design of vaccines and diagnostic antigens [ ] . other examples are the identification of immunodominant antigens from yersinia pestis [ ] and potential vaccine candidates from plasmodium falciparum (a prominent malaria pathogen) proteins [ ] . profiling autoantibodies has an important potential for biomarker discovery, with further uses in diagnostics and therapy selection. for example, the patterns of antibody reactivity in multiple sclerosis sera identified by antigen microarrays, comprising heat-shock proteins, lipid autoantigens and cns proteins, distinguished as different forms of the disease [ ] . a remarkable 'myelin proteome' antigen microarray has been used to identify 'epitope spreading' through profiling the evolution of autoantibody responses that, in turn, guided the design of tolerizing dna vaccines for encephalomyelitis [ ] . to address autoimmune events associated with neurological disorders, cerebrospinal fluid, rather than serum, has also been studied on antigen microarrays [ ] . as well as in the established autoimmune diseases themselves, profiling autoantibodies may be a future means of cancer diagnosis, and the antigens identified may also be potential immuno therapeutics [ ] . a conformational protein array containing folded and functional autoantigens was capable of detecting serum antibody profiles from non-small-cell lung cancer patients [ ] . reverse-phase antigen arrays, prepared by directly spotting various dilutions of lysates from human cancer cell lines, have led to the identification of two promising pathological markers distinguishing colon from ovarian adenocarcinomas [ ] . reverse-phase arrays allowed profiling of protein levels from as few as three cell protein equivalents in samples of primary leukemia and hematopoetic stem cells [ ] . antigen arrays are also an ideal method for profiling specificity and cross-reactivity of affinity reagents such as antibodies. a proteome microarray comprising different yeast proteins was used to screen polyclonal and monoclonal antibodies for a comparative profiling of antibody crossreactivity [ ] . a commercial human-protein array comprising approximately , recombinant proteins has been found to be particularly useful for the rapid characterization of antibody specificity [ ] . the activity of signal transduction pathways can be measured using arrays by determining the amounts and activation state of the signaling proteins downstream of receptors. this was demonstrated for the erbb receptor tyrosine kinase pathway using lysates of human tumor cell lines and antibody microarrays. this approach also enabled the characterization of the inhibitory profile of a small-molecule inhibitor on the pathway level [ ] . to facilitate our understanding of pathways and their changes in disease, the effects of drugs and growth factors on the ptms of specific proteins in normal and tumor cells have been studied by antibody micro arrays [ , ] . a reverse-phase micro array approach has been successfully applied to discover distinct pathway-activation differences among breast cancer cell lines, demonstrating the use of array-based ana lysis for multiple pharmacodynamic biomarkers responding to targeted kinase inhibitors [ ] . additional signaling pathways or defects can be ascertained by exposing tumor cells or other diseased tissues to cytokines, chemokines or other bioactive molecules, using antibody arrays [ ] or reverse-phase arrays [ ] . in the future, it should be possible to reconstitute the complex pathways connecting the genome and the proteome in normal and diseased states through combining protein microarrays with genomicprofiling technologies. however, undertaking multiplexed quantitative ana lysis of protein-ligand interactions on microarrays, even with purified individual domains of the same functional families, requires careful standardization to avoid pitfalls due to review stoevesandt, taussig & he dissimilar behavior of the proteins and variable densities of active proteins. these considerations and the danger of relying on single concentration determinations have been discussed with respect to binding of phosphopeptides by an arrayed set of sh domains [ ] . protein microarrays provide a high-throughput method for ana lysis of ptms such as glycosylation and phosphorylation. protein microarrays allow large numbers of different glyco proteins to be studied in a single experiment [ ] . currently, lectin microarrays are used for high-throughput identification of protein glyco sylation [ ] , while glycoprotein microarrays can be probed with a variety of lectins in turn to reveal glycosylation patterns in sera from healthy controls and patients with chronic pancreatitis and pancreatic cancer [ ] . while phosphorylation controls much of the protein activity in living cells, linking particular kinases with specific substrates has been problematic. with functional protein microarrays, novel kinase substrates have been identified. for example, an array of proteins from arabidopsis thaliana was screened with mpk and mpk , respectively, followed by antiphosphotyrosine antibodies, leading to the identification of and candidate substrates for mpk and mpk , respectively [ ] . in another study illustrating the power of functional yeast proteome arrays, substrate screening identified candidate targets for each of the yeast kinases [ ] . such discovery approaches are complemented by reverse-phase and antibody microarrays for the parallel ana lysis of known protein phosphorylations. using reverse-phase microarrays, site-specific phosphorylation of signaling proteins from il- -stimulated t cells was detected, revealing differential protein phosphorylation [ ] . antibody microarrays have been employed for the time-resolved monitoring of cell-signaling pathways by quantifying both the amount and the phosphorylation status of signaling proteins [ ] . activities of phosphatases, cysteine proteases and serine hydrolases have been studied on array surfaces [ ] and by using suicide inhibitors, which attach covalently to the active site [ ] . sulfation reactions can also be studied on a chip surface [ ] . significantly, enzymes constituting an entire functional pathway can be identified by enzyme arrays. this has been achieved by immobilizing mixtures of enzymes in different ratios on an array surface. activities and efficiencies of different enzymes were analyzed by product detection. two-step sequential reactions, catalyzed by either luciferase or nucleoside diphosphate kinase, and a five-step pathway for trehalose synthesis, have been demonstrated [ ] . this approach provides a means of optimizing enzyme proportions for maximal pathway-output efficiency. an interesting application using metabolizing enzyme arrays for toxicologic screening of drugs has been described with arrayed p isozymes [ ] . after exposing the array to a candidate drug, a cell monolayer was grown on the p drug array and analyzed for the proportion of living and dead cells. capture microarrays provide suitable systems for mapping protein epitopes recognized by antibodies or other affinity reagents. by arraying all overlapping peptides of defined length taken from a protein sequence, linear epitopes with the minimal sequence requirement for molecular recognition can be identified. individual amino acids within an epitope can be assessed for their contribution through the arraying of protein mutants. a libraryscanning strategy was described in which each individual position in a -mer peptide was randomized by the other amino acids to generate peptide libraries [ ] . all libraries were then spotted and probed by the antibody. the detected signals indicated the role of particular residues in epitope recognition. identification of protective epitopes is essential for the design of effective vaccines. as there is no in silico method that can accurately predict the target proteins or epitopes that stimulate protective antibody immunity, screening with protein microarrays is useful for identifying immunodominant antigens from complex pathogens. in one such example, the immunogenic epitopes of the sars coronavirus were identified after screening humansera samples from infected individuals on capture arrays of viral proteins [ , ] . although a variety of array formats are increasingly used in basic research, biotechnology and medical applications, protein microarrays have not yet reached their full potential, particularly in academic research, for a number of possible reasons. current commercial array production methods are generally based on separate expression and purification of proteins; thus, they are prohibitively expensive, with costs of approximately us$ per slide. typically, academic applications require customized arrays displaying proteins of interest rather than complete proteome arrays. moreover, protein arrays for species other than humans and the most widespread model organisms are not commercially available. another problem is their limited shelf life, especially for functional protein arrays. we expect that the in situ methods for protein-array generation will contribute to changing this situation, especially approaches such as pisa, nappa and dapa, which offer cheaper and more flexible methods for academic laboratories. while small numbers of customized array slides can be economically produced through in situ synthesis when required, conventional methods that rely on spotting purified proteins are only cost effective if large batches of arrays are produced. the dapa technique is particularly useful for laboratories without microarraying equipment as it can generate multiple copies of the same protein microarray using a single dna array template. in current clinical applications, antigen microarrays and reverse-phase arrays are widely applied for profiling antibody repertoires from individual patient samples. protein microarrays: high-throughput tools for proteomics protein microarray technology will continue to be optimized in the coming years. the growing collections of cloned genes or gene sequences will facilitate the generation of 'proteome arrays' for many species. ongoing improvements in high-throughput protein expression and purification methods will enhance protein quality and quantity, increasing the functionality of arrayed proteins, with cell-free systems becoming a widely used alternative. technologies for making protein arrays by in situ synthesis will improve and accelerate array generation. further optimization of surface chemistries and structures, immobilization methods and readout technologies will allow for the generation of arrays with densities in the submicrometer scale. in proteomic applications, important areas of use will include high-throughput functional in vitro screens of molecular interaction networks, while for medical applications, arrays will be developed for diagnostics and the identification of relevant biomarkers. the increasing availability of affinity reagents through international initiatives such as proteomebinders will provide exciting possibilities in profiling cancer proteomes, to monitor characteristic protein signatures in patient sera or tumor extracts, leading to earlier diagnosis and prognosis before and during therapy [ ] . it is expected that the multidisciplinary collaboration of scientists from biotechnology, biochemistry, material sciences, physics and bioinformatics will ensure the continued development of robust and reliable protein microarrays, their widespread uptake and novel applications. the babraham institute is an institute of the biotechnology and biological sciences research council (bbsrc), uk. this review is an activity of the proteomebinders consortium, a research infrastructure coordination action supported by the eu th framework programme. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. • high-throughput protein production is desirable for making protein arrays. • arraying proteins in a defined orientation is a preferred approach, as it retains the protein activity on the array surfaces. • while current array readouts are mostly based on fluorescence, label-free detection methods have particular areas of use. • an emerging alternative to conventional protein arraying is the in situ synthesis and arraying of proteins by cell-free systems, as in the protein in situ array, nucleic acid programmable protein array and dna array to protein array technologies. • functional protein microarrays are increasingly used for high-throughput screening of molecular interactions, monitoring post-translational modifications and analyzing protein functions. • antibody arrays are valuable in profiling protein expression, thereby enabling the identification of biomarkers. antigen arrays are efficient tools for mapping protein epitopes and assist in the identification of vaccine candidates. • reverse-phase protein arrays are particularly useful for the screening of changes in the expression level of biomarkers and pathway targets involved in disease. • enzyme arrays provide a means for the large-scale ana lysis of enzymatic activities. pathway output efficiency can be enhanced by controlling the enzyme proportions spotted onto the array surface. papers of special note have been highlighted as: •• reports the use of a cell-free system to express the human proteome. in total, , clones have been constructed, of which , were tested by a cell-free, wheatgerm expression system. gene expression response to misfolded protein as a screen for soluble recombinant protein protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein cell-free protein synthesis: applications in proteomics and biotechnology proteomescale purification of human proteins from bacteria improved affinity coupling for antibody microarrays: engineering of double-(his)- -tagged single framework recombinant antibody fragments protein and peptide arrays: recent trends and new directions a protein-domain microarray identifies novel protein-protein interactions multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins global ana lysis of protein activities using proteome chips -breaking paper that reports the first use of a yeast proteomic array for large-scale screening and identification of calmodulin and phospholipidbinding proteins versatile protein microarray based on carbohydrate-binding modules direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase • recent report describing the covalent immobilization of tagged proteins on solid surfaces from cell lysates semi-wet peptide/ protein array using supramolecular hydrogel systematic comparison of surface coatings for protein microarrays comparison of hydroxylated print additives on antibody microarray performance antibody microarrays: an evaluation of production parameters investigation of non-nucleophilic additives for the reduction of morphological anomalies in protein arrays non-contact protein microarray fabrication using a procedure based on liquid bridge formation advances in functional protein microarray technology in situ synthesis of protein arrays review protein microarrays: high-throughput tools for proteomics emerging tools for real-time label-free detection of interactions on functional protein microarrays antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers cell-free n-terminal protein labeling using initiator suppressor trna fluorescence labeling of the c-terminus of proteins with a puromycin analogue in cell-free translation systems rapid functional ana lysis of proteinprotein interactions by fluorescent c-terminal labeling and single-molecule imaging a network ana lysis of changes in molecular interactions in cellular signaling a new two-color fab labeling method for autoantigen protein microarrays proteomic profiling of the nci- cancer cell lines using new high-density reversephase lysate microarrays signal amplification by rolling circle amplification on dna microarrays multiplexed protein profiling on microarrays by rolling-circle amplification allergen-specific ige detection on microarrays using rolling circle amplification: correlation with in vitro assays for serum ige ligation-based molecular tools for lab-on-a-chip devices proximity ligation assays for sensitive and specific protein analyses high throughput quantification of protein expression of cancer antigens in tissue microarray using quantum dot nanocrystals protein microarrays with carbon nanotubes as multicolor raman labels analysis of protein interaction and function with a -dimensional maldi-ms protein array high-speed biomarker identification utilizing porous silicon nanovial arrays and maldi-tof mass spectrometry label-free reading of microarray-based immunoassays with surface plasmon resonance imaging present and future of surface plasmon resonance biosensors grating-coupled surface plasmon resonance: a cell and protein microarray platform label-free and dynamic detection of biomolecular interactions for highthroughput microarray applications high-throughput nanohole array based system to monitor multiple binding events in real time nanowire nanosensors for highly sensitive and selective detection of biological and chemical species protein recognition via surface molecularly imprinted polymer nanowires comprehensive identification of human bzip interactions with coiled-coil arrays differential binding of calmodulin-related proteins to their targets revealed through high-density arabidopsis protein microarrays a proteomic microarray approach for exploring ligand-initiated nuclear hormone receptor pharmacology, receptor selectivity, and heterodimer functionality a network-based ana lysis of polyanionbinding proteins utilizing human protein arrays high-throughput and multiplexed protein array technology: protein-dna and protein-protein interactions regulation of gene expression by a metabolic enzyme identifying protein interactions with metal-modified dna using microarray technology linking dna-binding proteins to their recognition sequences by using protein microarrays dissecting dna-protein and proteinprotein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection functional protein microarrays for parallel characterisation of p mutants printing proteins as microarrays for highthroughput function determination microarrays to characterize protein interactions on a whole-proteome scale membrane protein microarrays finding new components of the target of rapamycin (tor) signaling network through chemical genetics and proteome chips identification of small molecule targets on functional protein microarrays antibody microarrays as an experimental platform for the ana lysis of signal transduction networks advances in recombinant antibody microarrays antibody arrays for determination of relative protein abundances profiling of cancer cells using protein microarrays: discovery of novel radiationregulated proteins antibody protein array ana lysis of the tear film cytokines detection of pancreatic cancer using antibody microarray-based serum protein profiling comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma antibody suspension bead arrays within serum proteomics antibodypedia, a portal for sharing antibody and antigen validation data profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery proteome-wide ana lysis of the serological response to vaccinia and smallpox immunodominant francisella tularensis antigens identified using proteome microarray vaccinia virus h l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice protein microarray for profiling antibody responses to yersinia pestis live vaccine profiling humoral immune responses to p. falciparum infection with protein microarrays antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis protein microarrays guide tolerizing dna vaccine treatment of autoimmune encephalomyelitis autoantibody profiling on high-density protein microarrays for biomarker discovery in the cerebrospinal fluid immunotheranostics: breaking tolerance in immunotherapy using tumor autoantigens identified on protein microarrays seromic ana lysis of antibody responses in non-small cell lung cancer patients and healthy donors using conformational protein arrays reverse phase protein array: validation of a novel proteomic technology and utility for ana lysis of primary leukemia specimens and hematopoietic stem cells analyzing antibody specificity with whole proteome microarrays • first report using a proteome array comprising different yeast proteins to screen the specificity of antibodies rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays profiling receptor tyrosine kinase activation by using ab microarrays antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells panorama ab microarray cell signaling kit: a unique tool for protein expression ana lysis proteomic ana lysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays proteome chips for whole-organism assays a ratiometric lectin microarray approach to ana lysis of the dynamic mammalian glycome glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera high throughput identification of potential arabidopsis mitogen-activated protein kinases substrates global ana lysis of protein phosphorylation in yeast review protein microarrays: high-throughput tools for proteomics •• reports a large-scale ana lysis of protein phosphorylation by the use of proteome chips. over phosphorylation events protein microarrays for multiplex ana lysis of signal transduction pathways reports the first description of the use of reverse-phase protein microarrays to monitor site-specific phosphorylation of many signaling proteins from stimulated living cells quantitative protein microarrays for time-resolved measurements of protein phosphorylation developing a strategy for activity-based detection of enzymes in a protein microarray enzyme familyspecific and activity-based screening of chemical libraries using enzyme microarrays • describes an interesting thematic enzymemicroarray format to characterize family-specific enzymes on array surfaces enzymatic activity on a chip: the critical role of protein orientation a functional protein chip for pathway optimization and in vitro metabolic engineering reports the use of protein arrays to reconstitute an enzyme pathway, demonstrating that entire pathways can be studied by protein microarrays metabolizing enzyme toxicology assay chip (metachip) for high-throughput microscale toxicity analyses protein microarrays for antibody profiling: specificity and affinity determination on a chip screening of specific antigens for sars clinical diagnosis using a protein microarray antigenicity ana lysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein • review article for in situ synthesis of protein microarrays. key: cord- -tsx e authors: byrne, barry; stack, edwina; gilmartin, niamh; o'kennedy, richard title: antibody-based sensors: principles, problems and potential for detection of pathogens and associated toxins date: - - journal: sensors (basel) doi: . /s sha: doc_id: cord_uid: tsx e antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. a critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. particular emphasis is placed on the detection of foodborne bacterial pathogens, such as escherichia coli and listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided. pathogenic bacterial, fungal and viral cells are ubiquitous in nature and pose a considerable risk to human and animal health, in addition to severely compromising the quality of agricultural produce (table ) . therefore, the monitoring of these microorganisms is of paramount importance for the prevention of nosocomial infections, the maintenance of general public health and for ensuring open access compliance with legislative and quality standards. the rapid detection and identification of a pathogen is essential, in particular where food samples with short shelf-lives are being analysed, or where the urgent administration of a suitable antimicrobial agent is required to treat a potential fatal infection. virulent pathogens may often be present in low numbers in samples, demonstrating that high sensitivity and specificity are also absolute necessities. hence, developing suitable detection methods which permit accurate, rapid and sensitive analysis is essential for monitoring the distribution of pathogens and, most importantly, ensuring customer/patient safety. table this review provides a comprehensive summary of the principles, problems and potential of using immunosensor-based analytical platforms for pathogen detection. it describes the development of electrochemical, potentiometric, piezoelectric and optical platforms for the monitoring of foodborne bacterial pathogens by incorporating monoclonal, polyclonal or recombinant antibodies in a variety of different assay formats. the overall strategy adapted is shown in figure . the analysis of fungal cells, associated toxic secondary metabolites, viral and water-borne pathogens (toxins and parasites) is also outlined. finally, the advantages of using sensor-based methodologies as an alternative to more traditional methods of pathogen detection, namely bacteriological testing and nucleic acid-based analysis, and alternative sensor-based formats (e.g. biomimetic and plant sensors), will be discussed. the culturing of pathogenic and non-pathogenic prokaryotic strains involves the aseptic transfer of an innoculum from a source (soil, food etc.) to suitable growth medium which results in amplification of microbial cell numbers, subsequently permitting quantitative determination [ ] . this propagation may be performed in the presence of selective markers, such as antibiotics, to suppress the growth of other strains that may also reside in the innoculum. subsequent transfer to selective or differential media generates colonies that can be distinguished based on their distinctive colony morphologies by ocular inspection (table ) and their identification confirmed by rigorous biochemical (glucose utilisation etc.) or nucleic acid-based assays [ ] . table . three commonly encountered bacterial foodborne pathogens with their selective media and epidemiological relevance. figures obtained for annual estimated cases and infectious doses (*) are obtained from reference [ ] and are representative of figures calculated by the united states department of agriculture (usda) economic research service. key: cfu -colony-forming units. colony count estimation provides an inexpensive and user-friendly protocol for quantitative and qualitative bacterial pathogen detection, and one which is routinely employed in the development of hazard analysis and critical control point (haccp) systems within the food industry and for the establishment of risk assessments [ , ] . however, a major disadvantage of this approach is the lengthy times required to obtain visible colonies that can be identified. this may take up to days for l. monocytogenes cells, cultured using the nf en iso - protocol [ , ] , and over weeks for another important food-related pathogen, campylobacter fetus [ ] . further complications with using this methodology arise from the ability of some bacterial strains to be viable but non-culturable. this phenomenon, and its importance from the perspective of the food industry, has recently been discussed with reference to l. monocytogenes [ ] and e. coli o :h [ ] . an alternative method for pathogen detection, and one which is often used in conjunction with active culturing to provide sufficient biomass, involves the amplification and subsequent analysis of pathogen-specific nucleic acid by polymerase-chain reaction (pcr) and sequencing ( table ). the versatility of these methodologies is emphasised by the ability of real-time pcr to provide rapid data analysis of multiplex pcr to facilitate the simultaneous analysis of multiple pathogens and of reversetranscriptase pcr to differentiate between viable and non-viable cells. furthermore, the presence of bacterial rnas (mrna and tmrna) in food samples can be determined through the use of nucleicacid sequence-based amplification (nasba) [ , ] . however, the implementation of these methodologies for pathogen detection can be complicated by external factors. for example, strains may originate from complex sample matrices, e.g. food sources that often contain high levels of fats, carbohydrates and other entities which necessitate a sample clean-up stage prior to analysis. furthermore, as discussed by de boer and beumer [ ] , the amplification of nucleic acid from a pathogenic strain is indicative only of its presence in the sample of interest and cannot be used to monitor toxin production qualitatively or quantitatively. non-specific dna amplification may also be observed; the presence of 'naked' dna in analytical samples may act as a template for the amplification of these superfluous products [ ] which complicates fingerprint-based analysis. therefore, alternative methods of pathogen analysis (e.g. antibody-based) can be more useful. a schematic representation of a full-length antibody is shown in figure . polyclonal, monoclonal and recombinant antibodies have frequently been selected for a wide variety of applications, including immunodiagnostics and biomarker detection. their production involves the exploitation of the immune system of murine, leporine, ovine or avian hosts ( figure a ). for the production of bacterial pathogen-specific antibodies, these hosts may be immunised with cells which may [ ] or may not [ ] be heattreated (exceptions include naïve antibody phage display libraries which are constructed independently of immunisations; see below). these antigens are typically administered in the presence of a suitable adjuvant, and the immune response generated by the host after a series of immunisations can be determined by screening serial serum dilutions for recognition of the antigen in an enzyme-linked immunosorbent assay (elisa)-based format. a schematic representation of an igg antibody comprising of two heavy (green) and light (blue) chains. carbohydrate elements are attached via the asparagine amino acid residue. a more in-depth discussion of antibody glycosylation is provided in reference [ ] . key: v h -variable heavy, v l -variable light, c h -constant heavy, c lconstant light. polyclonal antibodies (pab) are typically raised in rabbits, goats or sheep [ ] , and their popularity is illustrated by the fact that they are frequently selected in immunosensor-based assays for pathogen detection. it should be stressed that the inherent nature of pabs means that a selection of different epitopes may often be recognised on a single cell. in cases where this is undesirable, such as in the case where high specificity is a requirement, monoclonal or recombinant antibodies may be more applicable. monoclonal antibodies are generated through the use of hybridoma technology [ , ] and murine hosts are commonly selected for immunisation. the bone marrow, primary lymph nodes and, most commonly, the spleen are selected as a source of antibody-producing b cells which are harvested and fused to immortal myeloma cells. the resulting hybrid cells (referred to as hybridomas) subsequently secrete full-length antibodies that are directed towards a single epitope. suitable candidates, identified by elisa-based analysis, are then 'cloned out' to ensure that a single cell, producing antibody specific for an individual epitope, is present and the antibody generated can be used for assay development. recombinant antibodies, generated through the use of phage display technology and the biopanning of antibody repertoires (libraries) against a target of interest [ , ] , have been selected for the detection of a range of structurally diverse antigens, including proteins [ ] , haptens [ ] and carbohydrate moieties [ ] . three types of library may be used as sources of antibody pools; namely synthetic, naïve and immune. synthetic libraries are produced by pcr-based randomisation of complementarity-determining regions (cdr) from heavy and/or light chains, and naïve antibody libraries are typically assembled from b-cells extracted from unimmunised human donors. immune libraries are constructed from rna isolated from spleenocytes or the bone marrow of a host (avian, murine, leporine etc.) immunised with an antigen that generates the required immune response. the rna acts as a source of complementary dna (cdna) which, in turn, serves as a template for the amplification of variable heavy (v h ) and variable light (v l ) gene sequences which can be fused through an overlapping-extension splicing pcr reaction and subsequently cloned into a suitable phage or phagemid vector ( figure a ) [note that in the case of fragment antigen binding (fab) construction (see below), this process involves the fusion of variable and constant regions]. the introduction of this construct into suppressor strains of e. coli (such as xl blue) by electroporation, in conjunction with the packaging of phage particles via the addition of helper phage (a process referred to as rescuing), allows the encoded antibody structure to be 'presented' on the exterior of a bacteriophage particle, as illustrated in figure b . two types of antibody fragments may be presented, namely the single-chain variable fragment (scfv) and the fab, and these are illustrated in figure c and d. the production of these fragments is dependent on the vector selected for harbouring the library [ ] . biopanning is used for the selection of binders from an antibody library which may contain between and different antibody-encoding gene sequences. to achieve this, the antigen is immobilised on solid phase (e.g. on a column or immunotube) or bead-conjugated (in solution phase) and the antibody pool is subjected to recurrent rounds of selection against the antigen with increasing levels of stringency in terms of binding ability. selected binders are retained and subjected to additional screening to increase their specificity for the target (affinity maturation), which can be monitored by elisa-based analysis. the production of soluble antibody fragments can be facilitated by infecting phage pools into non-suppressor e. coli strains, such as top- f' or hb , and inducing with isopropyl-β-d- -thiogalactopyranoside (iptg) in the presence of low concentrations of glucose. these hosts recognise the amber (aug) codon engineered between the scfv and giii gene [ ] , producing scfv or fab fragments independent of the phage coat proteins. the majority of the examples given in this review involving immunosensor-based pathogen detection incorporate monoclonal or polyclonal antibodies. however, recombinant antibodies are as yet not fully exploited in this field and have several significant advantages over conventional antibodies. the specificity and sensitivity of recombinant antibodies for a particular antigen can be significantly enhanced by the targeting of cdr regions using site-directed mutagenesis or chain shuffling [ , ] . further advantages include the capacity to incorporate tags (e.g. his or c-myc) for isolation and, subsequently, immobilisation, the ability to fuse various labels (e.g. green fluorescent protein or enzymes) directly to the antibody fragment facilitating and simplifying detection, and the availability of a range of antibody formats (e.g. scfv, fab, re-engineered igg, dimers etc.). avian hosts, in particular, have been shown to be useful for the production of high-affinity recombinant antibodies [ , , ] . . immunisation-related stages are represented by a red line, with those involving antibody production shown in black. a more in-depth discussion of the generation of recombinant antibodies, inclusive of fab fragments, can be found in reference [ ] . additional hosts may also be used for antibody production, including camels (camelid), sheep (ovine) and pigs (porcine). a filamentous phage displaying scfv antibody fragments when selecting monoclonal, polyclonal or recombinant antibodies for the detection of pathogens, certain characteristics are of great importance. firstly, the antibody should be able to detect and quantify very low cell numbers (sensitivity) and this may frequently be an issue for foodborne-related bacterial pathogens (table ) . secondly, it should be able to differentiate specific strains of interest from related microflora which may also reside in the sample (specificity). hence, the selection of a highly-specific epitope on the pathogen is a key consideration, since many bacterial strains share homologues of surface-presented proteins which can lead to the detection of multiple cell-types by a single antibody. it is therefore recommended that a constitutively-expressed antigen, which is species-specific, is targeted [ ] . where possible, the expression of this target antigen should not be highly dependent on the growth matrix of the pathogen. finally, the antibody should bind with its cognate target with sufficient strength to permit interrogation (high-affinity). the identification of an antibody candidate that satisfies these requirements can be facilitated through screening by elisa-based analysis to reduce the number of potential antibodies to a smaller number which can subsequently be screened by sensor-based analysis to identify the candidate with the best affinity for the target epitope. this antibody can then be further selected for incorporation on an immunosensor-based platform. antibodies can be successfully used to isolate and collect pathogens from complex matrices where numbers are low and the sample volume is large. immunomagnetic separation (ims) involves the coating of a pathogen-specific antibody on a magnetic bead which can be used to facilitate binding, concentration and removal of pathogenic cells from a complex sample media (provided that the antibody has sufficient specificity). retrieved cells can then be propagated on selective of differential media. the versatility of this technique is further illustrated by the ability to detect pathogen-antibody complexes with beads coated with a cognate secondary antibody, an indirect assay format demonstrated by torensma and co-workers [ ] for the detection of l. monocytogenes cells bound to a murine monoclonal antibody produced from the immunisation with whole bacterial cells. ims has also been applied for the detection of these cells in cheese [ ] , s. typhimurium in bovine faecal matter [ ] and e. coli o :h in beef carcasses [ , ] and bovine faeces [ ]. elisa-based analysis can be directly applied for the detection of foodborne pathogens. brooks and co-workers [ ] developed an elisa-based assay for the detection of c. fetus in bovine preputial washing and vaginal mucous samples. a sandwich assay format was developed by kerr et al. [ ] for the detection of a selection of different e. coli o :h strains from human and animal subjects by using a monoclonal antibody specific for a fimbrial antigen. the limit of detection was similar to that found by brooks et al . ( × cfu/ml). elisa-based assays have also been described for the detection of l. monocytogenes [ , ] and salmonella enterica spp. [ ] . a major drawback of these assay formats is that analysis times are often lengthy. typical elisa assays are comprised of a number of steps; namely blocking, washing, incubation of primary and secondary antibodies and substrate development. these can take several hours to complete and, understandably, this may be problematic in instances where rapid detection is a requisite. protein microarrays are excellent candidates for high-throughput analysis of biomolecular interactions in miniaturised assays formats [ ] . the implementation of antibodies on such platforms for pathogen detection offers a flexible approach for the screening of high number of bacterial isolates from numerous sample matrices. array formats typically consist of a panel of pathogen-specific antibodies spotted onto individual positions on a microarray slide by dedicated robotic handling (printing), with subsequent pathogen detection commonly employing sandwich elisa assay formats. gehring and colleagues [ ] printed a biotinylated antibody (caprine-derived) for e. coli o :h on a streptavidin-coated microarray slide. captured cells were further probed with a fluorescein-labelled secondary antibody and microarray spots were visualised through the use of fluorescent microscopy. this sandwich assay format had a linear range of detection of × - × cells/ml. cai et al. [ ] developed an antibody microarray capable of the parallel analysis of somatic (o) and flagellar (h) antigens on salmonella strains from twenty commonly encountered serovars, including typhimurium, heidelberg and enteritidis. thirty five polyclonal antibodies from rabbit antiserum were spotted in pairs on commercially obtained microarray slides and used for the capture of eosin yfluorescently labelled cells. over % of the strains selected for analysis were positively identified, with an additional strains partially serotyped. the ability of this assay format to selectively differentiate between related and unrelated salmonella strains was demonstrated by the analysis of an additional strains selected from a panel of almost forty non-target serovars. anjum et al. [ ] also targeted o-antigen groups of e. coli strains with rabbit polyclonal antisera. the ability of these microarray formats to screen numerous bacterial pathogens in parallel was further illustrated by the printing of individual antibodies in a -well polystyrene plate by an 'in-house' robotic printing system. this cost-effective array format, devised by gehring and colleagues, was selected for the simultaneous detection of e. coli ( × cells/ml) and salmonella typhimurium ( × cells/ml) in buffer and ground beef extract [ ] and further illustrates the versatility of such antibody-based formats. finally, antibody-based microarray platforms have been selected for the sensitive and parallel detection of structurally diverse pathogens. these include bacterial strains that pose a potential bioterrorism risk (e.g. burkholderia mallei, f. tularensis and y. pestis) and viral particles (e.g. west nile virus) [ ], foodborne pathogens (campylobacter jejuni ) and mycotoxins [ ] and, finally, sporeforming bacterial cells (b. globigii ) and toxins [ ] . the latter assays [ , ] implement the naval research laboratory (nrl) array biosensor. this elegant platform can simultaneously detect pathogenic bacterial cells and toxins, and can perform sandwich (as is the case with many of these examples) and competition immunoassays in parallel with an assay time of approximately minutes [ ]. toxin and virus-related pathogen detection is discussed in sections and with reference to immunosensor-based analysis. biosensors are analytical devices which combine a biological recognition ligand with physical or chemical signaling devices (transducers). the recorded biomolecular interactions are transformed into digital signals which are interpreted by a computer-aided readout, thereby providing the user with a representation of the interaction that occurs between the bound (ligand) and free (analyte) entities ( figure ). many different sensor formats have been utilised for pathogen analysis using antibodies; namely electrochemical, mass-based, magnetic and optical. the sensitivities of these assays are dependent on the properties of the transducer and the quality of the antibody. an overview of each sensor type and an explanation of how antibodies can be incorporated for pathogen detection follows. here, a full-length antibody is captured on protein a immobilised on a carboxymethylated dextran-coated sensor surface and is used for the capture of an analyte. this interaction produces a specific physicochemical change, such as a change in mass, temperature or electrical potential. this is then converted (via a transducer) to a signal which the user can interpret. the principle behind these assay formats is the coupling of a specific antibody with an electrode transducer which functions to convert a binding event into an electrical signal. in general, electrochemical biosensors can be based on four transducer types; namely amperometric, impedimetric, potentiometric and conductimetric. many amperometric biosensors utilise an enzyme-based system that generates an electroactive product which can be oxidised or reduced at a working electrode (carbon, gold etc.). the resultant current can then be detected. this format has several advantages, including the capacity to fabricate disposable and customised screen-printed electrochemical electrodes (screen-printed carbon electrodes) by depositing inks (carbon, silver etc.) in a pre-determined arrangement and thickness [ ]. these systems are economical, robust and sensitive and can be used in conjunction with mediators such as ferrocenedicarboxylic acid (fedc) or iodine to improve selectivity [ ] . furthermore, there is major potential to miniaturise these systems. this leads to smaller sample volume requirements. gehring et al . [ ] developed an amperometric assay for the detection of s. typhimurium cells which were captured with magnetic bead-conjugated antibodies and detected with an alkalinephosphatase (ap)-labelled goat anti-salmonella antibody. after deposition of the beads on graphite ink electrodes, the ap-catalysed production of electroactive para-aminophenol (pap) from its substrate, para-aminophenyl phosphate (papp), was monitored electrochemically and the generated signal was directly proportional to the number of captured bacterial cells. this assay had a sensitivity of × cells/ml. ivnitski and co-workers [ ] also applied this methodology for the detection of campylobacter. here, anti-campylobacter antibodies were embedded in a bilayer lipid membrane and, upon binding with free cells, a conformational change was introduced which allowed the transport of ions through the membrane. the resultant current was detected amperometrically via a stainless steel electrode. this rapid ( minutes) assay allowed the researchers to verify that ions could pass through the channel per second. this value was correlated with a theoretical value of one bacterial cell per sample. lin and co-workers [ ] recently immobilised a monoclonal antibody on screen-printed carbon electrodes (spce) for the capture of pure cultures of e. coli o :h , and implemented a horseradish peroxidase (hrp)-labelled polyclonal antibody for detection in an indirect sandwich assay format. it was noted that the attachment of gold nanoparticles and the use of fedc, as a mediator, resulted in a noticeable amplification of the response current generated. this enabled the detection of approximately × cfu/ml in hour. the assay had excellent selectivity and specificity, with minimal cross-reactivity observed when groups of other food pathogens were tested in parallel (l. monocytogenes, salmonella choleraesuis and vibrio paraheamolyticus ), thus illustrating the importance of having a selective biorecognition element. crowley and colleagues [ ] also selected a spce-based platform for l. monocytogenes detection. a direct sandwich assay format, consisting of a leporine polyclonal capture antibody and an ap-labelled detection antibody, could detect × cells/ml. comparable sensitivity was observed when polyclonal goat ( × cells/ml) andrabbit ( × cells/ml) antibodies were used for capturing cells in an indirect sandwich assay format. the direct immobilisation of l. monocytogenes cells on the spce provided a low response. impedimetric biosensors are often based on the fact that the metabolic redox reactions of microorganisms are detectable and quantifiable when performed in the presence of a suitable mediator [ ] . hence, viable microbial biomass can be determined by monitoring microbial 'metabolism' which, in turn, increases conductance and capacitance and results in a decrease in impedance. similarly to amperometric biosensors, several elegant antibody-based impedimetric assays have been used for pathogen detection. radke and alocilja [ ] developed a high-density microelectrode array for the sensitive detection of e. coli o :h ( × - × cfu/ml) using a goat anti-igg polyclonal antibody for capture. tully and colleagues recently implemented a biotinylated leporine polyclonal antibody for the detection of internalin b (inb), a l. monocytogenes cell-surface protein. when captured on avidin-coated planar carbon electrodes modified with polyaniline, a conductive polymer, the limit of detection for inb was found to be . pg/ml [ ] . the versatility of using this approach for the detection of this bacterium was also illustrated by wang et al. [ ] who adopted a different protocol by immobilising a monoclonal antibody on a titanium-dioxide nanowire to detect × cfu/ml. finally, su and li [ ] demonstrated how a quartz crystal microbalance (qcm) system using impedance could detect s. typhimurium in chicken meat. the implementation of magnetic beads resulted in a significant improvement in assay sensitivity, with a limit of detection of × cells/ml. minimal cross-reactivity was observed with e. coli. in potentiometric biosensors the conversion of a biorecognition process into a change in potential signal is detected by a reference electrode. potentiometric biosensor formats typically consist of a perm-selective outer layer and a bioactive element, such as urease, may be introduced to enhance the performance of the assay [ ] . a methodology that combines potentiometric and optical detection, namely the light-addressable potentiometric sensor (laps), was shown to be applicable for pathogen detection. gehring et al . [ ] implemented this technology in conjunction with an immune-ligand assay (ila) for the detection of e. coli o :h . the assay format devised involved the enumeration of cells by biotinylated polyclonal capture and fluorescein-labelled detection antibodies which were raised in caprine hosts through the administration of heat-killed cells. this 'sandwich' complex (in the presence of an additional urease-labelled anti-fluorescein antibody) was subsequently captured on a streptavidin-bovine serum albumin (bsa)-coated nitrocellulose membrane. urease enzymatic activity was monitored by the hydrolysis of urea to carbon dioxide and ammonia. the authors were able to detect approximately . × cells/ml and . × cells/ml of heat-killed and live cells of e. coli, respectively, in buffered solutions. dill and co-workers [ ] utilised a silicon chip-based laps assay to detect low levels ( cfu) of s. typhimurium . here, biotinylated and fluorescein-labelled anti-salmonella antibodies were selected as biorecognition elements. this assay format was subsequently applied for the monitoring of chicken carcass washings spiked with salmonella cells, and demonstrated a high recovery rate for cells ( %). the final electrochemical immunosensor format that will be discussed, with reference to the detection of e. coli and salmonella spp., is based on conductimetric detection [ ] . here, a biological signal is converted to an electrical signal via a conductive polymer, such as polyacetylene, polypyrrole or polyaniline. muhammad-tahir and alocilja [ ] developed a conductimetric biosensor incorporating a polyclonal antibody-based sandwich assay format in which the detection antibody was labelled with polyaniline. this sensor could detect approximately cfu/ml and cfu/ml of e. coli o :h and salmonella spp., respectively. this approach was also used for the detection of e. coli cells in a selection of different sample matrices, including lettuce and strawberries [ ] . the sensitivity recorded was cfu/ml. furthermore, the assay was rapid ( minutes) and could be generated in a disposable format. hnaiein and co-workers [ ] developed a novel conductimetric immunosensor for e. coli . a biotinylated polyclonal antibody was captured on streptavidin-coated magnetite nanoparticles. these were subsequently bound on a conductimetric electrode through the use of glutaraldehyde coupling. conductimetric measurements were facilitated through the application of an alternating-current (ac) voltage. the incorporation of nanoparticles facilitated an increase in conductivity, enabling . cfu/ml to be detected. a small amount of background was observed when s. epidermis cells were assayed in parallel. this was attributed to the use of a polyclonal capture antibody and reinforces the view that in some assay formats, monoclonal or recombinant antibodies may be more suitable. piezoelectric biosensors operate on the principle that a change in mass, resulting from the biomolecular interaction between two entities, such as an antibody and its respective antigenic determinant, can be determined [ ] . for example, in a quartz crystal, mass changes result in alterations in resonance frequency. piezoelectric immunosensors are affordable and disposable options for pathogen detection, and the implementation of qcm for the direct detection of analytes, such as bacterial cells, alleviates the need for labelled secondary antibodies [ ] . babacan and co-workers [ ] demonstrated that the use of protein a for the capture of a polyclonal antibody to s. typhimurium enhanced reproducibility and surface stability when compared to polyethyleneimine-glutaraldehyde (pei-ga) coupling. the resultant assay format permitted the detection of . × cfu/ml. fung and wong [ ] described how the use of ethyl-n`-( `dimethylaminopropyl)-carbodiimide hydrochloride (edc) and n-hydroxysuccinimide (nhs) coupling, a methodology routinely selected for the immobilisation of ligands on optical sensor platforms, allowed the capture of a monoclonal antibody for s. paratyphi a. the use of this surface immobilisation chemistry was shown to provide good stability and sensitivity, with a limit of detection of . × cells/ml. with respect to both of these formats and previously mentioned assays involving protein a immobilisation [ ] , the selection of a proper strategy for correctly orientating antibodies is conducive to enhanced sensitivity and selectivity. kim and co-workers [ ] used a qcm platform based on impedance measurement for the detection of s. typhimurium (the limit of detection was approximately × cfu/ml). su and li [ ] developed a piezoelectric sensor for detecting between × to × cfu/ml of e. coli o :h through the implementation of antibodies on a qcm via a -mercaptohexanedecanoic acid (mdha) monolayer. pohanka et al. [ ] used a polyclonal antibody linked to the piezoelectric crystal surface using glutaraldehyde to detect e. coli. the resulting assay was rapid, permitting analysis in ten minutes (inclusive of a regeneration step for re-analysis), and greater than ten assays could be performed without the need for re-calibration. this 'label-free' assay had a limit of detection of × cfu/ml. in therometric biosensors thermistors are frequently selected as temperature transducers [ ] . magnetic biosensors, in contrast, implement magnetic beads coated with a suitable ligand that can be detected within a magnetic field. from the perspective of bacterial pathogen detection, the latter platforms have been explored to a greater degree than their thermometric counterparts. magnetic systems offer distinct advantages. for example, when a sample selected for analysis does not contain any contaminating materials with magnetic properties, background signals (non-specific) are minimised. ruan and colleagues [ ] immobilised anti-e. coli antibodies on a magnetoelastic cantilever through the construction of a self-assembled monolayer (sam). the principle of this assay was the conversion of a substrate, -bromo- -chloro- -indolyl phosphate (bcip), to an oxidised and insoluble blue precipitate via an ap-catalysed reaction (secondary antibody). this product accumulated on the sensor surface, and the resulting changes in resonance frequency were recorded, facilitating the detection of × cells/ml of e. coli o :h . mujika et al. [ ] recently developed a magnetoresistive sensor for the detection of e. coli . it consisted of a sandwich assay whereby the bacterial cells were captured with a polyclonal antibody and detected using leporine polyclonal antibodies coated on superparamagnetic beads. the application of an external magnetic field was used for monitoring. this assay had a sensitivity of × cfu/ml of e. coli o :h . furthermore, minimal cross-reactivity was seen when s. choleraesuis was tested on this format. with reference to immobilisation strategies, when comparative analysis was performed between three different materials, silicon nitride was found to be more suitable than silicon dioxide (sio ) and su- for antibody capture. finally, this sensor format was hand-held, and these miniaturised formats demonstrate one approach for 'on-site' pathogen detection. in conclusion, electrochemical, piezoelectric and magnetic immunosensors can all be applied to foodborne pathogen detection. optical platforms also offer a powerful and 'label-free' methodology that permits 'real-time' pathogen detection, and these are discussed in section . surface-plasmon resonance (spr) is a phenomenon that results from the illumination of a metallic surface, such as gold, by visible or near-infrared radiation from a monochromatic light source via a hemispherical prism which exits to a detector (photodiode array) at an angle related to the refractive index (ri). the resultant oscillation of free electrons generates surface plasmons (electromagnetic waves) which resonate and absorb light. the specific wavelength/angle at which this occurs is a function of the ri in the proximity of the gold surface and relates to the mass on the chip surface. a change in mass, effected by the immobilisation of a ligand and, subsequently, further interactions which take place when analytes are passed over the modified sensor surface, causes a shift in the resonance to a longer wavelength and, hence, introduces a refractive index change ( figure ) . a large selection of commercially available optical biosensors can be directly applied for pathogen detection. wei et al. [ ] used the spreeta tm spr system (texas instruments) for the detection of campylobacter jejuni. here, biotinylated leporine polyclonal antibodies were immobilised directly on the sensor surface and the assay had a sensitivity of × cfu/ml. barlen and co-workers [ ] selected the plasmonic spr device (plasmonic biosensoren) for the detection of salmonella typhimurium ( . × cfu/ml) and s. enteritidis ( . × cfu/ml). mazumdar and colleagues also selected the same biosensor system for the detection of s. typhimurium ( . × cells/ml) in milk by implementing leporine polyclonal capture and detection antibodies [ ] . a range of other optical sensor platforms, including the proteon xpr (bio-rad) and sensíq (nomadics) and biacore tm (discussed below) also have the potential to be applied for pathogen monitoring. oh et al. [ ] devised a spr-based protein chip assay format with immobilised monoclonal antibodies against s. typhimurium , e. coli o :h , yersinia enterocolitica and legionella pneumophila . × cfu/ml of each pathogen could be specifically detected with their respective antibody. [ ] and [ ] . for illustrative purposes, a protein-a (green hexagon)-captured igg antibody is shown on a carboxymethylated dextran (cm ) sensor surface. the mass change introduced by the binding of an analyte of interest (blue circle) is shown as a change in refractive index (a to b) which can be determined through the use of dedicated software. koubová et al. [ ] were able to detect × cfu/ml of l. monocytogenes and s. enteritidis on an 'in-house' spr system, while taylor et al. [ ] devised an eight-channel spr sensor for permitting the detection of e. coli o :h ( . × cfu/ml), l. monocytogenes ( . × cfu/ml), c. jejuni ( . × cfu/ml) and s. choleraesuis ( . × cfu/ml) in buffer (pbs). rijal and colleagues [ ] applied a novel fibre-optic biosensor for the detection of e. coli o :h by immobilising a monoclonal antibody on a silanised ( -aminopropyl-triethoxysilane, aptes) silica fibre-tapered surface using edc/sulpho-nhs coupling. changes in light transmission ( nm) were introduced by pathogen binding, and the assay had a sensitivity of cells/ml. alternative fibre opticbased platforms that use fluorescent detection include the analyte , [ ] and the raptor biosensor. the latter is a portable device that utilises a sandwich elisa format for detecting pathogens. typically, a biotinylated capture antibody is immobilised on an avidin-coated fibre-optic waveguide. four such channels are housed within a plastic disposable 'coupon', thereby permitting parallel analysis to be performed with four different analytes. detection antibodies are labelled with a fluorophore, typically cyanine (cy ) [ ] or alexa fluor (af ) [ ] . fluorescently-tagged molecules that are located within - , nm of the waveguide surface are excited by a diode laser ( nm), and a percentage of the emitted fluorescence is detected by an optical probe and quantitated by a photodiode detector that collects emitted light at wavelengths of over nm [ ] . the raptor biosensor has been used for detecting foodborne pathogens, including s. typhimurium in spent water samples of spiked alfalfa seeds [ ] , l. monocytogenes in frankfurter meat [ ] and enterococcus faecalis from recreational water samples [ ] . pathogens can also be recovered and propagated by incubating waveguides containing bound bacterial cells in selective media post-analysis [ ] . these examples demonstrate the use of commercial and 'custom-built' spr systems. a more detailed discussion of biacore-based analytical approaches will now be provided, together with the problems encountered with these assay formats and methods for overcoming them. the versatility of biacore-based analytical platforms is demonstrated by the ability of the researcher to perform capture, sandwich or subtractive-inhibition assays, as shown in figure . hearty and colleagues [ ] produced a murine monoclonal antibody which was shown to be specific for the surface-located l. monocytogenes internalin a (ina) protein in native and recombinantly-expressed forms. when this antibody was immobilised on a cm surface through edc/nhs coupling, a limit of detection of × cfu/ml was observed when l. monocytogenes cells were tested. cross-reactivity studies clearly demonstrated the specificity of this monoclonal antibody, with minimal binding to e. coli, b. cereus and listeria innocua (the latter selected due to the non-expression of the ina protein) observed. this further illustrates the importance of this antibody as a species-specific reagent. sandwich assay formats are routinely selected for increasing sensitivity in elisa-based analytical platforms. this format was adapted for spr-based analysis of e. coli o :h and salmonella by fratamico and colleagues [ ] . they demonstrated that the sensitivity of a capture assay for e. coli o :h cells ( × cfu/ml) could be enhanced significantly by the subsequent addition of a caprine polyclonal antibody, which enabled the detection of between and × cfu/ml. another interesting observation deduced from this experimental work related to the immobilisation strategy. the initial experimental format implemented a capture assay format ( × cfu/ml). no apparent increase in sensitivity was observed when protein a was used to immobilise the mab. the ability of a sandwich assay format to enhance sensitivity was also described by bokken et al. [ ] for the detection of salmonella groups b, d and e. the original capture format permitted the detection of × cfu/ml. this sandwich format used a monoclonal capture and polyclonal detection antibody, the former immobilised through standard edc/nhs coupling. this assay format reduced the limit of detection to . × cfu/ml. while these assays clearly illustrate the potential that sandwich formats have for increasing assay sensitivity, it should also be mentioned that this is not always successful, as shown by the inability of two anti-l. monocytogenes polyclonal antibodies to enhance the signal in an assay format where cells were originally captured by a mouse monoclonal antibody [ ] . there are also additional concerns with using this sandwich format on biacore-based platforms due to the large size of the bacterial cells which exceeds the penetration depth of an evanescent wave (see section ). the subtraction inhibition assay (sia) is an extremely useful method for pathogen detection in spr-based immunoassays, and can be selected in instances where the user does not want to expose the system to pathogenic cells or to matrices which may have high viscosities. the principle of this assay format involves pre-incubating an antibody with a target pathogen and separating free from bound antibody. the quantity of free antibody is inversely proportional to the concentration of pathogen. haines and patel [ ] implemented this assay for the quantification of salmonella. a polyclonal antibody (specific for cell-wall epitopes) was incubated with freshly-prepared cells and subsequently passed through a syringe filter ( . m), enabling unbound antibody to be separated from antibodypathogen complexes. free antibody was then captured on an anti-fab-coated cm biacore chip. this novel assay format permitted five different strains to be detected at similar sensitivities ( × cfu/ml), and allowed comparative analysis with an additional ten unrelated strains at high concentrations ( × cfu/ml) to be performed. no response of any statistical relevance was observed, illustrating the versatility of the sia assay format to be used for selective analysis. leonard et al. [ ] developed a sia assay for l. monocytogenes but adopted a different approach. a polyclonal antibody, produced through the immunisation of a rabbit with heat-treated cells, was purified by protein g affinity chromatography and added to differing concentrations of heat-killed cells in phosphate buffered saline solution (pbs) and incubated at c for minutes. a centrifugation step was used as an alternative to filtration for the separation of free polyclonal antibody, and subsequent analysis was performed on a goat anti-rabbit polyclonal antibody immobilised on a cm surface. the efficacy of this assay format was illustrated by the low limit of detection ( × cells/ml), and by the short assay time required to obtain data ( minutes; excluding preparation of the sensor surface). a selection of immunosensor-based analytical platforms has also been developed for the detection of other bacterial pathogens, including yersinia pestis , vibrio cholerae , mycobacterium tuberculosis and brucella abortus (table ) . furthermore, an increase in public concern has resulted from the elevated numbers of nosocomial infections which have been caused by bacterial strains such as clostridium difficile and methicillin-resistant s. aureus (mrsa). the latter bacterial strain produces enterotoxins [ ] and several immunosensor platforms have enabled the detection of staphylococcal enterotoxin b (seb). harteveld and co-workers [ ] developed a piezoelectric immunosensor for detecting . g/ml of seb through the development of a competition assay. . ng/ml antigen [ ] the capture of a rabbit polyclonal antibody on the sensor surface did not permit the detection of free seb. the researchers, therefore, immobilised the toxin and subsequently passed over varying concentrations of antibody pre-incubated with the toxin. the response generated was inversely proportional to the concentration of free antigen in solution. a rapid (less than minutes) fibre-optic spr-based assay was also developed by slavík et al . [ ] , capable of detecting ng/ml of seb. immobilisation of antibodies (polyclonal) was facilitated by glutaraldehyde coupling. moreno-bondi and colleagues [ ] reported that it was possible to detect femtogram (fg) quantities of human serum antibodies against seb using an array biosensor (other antigens detected in this study included diphtheria toxin, hepatitis b surface antigen (hbsag) and tetanus toxin), while a spr-based assay, reported by subramanian et al . [ ] permitted the detection of whole s. aureus cells in direct and sandwich assay formats ( × cfu/ml). the sensor format used in this assay was the reichert sr , with alkane monothiol and dithiol dendritic tether-based sams used for the capture of polyclonal antibodies via edc/nhs coupling. the detection of fungal strains is of great importance, due to their association with crop spoilage and their involvement as human pathogens, and immunosensor-based technologies have been developed for their determination. a key consideration with analysing fungal cells relates to the fact that they are significantly larger than their bacterial counterparts, with fungal spores often over micrometers in diameter [ ] . this is understandably problematic in terms of system blockage. hence, sia assays, described earlier for bacterial strains [ , ] , may be used to circumvent this problem. skottrup et al. [ ] pre-incubated a murine monoclonal antibody with sporangia of p. infestans at c for hour. the separation of bound and unbound antibody was permitted by a five minute centrifugation ( g). free antibody was then passed over a surface containing bound goat-antimouse polyclonal antibody, allowing quantitation of free antibody. the limit of detection was . × sporangia/ml, and no cross-reactivity was seen when other fungal strains, such as melampsora euphorbia and botrytis cinerea were assayed in parallel. a similar assay was also used to detect spores of puccinia striiformis, using a polyclonal rabbit anti-mouse igm antibody to capture a mouse monoclonal igm antibody produced from immunisation with whole urediniospores. completion of the resultant assay took approximately minutes with a detection level of . × urediniospores/ml [ ] . additional immunosensor formats have also been used for the detection of human fungal pathogens. muramatsu et al . [ ] applied a piezoelectric sensor for detecting candida albicans through the immobilisation of an anti-candida antibody on a palladiumplated electrode. the recording of a loss in resonance frequency enabled the detection of × cfu/cm - . medyantseva and colleagues [ ] targeted an antigenic determinant on trichophyton rubrum with a polyclonal antiserum. their amperometric immunoassay had a sensitivity of × - mg/ml of antigen and was rapid ( minutes). the monitoring of the presence of aflatoxins, naturally occurring mycotoxins produced by several strains of aspergillus spp. , in fruit, vegetable and food produce, is also of great significance. aflatoxins can cause contamination of nuts (almonds, walnuts), cereals (rice, wheat, maize) and oilseeds (soybean and peanuts). while approximately structurally diverse aflatoxins have been reported, aflatoxins b , b , g and g and m (figure ) represent the greatest danger to human health [ ] . daly et al. [ ] used a rabbit polyclonal antibody to detect afb , which was conjugated to bsa and immobilised onto a cm biacore chip. a competition assay between free and bound afb permitted a linear range of detection of trace levels ( - ng/ml). daly and co-workers [ ] subsequently generated murine scfvs against afb by using a phage display format and incorporated these antibodies into a biacore-based inhibition assay. dunne et al. [ ] also developed a spr-based inhibition assay that incorporated monomeric and dimeric scfv antibody fragments for permitting the detection of between and , ppb and and , ppb of afb with monomeric and dimeric scfvs, respectively. adányi et al . [ ] developed an optical wavelength lightmode spectroscopy (owls)-based assay for the detection of afb and ochratoxin a. integrated optical wavelength sensors were used in conjunction with murine monoclonal antibodies, with the sensitive detection range for a competitive assay being between . and ng/ml. an indirect screening protocol was subsequently applied for the detection of these toxins in wheat and barley samples. the versatility of immunosensor-based analytical platforms for pathogen detection is further illustrated by the ability to develop assays for the sensitive detection of viral pathogens (table ) . these include hepatitis-c virus, the severe acute respiratory syndrome (sars) virus and bovine diarrhoeal virus, whose particles present an additional selection of structurally diverse epitopes which can be targeted by antibodies. single-celled phytoplankta play important roles in the aquatic environment by providing nourishment for a selection of heterotrophic marine animals. these include filter-feeding bivalve molluscs, such as mussels, clams and scallops. among the reported , species of marine phytoplankton, have been postulated to occur in high-numbers, causing harmful algal blooms (habs) or 'red-tide' events. approximately of these species produce secondary metabolites, collectively referred to as phycotoxins [ ] , that are structurally diverse and nonproteinaceous compounds which have low molecular weights (in contrast to whole cell pathogens). they pose a considerable risk to human health by causing respiratory, neurological or gastrointestinal problems at low concentrations. the primary route of infection is through the ingestion of contaminated shellfish meat or drinking water. furthermore, habs also have a devastating effect on the shellfish industry and algal blooms can also result in reduced tourist activity and concomitant economic losses. several countries have established regulations and specific concentration limits for phycotoxinlevelsin seafood [ ] . [ ] phycotoxin groups are classified according to the associated symptoms of infection, and a selection of structures is shown in figure . paralytic shellfish poisoning (psp)-associated toxins are water soluble, thermostable tetrahydropurine molecules which are subdivided into four structural categories, namely carbamate, n-sulphocarbamoyl, decarbamoyl and dideoxycarbamoyl. the most commonly encountered psp-causing toxins are gonyatoxin (gtx) and saxitoxin (stx). the latter is especially toxic, and over structural analogues with differing degrees of toxicity have been reported in nature [ ] . a causative agent of amnesic shellfish poisoning (asp) is domoic acid, a potent kainoid neuro-excitatory toxin which is synthesised by the marine diatom pseudo-nitzchia pungens [ ] and functions by binding to specialised receptors and inducing depolarisation of neuronal cells. diarrheic shellfish poisoning (dsp) originates from the consumption of shellfish material contaminated with the polycyclic ether toxins okadaic acid (oa), dinophysis-toxin (dtx ) and pectenotoxins (ptx). yessotoxin (ytx) is also classified under this grouping as it was isolated in from scallops associated with a dsp-related poisoning event. however, it was noted that the pharmacological properties of ytx differed from those of dsp toxins [ ] . okadaic acid, dtx and ptx are all produced by dinoflagellates belonging to the dinophysis and prorocentrum species, whereas ytxs are synthesised by protoceratium reticulatum [ ] . finally, cyanobacterial poisoning is caused by the hepatoxins microcystin (mc) and nodularin during red-tide events. several bacterial species have been identified as being causative agents, including members of the geni microcystis, anaebaena and planktothrix and consumption of contaminated water supplies is the primary route of infection [ ] . a small number of immunosensor-based formats for the monitoring of phycotoxins have been developed (table ) , and these have mainly focussed on btx, da, mc, oa and stx. several factors have contributed to this low number. a key factor is the scarcity of sufficiently pure toxin for antibody generation and the poor supply of reference material for assay development [ ] . this has been problematic in the development of immunosensor-based assays for detecting other important marine toxins. azaspiracid (azp) shellfish poisoning was first reported in in the netherlands and was attributed to the consumption of mussels (mytilus edulis) which were originally cultivated in killary harbour, ireland [ ] . a total of congeners of azp have been characterised [ ] , and the producing strain has been postulated to be the dinoflagellate protoperidinium spp. [ ] . no antibodies have been developed against this target in its natural state, although an elisa using an ovine polyclonal antibody against a synthetic aza hapten was reported [ ] . the availability of more reference material should permit additional assays to be developed for this and other marine toxins. another important aspect for antibody-based marine algal toxin detection relates to the structural similarity that exists between toxin congeners. furthermore, if a mixture of toxins is analysed in an immunosensor format, underestimation or overestimation of toxicity may occur as a result of an antibody being able to recognise multiple isomers of the same toxin molecule. this suggests that suitable antibody candidates have to be rigorously screened to ensure that cross-reactivity does not occur. finally, biosensors for marine toxins should permit the detection of a toxin in a complex matrix, such as shellfish meat. the formats described in table were developed in an attempt to replace the current regulated methods of marine toxin detection, including the mouse bioassay and highperformance liquid chromatography-mass spectrometry (hplc-ms). it remains to be seen whether they will be incorporated into legislation or routine monitoring programmes in the near future. immunosensor-based assay formats have allowed the detection of a selection of water-borne parasites. a piezoelectric assay was described by campbell and mutharasan [ ] for the detection of between and , oocysts/ml of cryptosporidium parvum, while kang and co-workers [ ] developed a biacore-based immunosensor assay which allowed the detection of between × - × oocysts/ml. the flexibility of using this methodology has also been illustrated by the ability to also detect other parasitic pathogens, including schistosoma japonicum [ ] [ ] [ ] [ ] and borrelia burgdorferi [ ] , which act as causative agents of schistosomaisis and lyme borreliosis, respectively. these assays use amperometric [ ] [ ] [ ] , piezoelectric [ ] and optical [ ] -based platforms. this review has outlined the principles and practices of antibody-based sensors for facilitating the detection of bacterial, fungal and viral species and toxins. a wide range of different applications have been highlighted involving the use of polyclonal and monoclonal antibodies (and, to a lesser extent, recombinant antibodies). however, it should also be emphasised that several problems may need to be addressed when developing related sensor-based assays, and these are now discussed. several of the aforementioned assays have also focused on a particular antigen. while this is also the most effective method for ensuring specificity, this may also be detrimental in instances where the exposure of a bacterial strain to stress, such as osmotic shock, alterations in ph or temperature fluctuations, or different growth media (e.g. different food matrices) may compromise the expression of a selective antigen. hahm and bhunia [ ] exposed cells of l. monocytogenes , salmonella spp. and e. coli o :h to a variety of stress conditions and noted that antibody-based responses were reduced. hearty and colleagues [ ] heat-treated l. monocytogenes cells and assayed these alongside untreated cells on a monoclonal antibody-immobilised biacore surface. a significant decrease in signal was observed when cells were treated at c for minutes, an observation putatively attributed to an alteration in the topography of the bacterial cell wall introduced by this treatment. these observations postulate that the sensitivity of an antibody may be compromised by an external factor, reiterating the importance of bacteriological propagation considerations. this point is particularly important in cases where antibodies are unable to differentiate between viable and nonviable cells, with active culturing able to circumvent this problem. several of the assays described in this review have been performed on spr-based analytical platforms, and have involved the detection of bacterial and fungal cells whose sizes are typically between - micrometers and in excess of micrometers, respectively [ ] . capture formats are typically used, involving the immobilisation of an antibody and the subsequent capture of a cell and, if deemed necessary, the addition of secondary antibody to enhance sensitivity [ , , ] . in biacorebased analytical systems, the depth at which a spr-produced evanescent wave can penetrate when tir occurs is . m [ , ] . hence, the direct immobilisation of large bacteria and fungal cells, whose diameters exceed this area, might compromise detection. conversely, in the cases where bacterial cells are captured on immobilised antibodies, the whole bacterial cell will not be contained within this area, implying that only a portion of the cell will be able to contribute towards a ri change. this observation may explain why shorter dextran chain lengths, such as those selected by bokken et al. [ ] (f or cm biacore sensor chips) may be more suitable as, in theory, the bacterial cell is spatially arranged closer to the surface and, hence, is more exposed to the evanescent wave field. it is also worth noting that biacore detection systems typically monitor spr angles on the sensor surface over an area of . mm [ ] . this implies that a reduced spr signal may arise from large cells sterically hindering each other if present in large amounts. this problem can be addressed by monitoring the sensor surface by atomic force microscopy (afm), as discussed by hearty et al . [ ] who were able to undock a cm chip, containing l. monocytogenes cells bound to a monoclonal antibody, incubate overnight in a glutaraldehyde-cacodylate buffer and fix in the presence of osmium tetroxide. dehydrated chips, treated with ethanol, could then be analysed to monitor surface topography. finally, it is worth mentioning that elisa and biacore-based assays differ from each other in that the former typically involves a 'static' incubation of antibody and pathogen, while biacore, and indeed several other assay formats, have additional considerations, including fluid forces. it is therefore of great importance that the antibody selected has sufficient affinity to allow cells to be captured and, most importantly, retained to permit further analysis [ ] . this limitation effect can be overcome thorough the use of low flow-rates, such as l/minute [ ] . biomimetic sensors (e.g. 'electronic noses' and 'electronic tongues') and plant sensors can be selected as alternative methodologies to immunosensors for detecting pathogens. electronic noses are comprised of sensor arrays that are capable of detecting a selection of compounds (e.g. ketones, aldehydes, aromatic and aliphatic compounds) produced during the growth stages of bacterial strains on a certain substrate. needham and colleagues [ ] were able to detect one bacterial (b. subtilis) and two fungal strains (penicillium verrucosum, pichia anomala) on bread before visible signs of spoilage were observed. lipoxygenase-based enzymatic spoilage could also be differentiated from microbial spoilage, and this methodology was coupled with gas chromatography-mass spectrometry (gc-ms) for characterisation of the 'volatiles' (e.g. -butanol, -butanone) produced during growth of these strains. alocilja et al. [ ] were able to differentiate strains of e. coli o :h from unrelated e. coli strains by monitoring the gaseous products produced when cells were propagated in a nutrient broth liquid culture. the electronic nose-based sensor chamber incorporated four metal-oxide gas sensors for the detection of volatile products of e. coli growth, such as amines, ketones and alcohols. this investigation allowed the researchers to demonstrate that e. coli o :h had a different gas signature pattern from the unrelated strains tested in parallel. furthermore, balasubramanian and co-workers were able to detect s. typhimurium in spiked vacuum-packed beef striploins ( . cfu/g beef) [ ] . in contrast, electronic tongues focus on the analysis of liquid samples, and are applicable for the analysis of food quality [ ] . this biomimetic sensor format was selected by lan and colleagues for the detection of s. typhimurium ( × cfu/ml) in chicken carcass samples [ ] . non-antibody biomimetic receptor molecules, including engineered proteins, peptides, aptamers (single stranded dna or rna), ribozymes or synzymes (synthetic enzymes), also have potential in the detection of pathogens and other food contaminants [ ] . a piezoelectric biosensor using oligopeptides designed to mimic the binding site of the aryl hydrocarbon receptor (dioxin receptor) protein was used to sensitively detect dioxins ( - ppb) [ ] . similarly, surface-immobilised antimicrobial peptides (e.g. polymyxins b and e) were used to detect s. typhimurium ( × ) and e. coli o :h ( × cfu/ml) in direct and sandwich assay formats [ ] . pan et al. [ ] reported the successful detection of s. enterica serovar typhi by using a single-stranded rna aptamer (s-ps . ) that bound to pili (type ivb) expressed on the bacterial cell that were instrumental in promoting pathogenesis. the ability of plants sensors (phytosensors) to detect environmental conditions and plant pathogens is still in its infancy in terms of sensor technology. a phytosensor capable of detecting plant pathogens at the molecular level was described by mazarei and co-workers [ ] . transgenic tobacco plants, containing an inducible plant defense mechanism linked to the β-glucuronidase reporter gene, inoculated with alfalfa mosaic virus showed increased β-glucuronidase expression. these examples demonstrate that the combination of synthetic receptors mimicking nature with desired transducers can be selected as an alternative to immunosensor-based analysis for pathogen detection, although further development will be needed before these alternative formats are selected above immunosensor platforms for pathogen analysis. the importance of antibodies as biorecognition elements for pathogen detection was discussed. antibody-based sensors can provide robust, sensitive and rapid analysis. in most cases the key element is the quality of the antibody used and recombinant antibodies have many advantages, including the ability to be genetically modified to improve selectivity, sensitivity and immobilisation. in practice, the development of these assays is simplified through the development of a suitable antibody and, subsequently, an assay format. while there are several problems associated with these methods, the potential for monitoring bacterial, fungal, viral and parasitic pathogens is immense. innovative recent developments, such as the hand-held device described recently by mujika et al. [ ] , signal the way forward for pathogen detection. future trends will continue to implement immunosensor-based technologies into microdevices, ultimately permitting on-site analysis to be performed in a rapid, reliable and sensitive manner. a review of conventional detection and enumeration methods for pathogenic bacteria in food biosensors and bio-based methods for the separation and detection of foodborne pathogens advances in biosensors for detection of pathogens in food and water accession date sel, a selective enrichment broth for simultaneous growth of salmonella enterica, escherichia coli o :h and listeria monocytogenes production, characterisation and potential application of novel monoclonal antibody for rapid identification of virulent listeria monocytogenes methodology for detection and typing of foodborne microorganisms rapid detection of listeria monocytogenes in food using culture enrichment combined with real-time pcr evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of campylobacter fetus in bovine preputial washing and vaginal mucus samples viable but nonculturable listeria monocytogenes on parsley leaves and absence of recovery to a culturable state proteomic characterization of enterohemorrhagic escherichia coli o :h in the oxidation-induced viable but non-culturable state development of nasba, a nucleic acid amplification system, for identification of listeria monocytogenes and comparison to elisa and a modified fda method a molecular beacon-based real time nasba assay for detection of listeria monocytogenes in food products: role of target mrna secondary structure on nasba design pcr and the detection of microbial pathogens in water and wastewater real-time pcr-based methods for detection of mycobacterium avium subsp. paratuberculosis in water and milk rapid detection of escherichia coli o :h by immunomagnetic separation and real-time pcr detection of methicillin-resistant staphylococcus 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plasmon resonance biosensor detection of food borne pathogens using bia detection of staphylococcal enterotoxin b employing a piezoelectric crystal immunosensor a miniature fiber optic surface plasmon resonance sensor for fast detection of staphylococcal enterotoxin b multiplexed measurement of serum antibodies using an array biosensor mono and dithiol surfaces on surface plasmon resonance biosensors for detection of staphylococcus aureus rapid detection of bacillus anthracis spores directly from powders from an evanescent wave fiber-optic biosensor method of measuring bacillus anthracis spores in the presence of copious amounts of bacillus thuringiensis and bacillus cereus a compact cmos biochip immunosensor towards the detection of a single bacteria rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system francisella tularensis detection using magnetic labels and a 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extracts using a portable surface plasmon resonance biosensor. harm development of evanescent wave all-fiber immunosensor for environmental water analysis label-free capacitive immunosensor for microcystin-lr using self-assembled thiourea monolayer incorporated with ag nanoparticles on gold electrode portable optical immunosensor for highly sensitive detection of microcystin-lr in water samples semi-automated membrane based on chemiluminescent immunosensor for flow injection analysis of okadaic acid in mussels immunosensor for okadaic acid using quartz crystal microbalance immunosensor for the determination of okadaic acid based on screen-printed electrode enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from irish mussels, mytilus edulis azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels, mytilus edulis , prior to analysis by liquid chromatography coupled with mass spectrometry antibodies with broad specificity to azaspiracids by use of synthetic haptens near real-time detection of cryptosporidium parvum oocyst by igm-functionalized piezoelectric-excited millimeter-sized cantilever biosensor surface plasmon resonance-based inhibition assay for real-time detection of cryptosporidium parvum oocyst an amperometric immunosensor based on a conducting immunocomposite electrode for the determination of schistosoma japonicum antigen an amperometric immunosensor based on nafionmodified electrode for the detection of schistosoma japonicum antibody silica sol-gel amperometric immunosensor for schistosoma japonicum antibody assay an amplified mass piezoelectric immunosensor for schistosoma japonicum serodiagnosis of lyme borreliosis infection using surface plasmon resonance effect of environmental stresses on antibody-based detection of escherichia coli o :h , salmonella enterica serotype enteritidis and listeria monocytogenes early detection and differentiation of spoilage of bakery products protocol development using an electronic nose for differentiating e. coli strains identification of salmonella-inoculated beef using a portable electronic nose system amperometric electronic tongue for food analysis using a surface plasmon resonance biosensor for rapid detection of salmonella typhimurium in chicken carcass nonantibody-based recognition: alternative molecules for detection of pathogens piezoelectric sensors for dioxins: a biomimetic approach antimicrobial peptidebased array for escherichia coli and salmonella screening aptamers that preferentially bind type ivb pili and inhibit human monocytic-cell invasion by salmonella enterica serovar typhi pathogen phytosensing: plants to report plant pathogens the financial support of the centre for bioanalytical sciences (cbas), the biomedical diagnostics institute (bdi), dublin city university, the industrial developmental agency (ida), ireland, science foundation ireland (sfi), grant no. /ce /b , enterprise ireland (ei), the marine institute and beaufort marine initiative, safefood biotoxin research network and the european union th framework programme (research for benefit of small medium enterprise, grant no. ) is gratefully acknowledged. key: cord- - i grvlr authors: yim, sung sun; bang, hyun bae; kim, young hwan; lee, yong jae; jeong, gu min; jeong, ki jun title: rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (facs) date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: i grvlr antibodies and their derivatives are the most important agents in therapeutics and diagnostics. even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (facs). first, we constructed a library of synthetic human antibody in which single-chain variable fragment (scfv) was expressed in the periplasm of escherichia coli. after labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. after repeating this sorting, the positive clones were completely enriched in several hours. thus, we screened the library against three viral antigens, including the h n influenza virus, hepatitis b virus, and foot-and-mouth disease virus. finally, the potential antibody candidates, which show k(d) values between and nm against the target antigens, could be successfully isolated even though the library was relatively small (∼ ( )). these results show that repeated facs screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. for the last two decades, monoclonal antibodies and antibody fragments have been proven to be effective as therapeutic and diagnostic agents, and have long been invaluable tools in various fields of biological research [ , ] . for the development of antigenspecific antibodies, hybridoma technology that relies on animal immunization has been traditionally employed [ ] . the recent progress in combinatorial technologies because of in vitro antibody repertoires and high-throughput screening methodologies has allowed the development of target-specific antibodies without animal immunization [ , ] . in these technologies, various protein display systems including phage display, ribosome display, and cell-surface display, have been widely used for the initial isolation of antibodies specific to antigens from huge libraries, as well as for engineering the antibodies towards desired functions, e.g., enhanced affinity and higher thermostability. [ , , ] . however, the most recent tools require repeated screenings in order to isolate potential candidates from the library, and consequently, they require relatively long time periods (several days to weeks) to complete the screening. the recent emergence and rapid dissemination of new viruses that cause serious human and animal diseases, such as sars coronavirus, swine flu h n virus, and avian influenza h n virus, has raised world concerns. the development of new tools to quickly isolate antibodies against rapidly spreading infectious viruses for treatment as well as early diagnosis is urgently required. currently, fluorescence-activated cell sorting (facs) has been used in high-throughput screening of huge libraries (generally bigger than cells) that are constructed in various display systems in bacteria or yeast as the host [ , [ ] [ ] [ ] . the following strategy is usually used for screening a recombinant antibody library: (i) cultivation of library cells; (ii) fluorescent-antigenpeptide or protein labeling of the library cells; (iii) facs sorting of the highly fluorescent population; (iv) regeneration of the sorted cells by regrowth or re-cloning of the sorted target genes; (v) repetition of steps i-iv until a highly fluorescent population is separated from the negative control population; and (vi) analysis of the individual clones. among these steps, the step determining the screening time is the regeneration of the sorted cells (step iv). in all of the current screening strategies, the sorted cells need to be regenerated for the next round of sorting, which can be done by cultivating the cells for at least one day [ , , ] or by re-cloning the genes, which takes several days [ ] . in addition to the regeneration time, contamination of the sorted cells by nonspecific clones also needs to be considered. during the cultivation for regeneration of sorted cells, differential growth rates among various clones (particularly non-specific clones) due to unregulated protein expression and differing cell viability can decrease the library screening efficiency, resulting in more rounds of sorting (longer duration) to isolate the potential antibody candidate [ ] . herein, we report the development of a new high-throughput screening strategy based on escherichia coli protein display and facs sorting, which allows the simple and rapid isolation of potential candidates from a huge library in one day. first, we constructed the fully synthetic human antibody library in which antibody fragments (single-chain variable fragment, scfv) were produced in the periplasm of e. coli. after library cultivation and permeabilization, the cells were labeled with fluorescent antigen probes, and the highly fluorescent cells were sorted by using a high-speed cell sorter. immediately after the first-round sorting, the sorted cells were reused in the next round of sorting, without regeneration of the sorted cells. this resorting was repeated until a highly fluorescent population became enriched as the major population and, using the high-speed facs sorter, the best candidates could be isolated in one day. the overall strategy of this rapid screening is illustrated in the figure . the proof of this concept was successfully demonstrated by the isolation of specific antibody fragments against three model viral antigens: h n influenza virus, hepatitis b virus (hbv), and foot-and-mouth disease virus (fmdv) serotype o. the whole facs screening rounds of the synthetic human antibody library against each viral antigen could be done in one day, and these results show that repeated facs screening without regeneration of the sorted cells can be a rapid and efficient method to isolate potential antibody candidates in case of urgent requirements. the bacterial strains and plasmids used in this study are listed in the table s . e. coli jude- was used as the main host for gene cloning and library screening. e. coli hm was used for the production and purification of the isolated antibodies (scfv). the e. coli cells were inoculated into luria-bertani (lb) medium ( g/l of tryptone, g/l of yeast extract, and g/l of nacl) containing % (w/v) glucose. after an overnight cultivation at uc and rpm, the cells were transferred into ml of fresh lb medium without glucose in a -ml flask and incubated at uc and rpm. when the cell density (od ) reached . , the cells were induced with mm isopropyl-b-d-thiogalactopyranoside (iptg) and were further cultivated at uc at rpm for h. the cells were harvested by centrifugation at , rpm for min at uc for further analysis. in all the cultivations, ampicillin ( mg/ml) was used as the sole antibiotic. for the facs screening of a human synthetic antibody (scfv) library, three fluorescent antigen probes were chemically synthesized: (i) fitc-crdnwhgsnrpw as an n epitope of h n influenza virus [ ] ; (ii) fitc-nsttfhqalldprvrglyf-pagg as a pres epitope of hbv [ ] ; and (iii) fitc-pvtnvrgdlqvlaqk as a vp epitope of fmdv [ ] . one aliquot ( ml) of the library stocks frozen at uc was thawed and inoculated into ml of lb medium containing % (w/v) glucose and ampicillin. after cultivation under the condition described above, the cells were harvested by centrifuging for min at , rpm and uc. for efficient labeling with fluorescent probes, cell pellets were resuspended in tris-kcl buffer ( mm tris-hcl at ph . and mm kcl), which dramatically increased the permeability of the e. coli outer membrane and allowed the fluorescent antigen probes to permeate into the periplasm [ , ] . the resuspended cells were incubated with mm of antigen peptides (n , pres , or vp epitope) conjugated with fitc for h at uc. the cells were then washed twice with the same buffer ( tris-kcl), and the fluorescent probe-labeled cells were sorted using a high-speed flow cytometer (moflo xdp, beckman coulter, miami, fl). in facs sorting, the cells were selected on the basis of high fluorescence intensity detection through a / band-pass filter for obtaining the fitc emission spectrum. ''purify mode'' was used as the sorting mode, which sorts only those drops that contain positive cells. all the e. coli cells sorted in each round of screening were immediately reused for the next round of facs sorting without regeneration. the sorting was repeated until the highly fluorescent population was fully enriched. to obtain the required sample volume, ml of sheath buffer was added to the sorted samples during each round. after the final sorting, the scfv genes were amplified from the sorted cells by performing pcr with primers, assembly-f and assembly-r. after digestion with sfii, the amplified scfv genes were cloned into pmopac and transformed into e. coli jude- for the further analysis of the single clones. for enrichment of cells producing flash tag (flnccpgccmep) from non-specific cells, cells harboring pmopac -mbp were diluted with non-specific cells harboring pmopac at : ratio. after labeling with with mm of flash-edt (invitrogen), the mixed cells were applied to facs and the fluorescent cells were sorted by the repeated facs screening as described above. for the production of the isolated antibody fragments, the plasmids recovered from the isolated clones were introduced into e. coli strain hm . after cultivation in ml of lb medium, the cells were harvested by centrifugation for min at , rpm and uc. the cells were then washed twice with pbs and resuspended in the same buffer. crude extracts of the cells were prepared by sonication ( min at % pulse and % amplitude), and the extracts were centrifuged for min at , rpm and uc to yield soluble lysates. the soluble lysates were filtered through a . -mm syringe filter, and the soluble lysates were poured into poly-prep chromatography columns (bio-rad, hercules, ca) filled with talon metal-affinity resin (clontech, mountain view, ca). the resin was washed twice with ml of washing buffer, and the -his tag fused to scfv was eluted using ml of the elution buffer. the purified scfvs were used for elisa analysis. for the preparation of gst-fused antigens (n of h n , pres of hbv, and vp of fmdv), three vectors were constructed. the primers used in the construction of the gst-fused antigens are listed in the table s . the plasmid pgex- t- containing the gst gene was used as the template, and the n epitope sequence (crdnwhgsnrpw) of the h n influenza virus [ ] was fused to the c-terminus of gst by performing pcr with the primer sets gst-f and gstn -r. for the synthesis of the pres epitope sequence (nsttfhqalldprvrglyfpagg) of hbv [ ] and the vp epitope sequence (pvtnvrgdlqvlaqk) of fmdv [ ] , the same pcr strategy was used, except that the reverse primers-gstpres -r and gstvp -r-were used for synthesizing the pres and vp epitope genes, respectively. the pcr products containing the gst-fused n sequence, pres sequence, or vp epitope sequence were digested using ndei and hindiii, and then were cloned into pmopac to yield pmopac -gst-n , pmopac -gst-pres , or pmopac -gst-vp , respectively. e. coli jude- harboring pmopac -gst-n , pmopac -gst-pres , or pmopac -gst-vp were cultivated in ml of lb medium in a -l shaking flask at uc at rpm. when the cell density (od ) reached . , the cells were induced with mm of iptg and were further cultivated at uc at rpm for h. the cells were harvested by centrifugation at , rpm for min at uc. the cells were then resuspended in ml of pbs and were lysed by sonication ( min at % pulse and % amplitude). after filtration with a . -mm syringe filter (sartorius stedim biotech, goettingen, germany) to remove the residual insoluble debris, the gst-fused antigens were purified by using glutathione sepharose resin (ge healthcare biosciences ab, uppsala, sweden) as per the method specified by the manufacturer. the gst-fused antigens were mixed with . m carbonatebicarbonate coating buffer (ph . ) to a final concentration of mm. the antigen solution ( ml) was loaded onto -well elisa plate, which was then incubated for h at uc. subsequently, each well was washed four times with pbs-t ( mm nacl, . mm kcl, . mm na hpo , . mm kh po , and . % tween- at ph . ) and filled with ml of % bsa solution, and the plate was incubated for h at uc. after washing with pbs-t four times, each scfv sample (soluble lysate or purified sample) was loaded on to the plate, and the plate was incubated overnight at uc. each well was washed four times with pbs-t, after which : , -diluted monoclonal anti-his antibody conjugated to horseradish peroxidase (hrp) (sigma-aldrich, st. louis, mo) was added, and the plates were incubated for h at uc. finally, the wells were washed with pbs-t, and tetramethylbenzidien (tmb) was added for the colorimetric detection of the bound scfv clones. the reaction was arrested by adding m of h so stop solution. the absorbance was measured at nm by using a tecan infinite m pro elisa plate reader (tecan group ltd., mä nnedorf, switzerland). to confirm binding activity of scfv against inactivated whole fmdv, an fmdv serotype o kit (priocheck fmdv type o, prionics, switzerland) was used; elisa was performed in the same manner with the exception of the antigencoating step. protein samples were analyzed by performing electrophoresis on a % (w/v) sds-polyacrylamide gel electrophoresis (sds-page) gel. for the immunodetection of the his-tag-fused scfv proteins, a monoclonal anti-his antibody conjugated to horseradish peroxidase (hrp) (sigma-aldrich) was used. an ecl kit (amersham ecl prime western blotting detection reagent, ge healthcare) was used for signal detection. to prove the concept of our strategy (repeated facs screening without regeneration) (fig. ) , we first conducted the enrichment of probe-specific cells from mixture with non-specific cells. in this enrichment experiment, flash-edt which can interact with flash tag (flnccpgccmep) and give fluorescent signal [ ] was used as probe for cell labeling. cells producing flash-tag fused maltose binding protein (mbp) were mixed with cells producing no-tag protein at : dilution ratio, and the enrichment of positive cells were performed by repeated facs screening strategy. from the mixture, the fluorescent cells (top , . % of the total population) were sorted and, immediately after the first round sorting, the sorted cells were applied to the next round of sorting by facs without cell regeneration. as shown in fig. , the fluorescent cells began to be enriched after second round and fully enriched after fourth round sorting. this successful enrichment of probe-specific clones clearly indicate the repeated facs screening strategy can be used for the rapid screening of antigen-specific antibody from library. a fully synthetic human antibody (scfv) library with six diversified complementarity-determining regions (cdrs) was constructed. the detailed procedures for synthetic library construction are described in the figure s . briefly, by following the kabat definition [ ] and using the database of human germline genes (http://www.bioinf.org.uk/abysis), the single chain variable fragment (scfv) was designed to have eight framework regions (frs) and six cdrs. regarding the frequency of usage and expression in e. coli, the dp- and dpk human germline genes were chosen for the framework region of vh and vl, respectively. for the diversity of each cdr loop, except cdr h , degenerate oligonucleotides were designed on the basis of the frequency of amino acids for each site in the human germline gene database (table s ). in the case of cdr h , much greater diversity in amino acid composition and length was introduced, unlike that in the case of the other cdrs, by using sets of degenerate oligonucleotides with an nnk random sequence and different lengths ( - amino acids) (fig. s ). each vh and vl gene was assembled by pcr and, to eliminate the clones containing stop codons or frameshifts in the vh or vl gene, each pcr product of vh and vl was cloned into a b-lactamase fusion system. in the selection system, the e. coli clones containing inframe genes of the each chain produced the functional b-lactamase fusion proteins in the periplasm and were selected using ampicillin-containing agar plates [ , ] . after selection, the inframe vh and vl genes were rescued, and the gene sequences were determined by sequencing. through sequencing experiments, we found that all the clones contained in-frame codons, and no stop codons were observed within the coding genes (data not shown). the rescued vh genes and vl genes were linked for a full antibody fragment (scfv) format and the in-frame scfv genes were also selected in the b-lactamase fusion system. from the selected clones, all in-frame scfv genes were rescued and cloned into pmopac for constructing a synthetic human antibody (scfv) library. from the library, clones were randomly chosen, and their sequences were analyzed, and it was clearly confirmed that all clones contained various sequences in the cdrs (fig. s ) . also the randomly selected clones showed good levels of scfv gene expression in e. coli (fig. s ) . the size of the final constructed library was about . , which is sufficient for the initial isolation of antibody candidates against the target antigens. with this synthetic library, we conducted the isolation of antibody candidates against three antigens: (i) n antigenic epitope of h n influenza virus; (ii) pres antigenic epitope of hbv; and (iii) vp antigenic epitope of fmdv. in order to isolate the n epitope-specific antibody (scfv), the synthetic library was screened as shown in the figure . after cultivation of library, cells were harvested and treated with tris-kcl buffer to improve the permeability of the outer membrane and to increase the labeling efficiency of the cells for fluorescent probes [ , ] . then, cells were mixed with mm of n antigenic peptide conjugated with fitc, and cell fluorescence was analyzed by facs. compared to the negative control (e. coli harboring pmopac vector only), the cells from the original library showed a slightly higher, but mostly similar fluorescence intensity (fig. a) . from the original library, the most fluorescent cells (top , . % of the total population) were sorted until the number of sorted events reached , . immediately after the first round of sorting, the sorted cells were applied to a second round of sorting by facs, and the most fluorescent population of the sorted cells (top , . % of total population) were sorted again. after repeating this sorting three more times, the highly fluorescent population became the major population in the final ( th ) round of sorting. the fluorescence of the sorted cells was clearly observed to be fully separated from that of the original black bar indicate signals from bsa coated wells, and grey bars indicate signals from gst-fused n antigen coated wells. (c) elisa analysis with the purified scfvs. symbols: circle, square and triangle represent the purified antibody from clones s , s and s , respectively. the closed and open symbols represent the coating of gst-fused n antigen and bsa on -well plates, respectively. doi: . /journal.pone. .g library (fig. a) . finally, , fluorescent cells were collected and used for analysis of individual clones. the sorting results are summarized in the table . after the final screening, the scfv genes of the , sorted cells were rescued by pcr, and the pcr product was cloned into pmopac for the periplasmic expression of scfvs. after transformation into e. coli jude- , a total of colonies were randomly picked from the agar plate, and the binding activities of the isolated antibodies were analyzed by performing elisa. most clones, except for one (s ), exhibited higher binding activity against the n peptide than the non-specific bsa control (fig. b) . from the sequencing experiments, we found that among the clones, six clones including s , s , s , s , s , and s , had the same dna sequences. two other clones, s and s , were also same clones, while another two clones, s and s , were same. the same clones with the same sequences showed similar levels of binding activity, except for s and s (fig. b) . among the clones, four, including s , s , s , and s , showed relatively higher binding activity than the other clones; therefore, we tried purification of scfvs to analyze their specific activities. the four scfv genes were transformed into the e. coli strain hm , which is protease-deficient and suitable for higher scfv production [ ] . after cultivation, scfvs of three clones s , s , and s could be purified with high purity, but we failed to purify scfv of clone s (data not shown). the specific activities of three purified scfvs (s , s , and s ) against the n epitope were analyzed by elisa and we observed that the binding activities of s and s were higher than that of s and a non-specific bsa control (fig. c) . the sequence information of three isolated scfv (s , s and s ) was provided in the figure s . to demonstrate the general use of our strategy for antibody screening, we also isolated a potential antibody against another target, pres of hbv. after cultivation and permeabilization of the library, cells were labeled with pres antigenic peptide conjugated with fitc, and the labeled library cells were sorted by facs. from the original library, the most fluorescent cells (top , . % of total population) were sorted until the number of sorted events reached , , . then, through repeated sorting without regeneration (three more times), highly fluorescent cells were enriched as a major population, and finally , cells were collected ( fig. a and table ). the scfv genes from the final , sorted cells were regenerated by pcr, cloned into pmopac , and then transformed into e. coli strain jude- . among the regenerated clones, total colonies from the plate were randomly picked, and their binding activities were analyzed by elisa. most clones exhibited good binding activity against antigen pres of hbv, but among the clones, four (sp , sp , sp , and sp ) exhibited relatively higher binding activity than the other clones (fig. b) . from the sequencing experiment, it was confirmed that two clones (sp and sp ) were identical, and so three clones (sp , sp , and sp ) were chosen for further analysis. after cultivation, each antibody was successfully purified (data not shown) and the specific binding activities of purified antibodies were evaluated by elisa. we observed that the scfvs purified from clones sp and sp had much higher binding activity than that from the sp clones (fig. c) . the sequence information of three isolated scfv (sp , sp , and sp ) was provided in the figure s . foot-and-mouth disease (fmd) is a highly contagious viral disease infecting cloven-hoofed animals, such as cattle and swine, and it has resulted in massive livestock losses around the world. the diagnosis of fmdv at an early-stage of contamination is crucial to the prevention of the contagion. for the development of an immunodiagnostic system, a highly specific antibody is necessary; therefore, we chose the vp antigenic epitope of fmdv as another target antigen for the application of the repeated facs screening system. the synthetic antibody library cells were cultivated and labeled with fitc-conjugated vp antigenic peptide, and the labeled library cells were sorted by facs. from the original library, the most fluorescent cells (top , . % of total population) were sorted until the number of sorting events reached , . after two more rounds of facs screening, the highly fluorescent cells were enriched as a major population, and finally , cells were collected (fig. a and table ). the scfv genes from the final , sorted cells were regenerated by pcr, cloned into pmopac , and then transformed into e. coli strain jude- . from the regenerated cells, we randomly picked clones and obtained four clones (sv , sv , sv , sv ) expressing active scfvs against the vp antigenic epitope (fig. b) . the four scfvs (sv , sv , sv , sv ) that showed high binding were purified for further analyses. in elisa, we observed that one scfv (sv ) has much higher binding activity than the others (fig. c) . finally, we also examined the binding activity of the isolated scfv (sv ) against whole (inactivated) fmdv. it was clearly observed that the isolated sv antibody fragment exhibited high binding activity against fmdv, while negative control m scfv, which can specifically bind to anthrax toxin pa [ ] , showed a negligible signal (fig. ) . the sequence information of four isolated scfv (sv , sv , sv and sv ) was provided in the figure s . binding affinity of the isolated antibody the binding activity (k d ) of isolated antibody against each antigen were also determined by spr analysis. each antigen was immobilized on cm chip and the purified antibodies were loaded and binding affinities were analyzed. the binding affinities (k d s) of isolated antibodies, s scfv against n epitope, sp scfv against pres , and sv scfv against were nm, . nm and . nm, respectively (fig. s ) , which were similar to those typically obtained using hybridoma technology and other antibody screening strategies including phage display etc. in spr analysis, the affinity can be overestimated due to the avidity effect of dimerized or multimerized antibodies. to check the presence of dimeric antibodies in purified samples, all purified antibodies were analyzed by size exclusion column chromatography and by sds-page analysis in non-reducing and reducing conditions. in all experiments, we clearly confirmed the purified samples were present mainly as a monomeric form (fig. s and fig. s ) and so the possible avidity effect in spr analysis could be excluded. and the specificity of the isolated scfvs were also confirmed further by western blot on complex protein mixtures containing wildtype gst or antigen-fused gst. as shown in the figure s , each scfv could bind specifically to its own viral antigen-fused gst in the soluble lysate of e. coli host. taken all these data, we concluded that, in our screening, each antibody could be isolated not by nonspecific binding of antibody on the cell surface (stickiness) but by the histograms of original library, st round, and nd round sorted cells are represented by red, orange, and green curves, respectively. the rd round sorted cells were used for regeneration of scfv genes by pcr, and its histogram is not shown here. (b) elisa with the soluble lysates of selected clones. black bar indicate signals from bsa coated wells, and grey bars indicate signals from gst-fused vp antigen coated wells. (c) elisa analysis with the purified scfvs. symbols: circle, square, triangle and diamond represent the purified antibody from clones sv , sv , sv , and sv , respectively. the signals were detected from the wells coated with gst-fused vp antigen on -well plates. doi: . /journal.pone. .g specific binding of antibody to antigen probe, and this means that our strategy is valid for the rapid isolation of antibody from library. screening libraries constructed by animal immunization (mainly mouse) with antigens has several benefits, but mouse antibodies give rise to a human anti-murine antibody response (hama) [ ] . to reduce the undesired immunogenicity of the isolated mouse antibodies, ''humanizing'' procedures are commonly required, in which the non-human cdrs are conjugated into a given human sequence [ ] . however, the exchange of the mouse framework with the human framework does not always guarantee similar affinity, and often leads to less immunogenicity; therefore, it is not suitable for the rapid isolation of effective antibodies. instead of an immune antibody library, non-immune synthetic antibody libraries have been developed that are generally constructed in human antibody frameworks [ , ] . in the synthetic library, the quality and diversity are of particular importance in the isolation of the antibody; therefore, the six complementarity-determining regions are fully randomized and inserted into a single human antibody framework. for the efficient and soluble production of antibodies in a bacterial host, as well as for less immunogenicity in the human body, the choice of framework in library construction also needs to be considered seriously, and therefore, the isolated clones from the synthetic library are likely to have favorable properties for production and further therapeutic applications. within the human genome, it is known that v h , v l , and v k gene segments exist. although all gene segments can produce a functional antibody, the level of gene expression and the stability of the antibody are very different [ ] . in our synthetic library, the dp- and dpk human germline genes, which are highly prevalent in human antibodies and are known to be well expressed in e. coli [ ] [ ] [ ] [ ] , were chosen for the framework region of vh and vl, respectively. the resulting antibody library showed diversity size of . . we know that this library size is relatively small compared to diversity of natural human antibody repertoire and, the construction of much bigger library (. ) is needed to increase the probability for the generation of antibodies against various antigens. however, although the size of library is not large, the quality of our library was enough to be used for the isolation of antigen-specific antibody. to reduce the incidence of frame shifts and stop codons during library construction, the libraries were subjected to in-frame selection by fusion with b-lactamase and ampicillin resistance selection. the quality of the library was validated by sequencing clones that were randomly selected from the library and by analysis of antibody production in e. coli (fig. s ). most clones selected randomly from the constructed library and isolated clones by screening exhibited good expression levels with high solubility in e. coli; therefore, we concluded that the high-quality human antibody library with high diversity in six cdrs was successfully synthesized and was suitable for the rapid isolation of antigen-specific antibodies. for the isolation of highly potential clones from these huge libraries, large microbial libraries need to be screened through multiple rounds of sorting due to the contamination of non-specific false-positive clones; however, in most strategies, this regeneration step takes a long time, which makes it difficult to rapidly isolate potential antibodies from a combinatorial library [ ] . our strategy, however, does not include time-limiting regeneration steps for the sorted population. the sorted population is used immediately for the next round of sorting, and this re-sorting process is repeated until the highly fluorescent population becomes separated from the negative population. without this regeneration step, highly fluorescent cells that produce potential antibody candidates could be collected in four hours through repeated sorting. for this sorting, we used high-speed facs sorter which can screen up to , cells per sec and, with this sorting rate, the first round to sort . to . million cells from library took approximately h and all repeated sorting ( - times) could be completed in hours. in the initial round of sorting, false-positive clones, where fluorescent antigen probes are non-specifically bound to antibodies on cells, can be found; however, those clones can be removed during the repeated sorting due to the fact that the binding of these antigen probes is not strong or cannot be maintained over repeated sorting for four hours. with this strategy, the overall procedure from cell cultivation to final round sorting could be completed in one day, with much time saved, unlike that in the previous strategies. in addition, we need to consider the effect of differing cell growth on the sorted cell population in the next round of sorting. stressful conditions during the facs screening process and antibody expression can severely affect cell viability [ ] . therefore, during overnight cultivation for regeneration, the more viable cells tend to overgrow and become the major population simply because of their viability, and not because of their binding activity. the use of this unfavorable population in the next round of screening makes it difficult to isolate the positive clones. however, by eliminating the need for regeneration, our strategy can minimize the risk of overgrowth of false-positive clones, as well as losing positive candidates during the sorting process. consequently, antibody candidates can be rapidly isolated in one day, as we successfully demonstrated with three antigen models (n antigenic epitope of h n influenza virus, pres antigenic epitope of hbv, and vp antigenic epitope of fmdv), where the isolated antibodies exhibited highly specific and strong activity against their antigen targets. although we could get successful screening results for three examined antigens, it does not mean that the use of our screening strategy guarantee the successful screening of antibody against various antigens. as one limitation, we can consider the variability of the performance of facs screening in respect of individual antigen. due to the different properties (stickiness, size and permeability) of individual antigen probes, cells can be nonspecifically labeled with probes and, unwanted screening results (isolation of non-specific and low-affinity antibody) can be obtained. to minimize the non-specific labeling of probe, we may need to modify the labeling conditions and display system according to the properties of probes. for example, in current display system, big size (. kda) probe cannot be used for screening because it cannot enter to periplasm of cell, and so our strategy is not suitable for antibody screening against big size antigens including whole virus. to accommodate big size probes, we can suggest the use of apex system [ , ] instead of current periplasmic expression system. in apex system, outer membrane can be partially removed by spheroplasting treatment which allows the entrance of big size antigens to periplasm. with apex system, it was already demonstrated the antibody can interact with antigen as large as kda in periplasm of e. coli [ ] . our strategy can be easily combined with apex system. using apex system, cells can be labeled with big size antigens, and the positively labeled cells can be isolated by our repeated sorting. like this, the display system and labeling conditions can be modified if necessary, and the isolation of positive clones can be done by repeated sorting strategy. in conclusion, we developed a new strategy for isolating antigenspecific antibodies in e. coli by simply repeating facs using a highspeed cell sorter. a fully synthetic human antibody library with six diversified cdrs was constructed in e. coli [ ] , and with repeated sorting, potential antibody candidates against three viral antigens (n of h n influenza virus, pres of hbv, and vp of fmdv) could be successfully isolated in one day. the isolated antibody candidates were then easily purified, and their high activities against each antigen were confirmed by performing elisa. in the case of fmdv vp , the final isolated antibody candidate (sv ) was also revealed to have high affinity against not only the vp antigenic peptide probe, but also the entire fmdv. therefore, this study has shown that repeated facs screening without regeneration of sorted cells can be an alternative strategy to isolate potential antibody candidates for therapeutic or diagnostic use in emergencies, and our screening strategy is expected to be used in situations requiring a rapid response to spreading disease [ ] . figure s amino acid sequence of the isolated antibody against three antigens. s , s and s scfvs are against n epitope of h n ; sp , sp , and sp scfv are against pres epitope of hbv; sv , sv , sv and sv scfvs are against vp of fmdv. strategies and challenges for the next generation of therapeutic antibodies high level production of a kringle domain variant by high-cell-density cultivation of escherichia coli advances in the production of human monoclonal antibodies selecting and screening recombinant antibody libraries synthetic therapeutic antibodies yeast surface display for screening combinatorial polypeptide libraries applications of display technology in protein analysis targeting recombinant antibodies to the surface of e. coli: fusion to a peptidoglycan associated lipoprotein isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (pecs) separation of e. coli expressing functional cell-wall bound antibody fragments by facs anchored periplasmic expression, a versatile technology for the isolation of highaffinity antibodies from escherichia coli-expressed libraries development of an optimized expression system for the screening of antibody libraries displayed on the e. coli surface nanogap field-effect transistor biosensors for electrical detection of avian influenza label-free optical diagnosis of hepatitis b virus with genetically engineered fusion proteins high-yield production of the vp structural protein epitope from serotype o foot-andmouth disease virus in escherichia coli directed evolution of a g protein-coupled receptor for expression, stability, and binding selectivity universal genetic assay for engineering extracellular protein expression kabat database and its applications: years after the first variability plot simultaneous mutagenesis of antibody cdr regions by overlap extension and pcr a vector for the removal of deletion mutants from antibody libraries secretory production of recombinant protein by a high cell density culture of a protease negative mutant escherichia coli strain development of human anti-murine antibody (hama) response in patients the immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the cdr regions fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides construction of a large synthetic human scfv library with six diversified cdrs and high functional diversity biophysical properties of human antibody variable domains rapid construction and characterization of synthetic antibody libraries without dna amplification recombining germline-derived cdr sequences for creating diverse singleframework antibody libraries a novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin flow cytometric screening of cellbased libraries apex -hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering circular polymerase extension cloning for highthroughput cloning of complex and combinatorial dna libraries isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis key: cord- -p qbxi authors: kitching, r. p.; salt, j. s. title: the interference by maternally-derived antibody with active immunization of farm animals against foot-and-mouth disease date: - - journal: british veterinary journal doi: . /s - ( ) - sha: doc_id: cord_uid: p qbxi summary foot-and-mouth disease (fmd) is a highly contagious disease affectingruminants and pigs. in countries in which control of fmd relies predominantly on vaccination, young stock ingest specific anti-fmd virus antibodies in the colostrum. this maternally-derived antibody (mda) provides immediate protection against infection with fmd virus, but also interferes with the development of active immunity following vaccination. however, susceptibility to infection precedes the ability to respond to vaccination in the presence of mda. currently available vaccines cannot overcome this inhibitory effect of mda, and protection of young stock can only be provided by their- isolation from fmd virus. neonates of all species are born into a hostile environment of physical and biological dangers. the obligate parasites in particular require to infect new susceptible individuals in order to ensure their own survival. the immune system has developed to respond to the various strategies employed by these pathogens to take advantage of their intended host, and consists of a number of different components which reflect the varied and diverse methods of enuy and establishment that have evolved. however, not only are the different species of farm animal born with their immune system at different levels of maturity, the different parts of the complete system do not become functional simultaneously. at birth, the calf can immunologically respond to bluetongue virus (maclachlan el al., ) and foot-and-mouth disease (fmd) virus (nicholls et al., ) by producing neutralizing antibody, but can only partially respond to infection with parainfluenza- virus (thorsen et al., ) . experimental infection of the foetal calf has been used to show how the developing immune system - / / - /$ . / © bailli~re tindall responds to different antigens during its ontogeny (see review by osburn, ). the immune system of the pig is less mature at birth; it can respond to hog cholera vaccination at week of age (precausta et al., ) but not effectively to fmd vaccination until weeks after birth (francis & black, ) . however, despite the development of a functionally competent immune system, there is inevitably a lag period between immunization and the development of active immunity. it is during this period that prevalent viruses can establish themselves in young animals. irnmediate immunity in the newborn pig or ruminant is provided in the colostrum of the dana. all classes of antibody are present, in concentrations greater than those present in the parental blood, together with immunologically active cells. the specificity of this antibody reflects the vaccination and disease history of the dana, and therefore, by boosting the antibody levels to those pathogens associated with neonatal mortality through vaccination, the newborn animal can receive from its mother high levels of specific protection. however, this maternally derived antibody (mda) is equally effective in preventing the response to active vaccination in the young animal as it is in providing protection against disease. unlike the situation in primates, transfer of immunoglobulins across the epitheliochorial placenta of farm animals during pregnancy is not possible. early passive immunity in these species is transferred to the offspring posl-partum by the absorption of colostrum through the gut mucosa. in the calf and piglet an indiscriminate uptake by the gut epithelium of macromolecules and immunocompetent cells in colostrum is possible for the first - h of life (logan el al, ) . the process is non-selective and is responsible for the rapid increase of iga, igg and igm in the circulation of the suckling neonate, with peak titres occurring at - h postpartum. the persistent detection of mda in the serum and secretions of the offspring is dependent upon the initial colostral uptake, which is determined by the immune status of the mother, and the half-life of antibody in the offspring, which is different for each isotype. mda specific for fmd virus has been found to persist for up to months in calves and months in piglets (mll & wittman, ) . estimations of the half-lives of individual mda isotypes vary. one set of values for the calf gives figures of . days for iga, . days for igm and days for igg (banks, ) . the corresponding figures for pigs are . - . days for iga, . - . days for igm and . - days for igg (curtis & bourne, ) . variability in these results is due to the range of methods used for naeasurement, and whether allowance has been made for the production of antibody by the neonatal immune system and the dilution effect of increasing blood volume during growth. indeed, francis and black ( ) concluded that the decay of serum neutralizing igg titres during the first days of life in the pig was largely a result of expanding blood volume and not catabolism of the antibody. there is ample evidence that in addition to the protective potential of mda, it is also responsible for marked antigen-specific immunosuppression in young ani-mals for periods that exceed the period of protection (francis & black, ) . this is demonstrated in vivo by interference with the serum antibody response to vaccination, and in vitro by a reduction in the reactivity of lymphocytes upon stimulation with antigen. in this way mda may interfere with the active immunization of young animals in a reversible fashion. in cattle and pigs, this phenomenon has been demonstrated for several infectious diseases including fmd. it has long been appreciated that the active immunization of vertebrates early in life is not as effective as in the mature individual. immaturity of the immune system at birth is partly responsible for this observation, although it has been shown in the domesticated species that good vaccination responses can be generated from the nd week of life (ahl & wittman, ) . the greatest suppressive influence on vaccination response in the neonate is mda. immediately after birth, there is a short period of generalized suppression of b and t lymphocyte responsiveness, which could be due to the transfer of immunosuppressive factors in colostrum such as cortisol, histamine and cytokines (clover & zarkower, ) . however, it has been clearly shown that the presence of mda in the offspring correlates with the antigen-specific suppression of antibody responses, an effect that can be mimicked by the passive administration of monoclonal antibodies to neonatal mice (xiang & ertl, ) . the mechanism by which mda causes this effect is not completely determined for any species. the current hypotheses build upon the proposed mechanisms for regulation of the immune response in adults. initially it was thought that mda interfered with the generation of an antibody response following vaccination of young animals purely by the enhanced removal of inactivated vaccine antigens or by neutralization of attenuated live vaccines. in both instances it was thought that vaccine antigen would be prevented from encountering the immunocompetent cells of the neonatal immune system by the effective 'antigen blockade' provided by mda to the specific microorganism. more recent evidence suggests that mda has a direct suppressive effect upon immunocompetent cells via a similar negative feedback system to the one that is thought to regulate the antibody response in mature animals (katz, ) . by the cross-linking of membrane-bound immunoglobulin fc-receptors with surface immunoglobulin, immune complexes comprised of mda and vaccinal antigen may deliver a negative signal to the b cell. this suppressive signal is antigenspecific and has the potential to be limited to particular mda isotypes (uhr & mohler, ) . mda could thus act directly on b lymphocytes to down-regulate the proliferation and differentiation required for antibody production. alternatively mda complexed with vaccinal antigen may act through a regulatory network to either suppress antigen-specific th cells or actively up-regulate the production of an antigen-specific t suppressor cell population (harte & playfair, ) . a third theoly of mda-related immunosuppression makes use of the 'idiotype-anti-idiotype' immunoregulatory network proposed by jerne ( ) . in this antigen-free system mda would induce antibody in the offspring which recognized its own antigen-binding site, or idiotype. the internal image antibody molecules, or anti-idiotypes, thus created could then interact with antigen receptors on the surface of b and t lymphocytes due to their similarity to the initiating antigen. this leaves the fc portion of the antibody molecule free to deliver a negative signal to the cell through the fc receptors. in reality the results of vaccination trials in young animals do not decisively confirm or refute any one of these theories. suppression of the antibody response to rabies virus vaccination in both the dog (aghomo et al, ) and the mouse (xiang & erfl, ) has been shown to occur for considerable periods after mda has fallen below detectable levels. in the mouse system the suppression of in vitro t lymphoproliferation responses was more prolonged than the effect on the antibody response. the authors concluded that in this experimental system mda-induced t suppressor cells were responsible for vaccination failure in these neonatal mice rather than a simple reduction in antigenic load. in addition to the mda-mediated effects on neonatal immunocompetence in the mouse, fujii and yamaguchi ( ) reported that immunization of pregnant mice induced cd + t lymphocytes which were capable of either producing a suppressive factor themselves or stimulating a population of t suppressor cells to secrete such a factor that could cross the placenta and induce specific humoral and cellular immunosuppression in the neonate. one of the authors has also previously reported the induction in mice of suppressor t cells in offspring following maternal immunization (koshimo et al., ) . imnaunocompetent cells and various factors are absorbed by the gut during the first h of life in the domesticated species and, therefore, it is possible that these may have a regulato~ t effect on the immature immune system in these species similar to the transplacental factors in mice (palmerly & beer, ) . in the case of fmd vaccination in pigs, francis and black ( ) concluded that the complete immunological unresponsiveness seen in the first weeks of life was due to immaturity of the immune system and antigen blockade by high titre mda, and as this titre declined an active suppression of t and/or b cells occurred to variable degrees. the degree of inhibition of the antibody response in these piglets was directly proportional to the mda titre at the time of vaccination. this was reflected in the outcome of the challenge experiment which showed that whereas partial protection was achieved by vaccination at weeks old, earlier vaccination conferred no protection to fmdv challenge after months. in this same study it was possible to elicit a primary antibody response, consisting of igm, at an age when iga and igg responses were totally suppressed. this observation supports the view that a period of t lymphocyte immunosuppression follows the initial state of unresponsiveness, and that this interferes with the generation of secondary antibody responses. in this case the suppression acts after the initial stage of antigenprocessing and recognition by b cells, and affects the regulation of the evolution of the antibody response by interference with clonal expansion and antibody isotype-switching, both of which are necessary for a secondary response and the generation of 'immunological memory', and thus long-term protection. similar results have been reported from studies on the effects of mda on vaccination of calves against several viral diseases (marshall & frank, ; kimman et al., ) . vaccination of calves with high mda titres at -week-old inhibited the active production of antibody to bovine coronavirus (heckert et al., ) . again the igm response was least susceptible to the suppressive effects of mda in these calves, whereas both the serum igg~ and secretory iga responses were markedly depressed. however, it is notable that the degree of suppression to the different coronavirus proteins was not uniform, and the attthors suggest that the more immunogenic viral proteins may be able to elicit antibody responses at mda titres that inhibit the response to other proteins. the reported success of oil-emulsion vaccines in the face of high mda titres may also be due to improved immunogenicity (morgan & mckercher, ) . this suggests that the duration of the interference of mda with antibody responses to vaccines may be specific to individual protein sub-units or even epitopes (van maanen et al., ) , which emphasizes the importance of vaccine potency in a situation where high mda titres are present. in support of this hypothesis that interference by mda could be specific to particular antigens are the results of kit et al. ( ) who were able to partially overcome the mda to pseudorabies with a live glycoprotein glii-deleted marker vaccine. ill many of the fmd endemic regions of the world cattle, and sometimes also pigs, are vaccinated between one and four times a year, against the prevalent serotypes of fmd virus. the half-life of mda to fmd virus in the calf is approximately days (nicholls et al., ) and days in the pig (francis & black, ) , although in the pig the decline in concentration was considered to be due more to the increase in body weight than degradation or excretion of the mda. the initial blood level of mda and its subsequent persistence relates directly to its level in the colostrum and the efficiency with which it is adsorbed. heifers tend to have lower levels of specific anti-fmd virus antibody in their colostrum than adult cows, and consequently their calves receive lower quantities of specific antibody than the calves of second or third lactation cows. this can result in an earlier susceptibility to fmd infection in endemic situations, and makes it more difficult to formulate vaccination schedules. in south america, nicholls el al. ( ) showed that mda against fmd was likely to persist for - months, and that calves with mda vaccinated against fmd not only failed to respond, but that vaccination depressed the serum titre of specific fmd virus antibody in these animals. they recommended that calves receive their prima w vaccination at - months of age. a similar situation exists in the large dairy herds in the middle east (kitching, unpublished observations). high yielding dairy cows have been imported from europe and north america into a hostile climate and an environment in which previously unencountered disease such as fmd and rinderpest are endemic. clinical fmd is kept under control by three or four times a year vaccination, but frequently it is the - month age group which develops disease in spite of this vaccination regime. many farm managers had become frustrated by the apparent ineffectiveness of the fmd vaccine, whereby they had been using a variety of calfhood vaccination protocols, almost all of which initiated vaccination before months of age, and in some instances as early as day old. the reason for the vac-cine failure was tile same as had been shown in south america a decade earlier, except in the middle east the cows were being vaccinated far more frequently and efficiently and the mda in their calves was at a very high level. the calves were reaching the age of or months, by which time they had received at least two ineffective vaccinations because of the mda, and were then becoming susceptible to infection with fmd virus. to compound the problem, if the calves developed clinical fmd, the environmental shedding of virus from these calves was frequently sufficient to overcome the immunity of other animals on the farm, resulting in a major outbreak and loss of production from the milking herd. a study was undertaken in which a number of the larger dairy herds participated, to examine different calfllood vaccination programlnes. the results of this previously unpublished study support many of the conclusions reached by nicholls et al, ( nicholls et al, ( , , but in addition addressed some of the problems more relevant to the middle east situation. assuming the level of management was sufficient to ensure that all calves received adequate colostrum, preferably pooled colostrum to include colosu'um from older cows, vaccination at day old or month of age, even if it was followed by a booster vaccination at days old or months of age, respectively, was ineffective. it could not be shown, however, that vaccination at these ages reduced the level of circulating antibody against fmd virus in calves, as there was no statistically significant difference between antibody levels in the vaccinated groups and unvaccinated control groups. when calves which had previously failed to respond to vaccination because of mda were revaccinated at an age at which mda no longer interfered with the response (i.e. at or months of age), there was no evidence that these calves had been primed by the first vaccination. they showed a similar response to vaccination as totally naive calves. this is contrary to the results of nicholls et al. ( ) , although it is acknowledged that comparisons between free ranging south american cattle and zero-grazed intensively-reared dairy animals in the middle east may not apply. calves with waning mda could become clinically infected with fmd virus prior to being able to respond to fmd vaccination. calves with antibody titres below : , measured using the liquid phase enzyme-linked imnmnosorbent assay (hamblin et al., ) , became susceptible to infection, depending on the level of fmd virus challenge. however, calves with titres greater than : failed to respond to vaccination. this situation accounted for the persistence of fmd on one large farm, where the -month-old age group would become infected, maintain infection in the group for approximately month and then pass infection to the next, month younger group with which it was in contact and which was just developing susceptibility to infection, in spite of this younger group receiving vaccination. at all times during the study the strain of fmd virus causing the outbreaks was monitored for changes in antigenic characteristics which could modify the interpretation of the results. surprisingly, the virus remained antigenically stable over the -year period of the study, even though the pressure to mutate in order to evade vaccinal immunity was very great. the conclusion of the study was that together with a number of management recommendations, the most effective vaccination programme for calves of well vaccinated dams, was vaccination of all calves at , and months of age, to ensure that all calves had good levels of protection. vaccination of pigs against fmd is less widely practised due to the rapid turnover in the pig population. in addition, the immaturity of the immune response of the young pig makes vaccination of the sow during pregnancy a more effective method by which to protect the litter. some response to vaccination in -week-old pigs in the presence of mda was shown by francis and black ( ) . however, it was the recommendation of francis that vaccination of pigs from immune sows be delayed until - weeks of age, although additional vaccination at - weeks of age could be justified in order to protect those piglets which had received insufficient mda (francis, ) . francis and black ( ) found no evidence in the pig that vaccination in the presence of mda depressed the specific antibody to fmd virus. the vaccines used in cattle outside of south america against fmd use a saponin and aluminium hydroxide adjuvant. these vaccines are less effective in pigs, and an oil adjuvant is used in pig fmd vaccines. however, in south america, oil adjuranted fmd vaccines are used in cattle, and it is claimed that these vaccines overcome the depressive effects of mda (sadir et al., ) . experimental results showed that calves over days of age, but still with mda, responded as well as adult cattle to the oil-emulsified fmd vaccine. however, there was a - day delayed response in mda free calves under days of age vaccinated with this vaccine, which was suggested to be due to high levels of cortical hormones (sadir et al., ) . in summal t, it has been shown for several of the domesticated species that mda can have a marked antigen-specific suppressive effect upon active antibody production in the young, the degree and duration of which is directly related to mda titres shortly after birth. there is a gradual recovery of immune function in which the individual can become partially responsive prior to the acquisition of full immunoconlpetence. the precise point or points in the immune system of the offspring at which maternally-derived factors interfere is not defined. some authors have described generalized cellular responsiveness (clover & zarkower, ) , and others describe antigen-specific reduction in both primary and secondary antibody responses (husband & lascelles, ) . however, it appears that individual lymphocyte subsets may be more or less susceptible to the effects of mda. wittman and ohlinger ( ) found that mda titres which interfered with the cytolytic t lymphocyte response to aujeszky's disease vaccination in piglets did not prevent the sensitization of lymphocytes for antibody production. interestingly, it has also been shown that high titres of mda in piglets suppressed the generation of 'memory cells' following vaccination against aujeszky's disease (kuiper et al., ) whereas lower mda titres that still suppressed the formation of antibody permitted the generation of immunological memory, which was apparent from the production of a secondary-type antibody response upon subsequent virus challenge (van oirschot & de leeuw, ) . it is also possible that anatomical compartments within the immune system are affected variously by mda, as has been shown to be the case for the mucosal immune response to respiratory syncytial virus vaccination in calves (kimman et al., ) . this study showed that a protec-tive mucosal antibody response could be effectively primed by the local application of attenuated virus in the face of mda titres that inhibited all primal-y antibody responses and the generation of systemic antibody memo w. thus, by taking advantage of these observed qualitative and quantitative variations in the degree of immunosuppression induced by mda, it may be possible to design vaccination su'ategies that offer better protection against infectious diseases of young livestock. control of fmd by vaccination has benefitted little fl'om the recent advances in vaccine technology (see review by kitching, ) . iinprovements in antigen production and concentration techniques have not resulted in significantly better vaccines, and fmd remains a disease that cannot be controlled by vaccination alone. the interference by mda in tile active immunization of young stock merely makes control of the disease even more difficult. cah,es with declining mda become susceptible to infection with live virus before they are able to respond to an inactivated vaccine. this observation suggests that the), would also respond to a live vaccine at a higher serum level of mda than they would using a dead vaccine. however, previous attempts to develop a live attenuated fmd vaccine were unsuccessful as the virus quickly reverted to virulence. the genome sequences associated with virulence of fmd virus are not known, but stocks of the original attenuated vaccine still exist. if it was possible to fix the mutations responsible for attenuation of fmd virus, vaccination with live virus could again become an option, although the reputation that live fmd vaccines have acquired may exclude their use under any circumstances. if a live vaccine can partially overcome interference by mda, an alternative approach might be to insert fmd virus genes into a live vector. the glycoprotein gene of rabies virus has been placed in the vaccinia virus genome (pastoret el al., ) and the f and h genes of rinderpest virus have been placed in the genomes ofvaccinia virus (yihna el al, ) and capripoxvirus (romero et al, ) to produce effective live vaccines against rabies and rinderpest, respectively. these recombinant vaccines will replicate in the presence of mda to rabies or rinderpest as there ufill be no inhibition of the pox virus vector. initial attempts to do the same for fmd xdrus have so far been unsuccessful. similarly, peptide vaccines have not replaced conventional fmd vaccines in spite of considerable research effort. this has in part been blamed on their failure to stimulate appropriate b and t cell populations. if, as has been suggested, mda selectively suppresses antigen-specific responses, there would be little potential for peptide vaccines to overcome mda. however, lipford et al. ( ) have recently reported the development of immunostimulating complexes (iscoms) containing lipids and the saponin quil a, which induce antigen-specific cytolytic t cells. the incorporation of fmd xdrus antigens into an iscom could improve peptide immunogenicity. there is at present no completely effective strategy to protect young stock against fmd by vaccination in situations in which fmd is endemic. only by isolating them completely from challenge with field virus until their mda has declined sufficiently for them to respond to conventional vaccines can disease be prevented, but such an approach in many countries may be too expensive to contemplate. the serological response of young dogs to the flul t lep strain of rabies virus vaccine. veterina o protection of young animals against foot and mouth disease host defence in the newborn animal immnnologic responses in colostrum-fed and colostrum-deprived calves half-lives of immunoglobulins igg, iga and igm in the sermn of new-born pigs. hnmunolo~ the immoral immune response of young animals to foot-and-mouth disease vaccination. foot-and-mouth disease bulletin effect of the sow vaccination regimen on the decay rate of maternally derived foot and mouth disease antibodies in piglets response of young pigs to fmd oil emulsion vaccination in the presence and absence of maternally derived neutralising antibodies maternal t cells of immunised pregnant mice induce imnmne suppression in their offspring enzylne-linked immunosorbent assay (elisa) for the detection of antibodies against foot-and-month disease virus. iii. evaluation of antibodies after infection. epidemiolo~, a~td h~fection failure of malaria vaccination in mice born to immtme mothers. ii. induction of specific suppressor cells by maternal igg mucosal and systemic antibody responses to bovine coronavirus structural proteins in experimentally challengeexposed calves fed low or high amonnts of colostral antibodies antibody response to neonatal vaccination in calves towards a nenvork theory of the immune system adaptive differentiation of lymphocytes: theoretical implications for mechanisms of cell-cell recognition and regulation of imnmne responses printing for local and systemic antibody memory responses to bovine respirato w syncitial virus: effect of amotmt of virus, virus replication, route of administration and maternal antibodies circumvention of maternal antibody of newborn pigs with glycoprotein giii-deleted marker vaccine tile application of biotechnology to the conu-ol of foot-and-mouth disease virus maternal antigenic stimulation actively produces suppressor activity in offspring sensitisation studies with the intranasal (i.n.) vaccine (s~vax ,n) against aujeszky's disease based on the bartha strain vaccination with inamunodonfinant peptides encapsulated in quil a-containing liposomes induces peptide-specific primary cd + cytotoxic t cells changes in the serum immtmoglobulin levels of colostrum-fed calves during the first weeks post-partum anti-xdral responses of bluetongue virus-inoculated bovine foetuses and their dams clinical and immunologic responses of calves with colostrally acquired maternal antibody against parainfluenza- virus to homologous viral infection hnmune response of neonatal swine to inactivated fmdv vaccine with oil adjuvant. i. influence of colostral antibody the effect of maternally derived antibodies on the response of calves to vaccination against foot-and-mouth disease effect of age on response of cattle to vaccination against foot-and-mouth disease ontogeny of inmaune responses in cattle colostral cell-mediated immulfity and the concept of a common secretol-y inamune system a recombinant vaccinia-rabies virus for oral vaccination of wildlife against rabies single capripoxvirus recombinant vaccine for the protection of cattle against rinderpest and lumpy skin disease response to foot-and-mouth disease vaccines in newborn calves. influence of age, colostral antibodies and adjuvants bovine parainfluenza- vaccine studies regulatory effect of antibody on the intmtme response interference of maternal antibodies with the immnne response of foals after vaccination against equine influenza intranasal vaccination of pigs against aujeszky's disease. comparison with one or two doses of an inactivated vaccine in pigs with moderate maternal antibody titres aujeszky's disease vaccination and infection of pigs with maternal immunity: effects on cell-and antibody-mediated immunity transfer of maternal antibodies results in inhibition of specific immune responses in the offspring protection of cattle against rinderpest with infectious vaccine virus recombinant expressing the ha or f gene key: cord- -d n aokl authors: natesan, mohan; ulrich, robert g. title: protein microarrays and biomarkers of infectious disease date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: d n aokl protein microarrays are powerful tools that are widely used in systems biology research. for infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. new developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers. large-scale genome sequencing projects first advanced knowledge of the theoretical composition of proteomes and led to the development of dna microarrays for studying gene transcription at the organism-scale. using genome sequence data to guide the direct examination of proteomes then enabled the study of host-pathogen interactions occurring beyond the level of gene transcription. it soon became evident that direct correlations between gene expression and protein abundance were rare [ , ] , driving the development of new approaches to study complex proteomes. protein microarrays are ideally suited to serve this purpose and have enormous potential applications in biomarker discovery, diagnosis, vaccine development, and drug discovery for infectious diseases. fluorescence- based detection of protein interactions is similar to gene array methods, and data analysis often employs approaches previously developed for genome and transcription studies. the number of proteins that can be printed on a single microarray surface also approaches the same upper limits as nucleic-acid based systems. multiplexing of fluorescent probes is limited by the ability to separate signals of overlapping emission spectra. generally, pair-wise comparisons of - differentially labeled, experimental and control samples can be analyzed. for example, serum igg and igm binding to arrayed proteins can be independently probed by fluorescently labeled secondary antibodies, and detected by a confocal laser scanner (such as genepix, molecular devices, sunnyvale, ca, usa). a complete high-throughput screening of thousands of interactions can be performed accurately and rapidly ( figure ). direct labeling of probes may also be used, though structure and function may be adversely affected. most label-free techniques [ ] provide real-time measurements and in some cases yield kinetics (figure ), providing further insight into molecular interactions. surface plasmon resonance (spr) [ ] , nanowire surfaces [ ] , and mass spectrometry [ ] are examples of label-free methods that are applied in the field of protein microarrays. of these, only a limited number of spr-based instruments are currently available for analysis of complex protein microarrays. spr imaging (spri) is a more recent and promising development [ , ] . while planar arrays that use slides or chips for immobilizing capture molecules are most common, suspension arrays based on microbeads have many important applications. the xmap technology developed by luminex corporation (austin, tx, usa) uses . micrometer polymer beads infused with two fluorescent dyes at different ratios to yield up to distinct bead sets. the binding measurements are performed by flow cytometry with two lasers, one for the identification of the bead and the other for the sample. bead-based arrays are very useful for standardizing assays, with an upper limit of independent immobilized probes. protein microarrays are increasingly used in infectious disease research to identify new biomarkers that are involved in the disease process or that are targets of immune responses [ ] [ ] [ ] [ ] [ ] [ ] . in contrast with other proteomic techniques such as -d gel electrophoresis and mass spectrometry, the arrayed probes are known, conforming easily to high-throughput applications that require examination of the entire proteome of an infectious agent. a variety of formats have been developed for the study of pathogen proteomes. capture and reverse-phase are examples of abundance-based microarrays produced by spotting either antibodies [ ] or antigens [ ] on a substrate. the arrays can be probed with serum, plasma, and other biological samples to detect binding interactions. microarrays based on wellcharacterized antibodies are specifically useful for identifying biomarkers and quantification of proteins. aptamers [ ] , affibodies [ ] , or engineered antibody fragments [ ] are alternative capture molecules. antigen arrays find applications in serodiagnosis of disease, antibody-response profiling, and evaluation of immunity after vaccination. in reverse-phase protein microarrays (rppms), small amounts of biological samples from cells, tissues, sera, or plasmas are directly printed on the slide [ , ] . these arrays are probed with a single protein of interest that is detected by labeled antibodies. rppms were successfully used for the screening of cell-signaling pathways and posttranslational modifications of proteins [ ] [ ] [ ] . the biochemical activities of arrayed proteins have also been analyzed [ ] by enzyme activity or substrate activity, and protein interaction with other proteins, nucleic acids, or drugs [ ] [ ] [ ] . the host antibody response provides a signature of pathogen proteins displayed by infectious diseases that can be captured and detected by microarrays. our studies with vaccinia virus [ ] and the bacterium yersinia pestis [ ] exemplify the application of antigen microarrays for the analysis of immune responses ( figure ). vaccinia is a large dna virus (orthopoxviridae) that replicates in the cytoplasm of host cells from genomes encoding - proteins. the virion is comprised of approximately proteins. most phenotypic variability occurs in proteins encoded in the terminal regions of the genome that are associated with host virulence or immune evasion. some of these terminal-region proteins are secreted during cell infection and interfere with host immunity by binding complement factors, cytokines, and chemokines, while others interfere with signaling pathways regulating host gene expression and apoptosis. to construct a proteome microarray, genomic dna of the copenhagen (nc_ . ) vaccine strain (dryvax) was used as the template for pcr amplification of the open reading frames (orf). gateway recombination cloning (invitrogen, carlsbad, ca, usa) was employed to facilitate high-throughput production of proteins from all orf clones. all dna clones were sequence-verified through the entire length of their inserts. baculovirusbased expression was used to produce the recombinant viral proteins as gst-tagged fusions to ensure high yield of properly folded proteins with posttranslational modifications that are similar to those encountered in the human host. the gst-tagged proteins from cell lysates were affinity purified to % homogeneity in a single step by using glutathione-agarose in -well plates and analyzed for correct size and abundance by western blots. virus and control proteins were printed onto glass slides coated with a thin layer of nitrocellulose. ultimately, % of the proteome was successfully expressed, purified, and arrayed. a standard assay for measuring antibody interactions with proteins of the vaccinia proteome microarray was first developed with a pool of therapeutic human sera collected from vaccinia-immune individuals (vig) and this data was compared to results obtained from individuals vaccinated against smallpox using dryvax. the assay consisted of incubating a dilution of serum on the microarray surface, washes, and finally detection with a fluorescently labeled anti-ig antibody. fluorescent images were captured by scanning with a confocal laser (genepix b; molecular devices). because only a very small sample volume ( - l) is required, this approach is ideal for limiting the amount of sample consumed, often necessary with clinical material. a high level of reproducibility with a very low background was apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. incubation of the microarray with vig identified nine proteins that consistently bound antibody, while antibody interactions with all other proteins were insignificant, requiring no further treatment to suppress non-specific signals. these antigens were also recognized by antibodies from individual subjects following primary smallpox vaccination and were diverse in function, consisting of regulatory, surface, core, and secreted proteins. the identical vaccinia proteins clustered into proteins recognized by primary and secondary vaccinated subjects based solely on signal intensities. the vaccinia proteins c l and i l were not previously reported as antibody-recognized antigens. the nine antigenic proteins we identified did not bind antibody from non-vaccinated sera, confirming the specificity of these antibody-antigen interactions. however, o l and h r were reactive with antibodies from both vig and non-vaccinated control sera, suggesting that these were cross-reactive or non-specific interactions. these results indicated that only a small subset of proteins present within the complex orthopoxvirus proteome was associated with antibody responses. in addition, the human response to individual proteins was variable as sera from more than half of the subjects contained igg that recognized > vaccinia proteins, while the remaining samples recognized one to three proteins. further, antibodies from most individuals recognized a greater number of viral proteins after a boost vaccination compared to a single vaccination, suggesting that repeated infection expands the total number of proteins recognized by igg. a previous report [ ] similar to our study on vaccinia [ ] , analyzed the serological response to vaccinia and smallpox, observing that a significant amount of the antibody response to vaccinia was not involved in the virus neutralization, but could be used as potential markers for diagnostic and vaccine development. a later report [ ] evaluated an attenuated smallpox modified vaccinia virus ankara (mva) an alternative to dryvax vaccine by comparing antibody profiles in the sera of humans and monkeys. the overall immunogenicity of mva was reported to be comparable to that of the dryvax vaccine and the study concluded that mva may be a useful alternative to dryvax. our vaccinia virus microarray was expanded to allow analysis of additional poxviruses. monkeypox is a zoonotic viral disease that occurs primarily in central and west africa. immunity to monkeypox is provided by vaccination against smallpox caused by the closely related variola virus. to differentiate antibody responses to monkeypox virus infection from human smallpox vaccination, we developed a protein microarray covering - % ( - proteins) of representative proteomes from monkeypox viral clades of central and west africa, including % coverage ( proteins) of the vaccinia virus proteome as a reference orthopox vaccine. serum igg of cynomolgus macaques that recovered from monkeypox recognized at least separate proteins within the orthopox meta-proteome, while only of these proteins were recognized by igg from vaccinated humans. there were of antigens detected by sera of human vaccinees that were also recognized by igg from convalescent macaques. the greatest level of igg binding for macaques occurred with the structural proteins f l and a r, and the membrane scaffold protein d l. significant igm responses directed towards b r, f l, and a r of monkeypox virus were detected before onset of clinical symptoms in macaques. results from this study suggested that antibodies from vaccination recognized a small number of proteins shared with pathogenic viral strains, while recovery from infection also involved humoral immunity to antigens uniquely recognized within the monkeypox virus proteome (unpublished data). to identify antibody biomarkers that could distinguish plague from infections caused by other bacterial pathogens [ ] , we developed a microarray comprised of the proteome from the bacterium yersinia pestis. the chromosome of y. pestis co encodes approximately proteins, while an additional are episomally expressed by pcd , pmt , and ppcp . for comparison, the proteome of y. pestis kim contains individual proteins, % in common with co , and the closely related enteric pathogen y. pseudotuberculosis contains approximately proteins (chromosome plus plasmids). the microarray was comprised of proteins representative of over % of the orfs present within the y. pestis genome. in a manner similar to the vaccinia microarray, the y. pestis proteins were produced as full-length polypeptides fused to gst as an affinity isolation tag. an in vitro translation method, based on e. coli lysates, was used to express the proteins in a gram-negative background. the orf clones were fully sequenced to confirm quality and identity. the affinity-purified proteins were characterized by sds gels and western blots probed with an anti-gst antibody. different approaches for studying the antibody repertoire for plague in rabbits and non-human primates were investigated. based on results from experiments using the y. pestis proteome microarray, we identified new candidates for antibody biomarkers of bacterial infections and patterns of cross-reactivity with a panel of other gram-negative pathogens [ ] . we were able to cluster the proteins recognized by the antibodies into three groups: common proteins of all gram-negative bacteria, combinations of proteins unique to one pathogen, and proteins unique to one pathogen. this example demonstrates the power of a high-density proteome microarray to dissect the complex immunological relationships among a group of related human pathogens. convalescence sera from vaccinated non-human primates that survived an otherwise lethal aerosol challenge with y. pestis co (plague) or bacillus anthracis ames spores (anthrax) were also examined. in addition to the caf and lcrv proteins comprising the vaccine components, a subset of proteins from the y. pestis proteome were recognized by antibodies from plague survivors and none of these were detected with antibodies from anthrax survivors or non-challenged controls. signature patterns of antibody recognition were identified that reflected the orthologous relationships among proteomes of these bacteria. a high degree of cross-reactivity in the antibody response to burkholderia and related bacteria was also previously reported [ ] . further, a protein microarray consisting of % of y. pestis proteins was used by li et al. to profile antibody responses to live plague vaccine in rabbits [ ] , sentinel animals [ ] , and plague patients [ ] . predominant responses were observed for proteins in addition to f and v antigens in rabbits. in plague patients, they identified proteins that were potential serodiagnostic biomarker candidates and found antibody responses from different plague foci responsible for different clinical symptoms. additional microarray studies of antibody responses to bacterial pathogens were reported. sundaresh et al. [ ] developed a protein microarray with of the most reactive francisella tularensis proteins derived from a larger chip. in this study, they identified a set of immunodominant antigens from f. tularensis patients suitable for diagnosis development. in another study [ ] , a similar protein microarray approach was employed to find immunodominant antigens in mice vaccinated with killed f. tularensis vaccine and challenged with a virulent f. tularensis strain. the study found antigens in addition to known immunodominant antigens of f. tularensis discovered by other methods. this was also the first published report to use protein microarrays for studying protective immune responses to an infectious agent. immunoreactive antigens of coxiella burnetti, the causative agent of q fever, were identified using a protein microarray consisting of % of the c. burnetti proteome assembled by using a cell free protein expression system [ ] . serum from q fever patients and individuals vaccinated with q-vax vaccine strongly reacted to antigens of c. burnetti that were confirmed by elisa including antigens isolated from an e. coli expression system. in yet another study, human and goat antibody responses to brucella melitensis infection were analyzed [ ] , finding that there was differential recognition of antigens by the two host species. the serodiagnostic proteins were of potential value for diagnostic purposes as brucellosis is diagnosed by measuring the antibodies to lipopolysaccharide (lps), which cannot distinguish between past and present infection in endemic areas. antibody response to lyme borreliosis disease in humans and mice was studied by barbour et al. [ ] , using a protein microarray comprising % of the proteins from borrelia burgdorferi. a comprehensive analysis of the sera from b. burgdorferi patients and rodents revealed that only a small set of antigens elicited antibody responses in both hosts, while the majority of b. burgdorferi proteins did not induce antibodies. it is also possible to focus on specific subsets of antigens identified by bioinformatics. for example, a protein array produced from the outer membrane proteins of pseudomonas aeruginosa was constructed [ ] to study the immune response in patients, and several antibody-binding antigens were identified by this group as potential diagnostic markers. in another example, a recombinant niesseria meningitidis protein array was used for screening serum from meningitis patients [ ] for immune responses to phase-variable expressed proteins. the development of new proteomics methods has been driven in part by the need to identify new types of infection biomarkers. though studies of antibody responses dominate the field, protein microarrays should be considered as an alternative approach to examine other catagories of host-pathogen interactions. research areas with well-established cell proteomics data are prime targets. for example, because many of the host factors associated with phagosomes have been identified [ ] , protein microarrays may provide a convenient model system to examine interactions with pathogen proteomes associated with this portal of entry. in a similar manner, targeted microarrays comprised of pathogen proteins may be used to examine host interactions. the few published examples from this developing field of study should be mentioned. in one report, rppm along with other techniques [ ] were used to examine phosphoprotein signaling pathway in primary human small airway epithelial cells infected with bacillus anthracis, identifying reduced akt (protein kinase b) phosphorylation as a contributor to increased bacterial survival and pathogenicity. in another study [ ] , proteins from group a and b streptococci were arrayed on a chip and probed with human fibronectin, fibrinogen, and c binding protein, all well-known targets of gram-positive bacteria. the use of purified and well-characterized proteins to assemble the microarray will insure the highest quality and most reproducible results. however, the high level of quality control required to sequence, purify, and characterize elements of a proteome microarray in the examples we described may not be as important for provisional screening purposes. a coupled transcription/translation reaction was previously reported as a rapid method to develop protein microarrays against a number of infectious organisms [ , ] , bypassing sequencing and purification steps. in this technique, both transcription and translation occur simultaneously to synthesize proteins in parallel from pcr products, directly on the chip. presumably, results obtained by these methods will require follow-up studies with more rigorous controls for confirmation. the protein in situ array (pisa) developed by he and taussig [ ] first produces dna constructs from a pcr of the protein of interest and a t promoter for translational initiation followed by immobilization using a n or c terminal tag sequence. another approach [ , ] called nucleic acid programmable protein array (nappa) included slides coated with avidin to capture biotinylated plasmid dna for synthesizing proteins. proteins with fused tags were synthesized and immobilized on the chips coated with molecules to bind the tags [ ] . he et al. [ ] described a technique called dna array to protein array (dapa) where they utilized a single dna array to make multiple copies of the same protein array. in one example, a self-assembled nappa-based on a cell-free expression system was used for constructing a protein microarray of all proteins produced by varicella zoster virus (vzv), the cause of chickenpox [ ] . three sets of antigens in the vzv proteome were identified from human sera that may be useful in defining the clinical status of the infection and diagnostics. another vzv protein array produced from an e. coli expression system was later used to detect antibodies in human sera reactive to vzv viral proteins [ ] . the orf (ge) antigen identified in the study showed high-confidence for determination of the serological response to vzv. although these alternative in situ synthesis methods are rapid, they are not without significant shortcomings. it is difficult to characterize proteins expressed on the chip in contrast to direct spotting of purified proteins. two studies investigated the antibody response to vzv by protein arrays made from either nappa cell-free expression system [ ] or e. coli expression system [ ] . antibodies from the sera of patients recognized three microarrayed vzv proteins produced recombinantly with e. coli, whereas these identical proteins were not recognized on a nappa protein expression-based protein microarray. interestingly, these proteins were abundantly expressed in the nappa protein microarrays, suggesting that they did not fold correctly to expose their linear epitopes for binding. despite these limitations, microarrays based on in situ synthesis offer simplicity, efficiency, and highthroughput capabilities. the relatively small proteomes of most viruses increase in diversity by considering variations in clinical isolates. for example, while human papilloma virus produces only a few proteins, sequences for these may diverge in the more than strains of the virus. a protein microarray developed with human papilloma virus types [ ] was used to examine patient sera compared to asymptomatic subjects. the study identified e proteins as the most reactive antigens in the patient group. however, the e proteins were not useful as a diagnostic biomarker because the differences between patients and asymptomatic subjects were not significant. in another report, a bead suspension multiplex array (luminex xmap) was used to study serological responses to antigens of human papilloma virus [ ] . the data correlated well with elisa results and in addition the bead array was able to measure weak antibody responses. a protein array constructed from six coronaviruses including severe acute respiratory syndrome (sars) was used for screening serum samples for virus-specific antibodies [ ] . the microarray data predicted sars infection in % of the patients correctly with a specificity of %. the high efficiency, low cost, and high-throughput nature of chemically synthesized peptides are significant advantages compared to production of recombinant polypeptides. therefore, peptide microarrays have been explored as another alternative to recombinant proteins, with a limited degree of success. a chemically synthesized peptide microarray representing the major antigens of hepatitis b and c viruses, human immunodeficiency virus, epstein-barr virus, and syphilis was constructed on a glass slide and antibody responses were simultaneously detected [ ] . the assay showed very high sensitivity and specificity for the diagnostic identification of these viruses. another proof of principle study [ ] was reported to identify diagnostically relevant peptides using a peptide array deduced from bioinformatics data for echinococcus granulosa (tapeworm) infection, achieving only % sensitivity and % specificity compared to elisa. as an alternative to full-length polypeptides, expression and purification of protein domains may improve yield of stable products for use in microarrays. in one reported study, a total of protein domains (sh , sh , pdz etc.) were immobilized on nitrocellulose and screened with peptides [ ] . the results proved that the immobilized domains were stable and could be used for binding studies. kaushansky et al. [ ] constructed an array based on protein interaction domains such as src homology, phosphotyrosine binding domains, and mouse pdz domains produced from recombinant e. coli. these arrays required low sample consumption and may reduce false positive rates. advanced knowledge of the specific recognition events will be required to substitute peptides or single protein domains for full-length proteins to ensure that important epitopes are not missed for the purpose of studying antibody interactions. protein stability is an important and particularly challenging factor in using protein microarrays. the high-throughput nature of construction tends to sacrifice detailed knowledge about individual proteins. there are few methods available to assess folding and functionality of all arrayed proteins immediately before use. recombinant proteins constructed with fusion tags to facilitate folding during translation offer a partial solution, especially if proper folding of the fusion protein can be readily assessed. for example, unfolded glutathione-s-transferase (gst) will not bind to glutathione, the tripeptide ligand of gst, hence purification schemes employing gst fusions will favor proteins with stable structures. fusion tags also provide a convenient marker for monitoring quality control of protein spotting (figure ) . however, the fusion tag may hinder protein accessibility or alter function in other ways. because proteins may denature during printing and storage, enzymes or other denaturing-sensitive proteins that can be assessed after printing should be included in the microarray. antibodies are a natural choice to be used as capture molecules due to their specificity, affinity, ease of production, and potential for engineering. for probe development, the analysis of antibody specificity and cross-reactivity [ ] [ ] [ ] can be simplified by screening against target or off-target proteins printed as microarrays. antibody arrays are routinely used in the field of biomarker discovery and validation. the detection of binding molecules to an antibody array is usually done by direct labeling of proteins in the sample (serum, cell, or tissue) or by a sandwich format similar to elisa. for example, protein abundance in cells was determined by haab et al. [ ] , using antigen and antibody reactions. this was the first published report to use a two-color differential labeling of samples in a protein microarray. the microarray format is also convenient for standardizing the routine analysis of analytes present in screening assays, such as cytokines released by cell cultures [ ] . an antibody-based microarray for the multiplexed detection of cholera, diphtheria, staphylococcal enterotoxin b, tetanus toxins, anthrax protective antigen, and lethal factor was reported [ ] . a competition assay between labeled and unlabeled toxins in serum was used to simultaneously detect all the toxins in the antibody microarray. in another report, a sandwich fluorescence immunoassay based on an antibody microarray format was developed for the capture and detection of e. coli o .h [ ] , and later to screen large number of food samples in a high-throughput manner to simultaneously detect e coli o :h and salmonella typhimurium [ ] . the major drawback of capture microarrays is that few well-defined and high-quality antibodies against microorganisms are available. engineering antibody fragments is a potential alternate approach to accelerate the development of antigen-capture molecules. antibody fragments such as the fab portion of igg [ ] , phage display libraries or recombinant single chain variable fragment (scfv) containing the antigen-binding motif can be used in place of antibodies in protein microarray development [ , ] . a decreased cross-reactivity was also observed with fragments as compared to full-length antibodies. thin-layered nitrocellulose or chemically modified -d surfaces (glass, silicon, gold, and polymer) are generally used for immobilization of microarrayed proteins. buffer additives like glycerol, polyethylene glycol, or sugars that prevent drying of the proteins and choice of buffers should be carefully selected to match the type of surfaces used for arraying. alternatively, gel-based -d surfaces made from polyacrylamide or agarose provide a hydrophilic environment [ ] . regardless of the printing substrate selected, microarrayed proteins must retain functionality and stability during preparation and use. though it may not be possible to directly examine all elements of an array, it is important to include printing controls of interacting proteins that can be tested for functionality before use. there are several methods by which proteins are spotted onto substrates. the choice of technology used impacts spotting consistency, speed, spot diameter, and ease of use. in turn, the final spot quality required for printed proteins will depend on the method of detection. control of the laboratory environment to maintain constant temperature, humidity, and clean-room conditions will vastly improve printing consistency. printing with solid pins (for example stealth pin, arrayit corportion, sunnyvale, ca, usa) relies on capillary forces to release spots on contact with the surface, resulting in - m diameter spots depending on buffer composition and pin diameter. dip-pen lithography (dpn) uses atomic force microscopy microcantilevers to deposit spots in the range of - m diameter (nano enabler, bioforce nanosciences, ames, ia, usa) and inkjet printers (for example arrayjet, roslin, scotland) use piezoelectric elements to transfer the protein solution in the form of droplets to the target surface, resulting in spot diameters of - m. both pin and inkjet spotting methods deposit a very small amount of protein, requiring highly concentrated samples. unfortunately, highly concentrated samples may fail to adsorb completely on the surface and tend to spread during hydration. a new continuous flow microspotting method (wasatch microfluidics, taylorsville, ut, usa) uses microfluidic channel networks to continuously circulate protein samples over a spot to achieve uniform and maximum protein adsorption [ ] . this technique may be especially useful for dilute and crude protein sample arraying. fluorescence-based labeling of probes is most commonly used to detect binding events, due primarily to simplicity and sensitivity. examples are amine-reactive dyes such as cyanines (cy and cy ) or labeling probes with biotin followed by detection with an avidin-fluorescent dye conjugate. ccd cameras or fluorescent slide scanners are used for detection of the labeled probes within defined grid areas. sandwich immunoassay methods provide increased sensitivity and at the same time reduce non-specific binding, but their use is limited by the availability of paired antibodies. using a rolling circle amplification (rca) method can dramatically increase fluorescent signal intensity [ ] , thus increasing sensitivity in protein microarray detection methods. in this scheme, proteins are labeled with biotin and recognized by an antibody conjugated with a primer to which a circular dna is hybridized. fluorescently labeled oligonucleotides are used for elongation of dna and detection. a universal rca signal amplification scheme was used [ ] for measuring cytokines in a multiplexed format. a total of cytokines were simultaneously measured on an antibody microarray, demonstrating that even larger number of analytes can be measured by this technology. thus, rca is a powerful tool for improving the detection limits of protein microarrays by signal amplification. current fluorescence-based detection in protein microarrays is limited to two to three independent probes because broad emission profiles exhibited by the dyes used for detection often overlap with each other and with the background fluorescence emitted by substrates, such as the commonly used nitrocellulose. also, multiple excitation and emission channels are required to detect combinations of fluorescent markers. quantum dot (qds) nanoparticles are one alternative to fluorescent dyes. the qds are semiconducting fluorophores that are extremely bright, resist photobleaching, and have multiplexing capability. each contains an inorganic fluorescent core, commonly cdse, coated with a shell of another semiconductor such as cds or zns. the most important property of qds with reference to protein microarray detection is that they absorb light at broad wavelengths, from uv to visible range and emit light at a very narrow bandwidth, in contrast to fluorescent dyes. additionally, the qd surfaces may be modified with suitable chemistries for immobilizing biomolecules. streptavidin-coated qds in a protein microarray format were used to detect six different cytokines at picomolar concentrations [ ] , currently the practical limit for multiplexing with qds. additional multiplexing strategies have been previously described in detail [ ] . bead-based microarrays offer one solution to multiplexing. most commercially available methods were developed by luminex (xmap platform). the xmap technology is based on polymer beads incorporated with fluorescent dyes that produce unique signatures. these beads can be conjugated to different molecules to perform multiplexed assays. up to discrete interactions can be monitored simultaneously using this technology [ ] , perhaps extended to bead sets in the near future. antigens or peptides have been attached to the luminex beads and antibody responses to papillomavirus [ ] , epstein-barr virus [ , ] , and mycobacterium tuberculosis [ ] were reported. becton dickinson (franklin lakes, nj, usa) markets a cytometric bead array (cba) system, which is another multiplexed bead-based assay that includes flow cytometry for cytokine, chemokine, mouse isotyping, and signaling molecule analysis. label-free detection methods are used for real-time monitoring of binding events and to minimize artifacts caused by using labeled probes. spr imaging technology is widely used in a microarray format for many applications. spr is label-free, allows real-time monitoring, and has the additional ability to measure kinetics of the molecular interactions ( figure ). spr imaging was developed to simultaneously measure spr dip changes in an array format, recorded with a ccd camera. grating-coupled (flexchip, ge/biacore, piscataway, nj, usa) and prism-coupled (spri-plex, horiba scientific, edison, nj, usa, proteon xpr , bio-rad, hercules, ca, usa) spri systems are some examples of commercially available spri microarray systems. a model protein-protein interaction system was reported [ ] , consisting of the e protein of human papillomavirus complexing with the proteins e ap and p using a spri system. in another example, an array-based spectral spr system was used for measuring antibody response to mumps virus infection [ ] . in this system the reflected light from the array was collected into a fiber optic spectrometer for analysis. our laboratory routinely uses spri microarrays to validate interactions that were detected during primary microarray screens from a fluorescent probe read-out. because spot sizes must be large enough for resolution by the commonly used ccd imaging, practical array densities are currently less than independent immobilized probes. additional label-free detection methods have been described. surface-enhanced laser desorption/ionization time of flight mass spectrometry (seldi-tof-ms) has been used as a low throughput method for on-chip purification of proteins, ionization, and detection [ ] . however, this approach was not very successful in identifying biomarkers in parasitic diseases, and most markers found in the study were intact host proteins [ , ] . matrix-assisted laser desorption/ionization (maldi)-ms in combination with -d gel surfaces [ ] and silicon nanoporous nanovials [ ] were also used for biomarker discovery. the ms-based detection systems are important for specific applications, but there have been only limited advancements in the development of microarray formats, currently limiting use as a primary screening tool. nanowire-based systems detect changes in conductivity as a result of molecular binding [ , ] . it is possible to make an array of nanowires such that each nanowire is coated with a specific capture molecule. gantelius et al. [ ] reported a magnetic bead-based multiplexing format for the detection of auto-antibodies to antigens, comparing the assay results to fluorescence-based detection system. although the magnetic bead-based system was rapid, it was not sufficiently sensitive for routine use. despite the rapid progress in protein microarray development standardized methods for data handling and analysis are not universally accepted. fluorescence scanners made by different commercial vendors use stand-alone software to analyze data, while assay protocols used by investigators may vary widely. hence, it is often difficult to compare studies reported by different groups, and there is a need to formulate standardized methods for protein microarray data handling and analysis. a number of reports have outlined ways to normalize protein microarray data, mainly for different approaches to antibody microarrays. these normalization methods include concentrationdependant analysis [ , ] , spiked internal standards [ ] , and algorithm-based [ ] , and all have significant pros and cons. invitrogen/life technologies (carlsbad, ca, usa) developed a data analysis package (protoarray prospector) specifically designed for protein antigen microarrays, including a suite of statistical methods. bioarray software environment (base), a software package for microarray data management and analysis (http://base.thep.lu.se) developed by lund university, sweden, also addresses some of the issues we have outlined. however, it remains to be seen how rapidly standards will be adopted by the research community. antibodies are primary biomarkers of infection that are commonly used for diagnosis, measuring vaccine efficacy, and discovery of disease interventions. protein microarrays are important tools for measuring these complex antigen-antibody interactions and other host-pathogen interactions at different stages of the disease. the major impediments to development of protein microarrays are the inherent complexity of proteins themselves, availability of well-characterized antibodies to infectious agents, and standardized statistical methods for analysis of data. however, we expect many of these issues to be resolved in the coming years as these tools are used more frequently in infectious disease research. quantifying e. coli proteome and transcriptome with single-molecule sensitivity in single cells correlation between protein and mrna abundance in yeast label-free detection methods for protein microarrays present and future of surface plasmon resonance biosensors multiplexed electrical detection of cancer markers with nanowire sensor arrays high-speed biomarker identification utilizing porous silicon nanovial arrays and maldi-tof mass spectrometry protein and antibody microarrays: clues towards biomarker discovery. front. drug des protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis defining the humoral immune response to infectious agents using high-density protein microarrays design of high-density antibody microarrays for disease proteomics: key technological issues extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis analysis of the human immune response to vacccina by use of novel protein microarray suggests that antibodies recognize less than % of the total viral proteome applications of protein microarrays for biomarker discovery protein microarrays: high-throughput tools for proteomics advances and perspectives in aptamer arrays affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats use of a single-chain antibody library for ovarian cancer biomarker discovery reversephase protein microarrays: application to biomarker discovery and translational medicine development of reverse phase protein microarrays for clinical applications and patient-tailored therapy reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front analysis of signaling pathways in cancer cell lines by protein lysate array reverse-phase protein lysate microarrays for cell signaling analysis multiplex approaches in protein microarray technology printing proteins as microarrays for high-throughput function determination self-assembling protein microarrays global analysis of protein activities using proteome chips proteome-wide analysis of the serological response to vaccinia and smallpox antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of dryvax a burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens protein microarray for profiling antibody responses to yersinia pestis live vaccine serologic survey of the sentinel animals for plague surveillance and screening for complementary diagnostic markers to f antigen by protein microarray antibody profiling in plague patients by protein microarray from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis immunodominant francisella tularensis antigens identified using proteome microarray candidate antigens for q fever serodiagnosis revealed by immunoscreening of a coxiella burnetii protein microarray large scale immune profiling of infected humans and goats reveals differential recognition of brucella melitensis antigens a genome-wide proteome array reveals a limited set of immunogens in natural infections of humans and white-footed mice with borrelia burgdorferi genome-wide study of pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays bacterial protein microarrays for identification of new potential diagnostic markers for neisseria meningitidis infections analysis of phagosomal proteomes: from latex-bead to bacterial phagosomes anthrax infection inhibits the akt signaling involved in the e-cadherin-mediated adhesion of lung epithelial cells capturing host-pathogen interactions by protein microarrays: identification of novel streptococcal proteins binding to human fibronectin, fibrinogen, and c bp profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery identification of humoral immune responses in protein microarrays using dna microarray data analysis techniques single step generation of protein arrays from dna by cell-free expression and in situ immobilisation (pisa method) next-generation high-density self-assembling functional protein arrays printing protein arrays from dna arrays systematic analysis of the igg antibody immune response against varicella zoster virus (vzv) using a self-assembled protein microarray a systematic approach for the identification of novel, serologically reactive recombinant varicella-zoster virus (vzv) antigens high-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins severe acute respiratory syndrome diagnostics using a coronavirus protein microarray peptide-protein microarrays for the simultaneous detection of pathogen infections felger, i. serodiagnosis of echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray a protein-domain microarray identifies novel protein-protein interactions quantifying protein-protein interactions in high throughput using protein domain microarrays protein arrays as tools for serum autoantibody marker discovery in cancer protein microarrays: a new tool for profiling antibody cross-reactivity analyzing antibody specificity with whole proteome microarrays protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin b antibody microarrays for native toxin detection antibody microarray detection of escherichia coli o :h : quantification, assay limitations, and capture efficiency an antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules protein microarrays for antibody profiling: specificity and affinity determination on a chip evaluation of antibodies and microarray coatings as a prerequisite for the generation of optimized antibody microarrays microarrays based on affinity-tagged single-chain fv antibodies: sensitive detection of analyte in complex proteomes why -d? gel-based microarrays in proteomics continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging mutation detection and single-molecule counting using isothermal rolling-circle amplification multiplexed protein profiling on microarrays by rolling-circle amplification protein microarrays and quantum dot probes for early cancer detection antibody suspension bead arrays within serum proteomics serologic response to oncogenic human papillomavirus types in male and female university students in evaluation of three different assays for the assessment of epstein barr-virus immunological status evaluation of a multiplexed bead assay for assessment of epstein-barr virus immunologic status profiling antibodies to mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of hpv, e , e ap, and p array-based spectral spr biosensor: analysis of mumps virus infection is seldi-tof a valid tool for diagnostic biomarkers? identification of novel diagnostic serum biomarkers for chagas' disease in asymptomatic subjects by mass spectrometric profiling use of proteinchip technology for identifying biomarkers of parasitic diseases: the example of porcine cysticercosis (taenia solium) analysis of protein interaction and function with a -dimensional maldi-ms protein array nanowire nanosensors for highly sensitive and selective detection of biological and chemical species protein recognition via surface molecularly imprinted polymer nanowires magnetic bead-based detection of autoimmune responses using protein microarrays a concentration-dependent analysis method for high density protein microarrays development of an internally controlled antibody microarray optimized normalization for antibody microarrays and application to serum-protein profiling determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays the authors appreciate the many contributions of sarah keasey, christine pugh, emily cisney, stefan fernandez, and other members of the riid protein interactions team, the contributions of barry schweitzer and our other collaborations at life technologies for pioneering studies in protein microarray technology, and justin rodrigo for critical reading of the manuscript. opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the united states army. key: cord- -xx imhzb authors: lambour, jennifer; naranjo-gomez, mar; piechaczyk, marc; pelegrin, mireia title: converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: xx imhzb monoclonal antibodies (mabs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. indeed, the number of antiviral mabs developed in recent years has grown exponentially. although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. the ability of antiviral mabs to modulate antiviral immune responses in infected organisms has recently been revealed. more specifically, upon recognition of their cognate antigens, mabs form immune complexes (ics) that can be recognized by the fc receptors expressed on different immune cells of infected individuals. this binding may be followed by the modulation of the host immune responses. harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mabs. this review focuses on the role of ics formed with different viral determinants and mabs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. potential deleterious effects of ics on the host immune response are also discussed. therapeutic potential of antiviral monoclonal antibodies monoclonal antibodies (mabs) have gained an important place in the therapeutic arsenal against severe human diseases. more than mabs have been approved or are under review for human use, and several hundred are currently being tested in the clinic, , most of them to treat patients suffering from a variety of cancers or inflammatory diseases. concerning antiviral mabs, only one, directed against respiratory syncytial virus (rsv), has been approved for the prophylactic treatment of pediatric infections. however, employing mabs as antiviral drugs is under consideration for the treatment of several chronic and acute severe viral infections, especially to address the public health emergencies such as the recent ebola virus and middle east respiratory syndrome coronavirus outbreaks. [ ] [ ] [ ] [ ] [ ] illustrating this trend, the number of antiviral mabs developed and tested in preclinical and clinical trials has grown exponentially in the past years and includes mabs directed against life-threatening agents, such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv), influenza virus, dengue virus, ebola virus and severe acute respiratory syndrome virus coronavirus, among others. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] importantly, recent clinical data have also demonstrated the efficacy of anti-hiv mabs in controlling viremia, when administered to hiv-infected patients, lending strong support to the idea that mabs could broaden the therapeutic arsenal against severe viral infections. , their use as antiviral agents is all the more likely to be considered given that multiple biological activities may account for their therapeutic effects. although a few mabs have been developed to inhibit the recognition of viral receptors or co-receptors on the surface of target cells, most antiviral mabs have been selected for their ability to neutralize virions through the binding of their antigen-binding (fab) fragment to viral surface antigens essential for entry into host cells. however, the biological activity of antibodies is also mediated by the fragment crystallizable region (fc) moiety. thus, it is interesting to note that most antiviral mabs in use are immunoglobulin (ig)-gs displaying a variety of effector functions, including binding to both complement and fcγ receptors (fcγrs). different types of fcγrs are expressed in a regulated manner by many cells of the immune system, including b cells, natural killer cells, dendritic cells (dcs), monocytes/macrophages, granulocytes and mast cells, and their engagement by the fc antibody moiety is essential for regulating the antibody effector functions. , upon recognition of their target antigens, antiviral mabs can facilitate virus elimination via two types of complementmediated mechanisms: (i) inactivation of viral particles and/or phagocytosis of opsonized virus mediated by cells of the innate immune system ( figure ) and (ii) opsonization and subsequent lysis of infected cells, when viral antigens are also expressed on the cell surface (for example, envelope (env) glycoprotein of lentiviruses such as hiv) via complement-dependent cytotoxicity. in addition to complementmediated actions, recognition of fcγrs can entail antibody-dependent cellular phagocytosis and antibody-dependent cell-mediated cytotoxicity ( figure ). , [ ] [ ] [ ] [ ] finally, antiviral mabs also have a role in viral blunting by inhibiting cell-to-cell viral transmission. in addition to controlling the viral propagation by these mechanisms, the opsonization of viral particles and/or infected cells by therapeutic antiviral mabs of the igg type leads to the formation of immune complexes (ics) recognizable by the fcγrs expressed on antigen-presenting cells (apcs) such as dcs. this can potentially affect the endogenous antiviral adaptive immune response of passive immunotherapy-treated individuals. despite the fact that the immunoregulatory functions of antibodies (as well as ics) have been known for a long time, and have been reported in different experimental settings and physiopathological situations, , - the immunomodulatory role of mabs with clinical potential as antiviral drugs has only recently been considered. this review mainly focuses on the induction of antiviral immune responses by ics in both passive immunotherapies and vaccination strategies. the potential deleterious effects of antiviral antibodies on the host immune dysfunction and/or viral propagation are also discussed. only recently have studies addressed whether and how passive immunotherapies utilizing antiviral mabs are able to enhance the antiviral immunity in infected individuals. this is largely due to the limited availability of suitable immunocompetent animal models of viral infection that allow in-depth investigations of the endogenous immune response. the concept that passive immunotherapies utilizing antiviral mabs can induce long-term protective immunity has recently been established using an immunocompetent mouse model, consisting of short immunotherapies given to young animals infected with the frcase murine leukemia virus. the induction of such 'vaccine-like' effects by antiviral mabs, as well as some of the mechanisms involved, are reviewed in detail elsewhere. in brief, the inoculation of mouse pups with frcase is fatal because the antiviral immune response is too weak to control the viral propagation. in contrast, treatment with a neutralizing mab for several days shortly after infection blunts viral propagation and induces a lifelong protective antiviral immunity composed of both a highly neutralizing humoral response and a cytotoxic cd + t-cell response. [ ] [ ] [ ] [ ] [ ] [ ] this induction of protective immunity strictly depends on the fc fragment of the neutralizing mabs. , moreover, the formation of ics composed of the administered mabs and infected cells rather than virions is crucial for the enhanced antiviral immune response. such ics are recognized by the fcγrs expressed by dcs, which facilitate ics internalization and lead to stronger activation and more efficient antigen presentation by dcs, eventually leading to stronger cytotoxic t-lymphocyte (ctl) responses. an fc-mediated effect that occurs concurrently is the inhibition of regulatory t-cell (treg) expansion. this depends on the mab effector functions and occurs rapidly. moreover, it is necessary for the development of the protective humoral and cellular responses, as treg-mediated immunosuppression is observed in all cases of chronic viral infections, where it dampens antiviral immune responses, thereby permitting the establishment of chronicity. finally, breastfeeding and placental transfer of maternal anti-frcase igs induced by mab immunotherapy not only led to the viral propagation blunting in infected pups, but also to the induction of long-lasting protective humoral immunity in these animals. this is a particularly interesting observation when one considers that the frcase model is reminiscent of perinatal infection by hiv, including breastfeeding-mediated mother-to-child virus transmission. other evidence for the induction of 'vaccine-like' effects by antiviral mabs comes from studies in several preclinical models of human viral infections and from hiv-infected patients. in a mouse model of rsv infection, the administration of a neutralizing mab directed against the virus attachment protein g induced a shift in the adaptive immune response from th -to th -type, leading to sustained and enhanced humoral and cd + t-cell responses. however, this effect was not fc-dependent, but rather due to the ability of the therapeutic mab to counteract the intrinsic immunosuppressive activity of the rsv g protein. mab-driven enhancement of the humoral response has also been reported in two preclinical models of henipavirus infection in african green monkeys. , recovery from both hendra and nipah virusinduced disease correlated with the development of host antibody responses consequent to the administration of the highly neutralizing . mab. this hendra and nipah virus cross-reactive mab is currently being considered for human use. finally, anti-hiv antibodies can modulate immune responses in infected organisms. such effects were initially reported in several nonhuman primate models of hiv infection and then observed in infected humans. macaques were infected with different strains of simian immunodeficiency virus (siv) or simian hiv (shiv, a chimeric virus in which hiv env substitutes for that of siv and allows for the assessment of the antiviral effects of anti-hiv antibodies) following different protocols. these experiments showed that the administration of highly neutralizing antibodies (either mabs or polyclonal igs) enhanced both the humoral and cellular antiviral immune responses of treated animals. , [ ] [ ] [ ] interestingly, recent clinical data describe the elicitation of host humoral responses in viremic subjects upon single injection of the potent bnc anti-hiv mab. however, the mechanisms leading to the stimulation of antiviral immune responses in these preclinical models of hiv infection or in infected patients remain uncharacterized. moreover, it is unknown whether these antiviral responses have genuine protective vaccine-like effects. in any case, these important observations open new avenues for the improvement of mab-based antiviral hiv therapies. moreover, as the in vivo activity of anti-hiv- bnabs, including viral load control, was recently shown to crucially depend on fc effector functions, , an important issue is identifying that fc-fcγrs interactions are involved in the induction of vaccinelike effects by antiviral mabs. to understand the mechanisms underlying the enhancement of antiviral responses by ics, several in vitro studies have addressed whether antibody-mediated viral uptake by dcs could lead to stronger activation of these cells and the development of stronger virus-specific cd + and cd + t-cell responses in an fc-dependent manner. such an increase in the cellular immune response has been reported in different infectious settings using ics made with different types of antigens, including recombinant viral proteins and whole virions, as well as infected cells (table ) . concerning ics made with viral proteins, several reports have shown that ics made up of anti-hbv mabs and the hepatitis b surface antigen (hbsag) can affect dc function and enhance t-cell responses. hbsag/anti-hbv ics significantly increased the uptake of the immunocomplexed hbsag antigen, and augmented the in vitro proliferation of virus-specific t cells and their production of interferon (ifn)-γ. moreover, dcs from hbv-infected patients incubated with hbsag/anti-hbv ics showed higher expression of major histocompatibility complex (mhc)-ii molecules and higher production of interleukin (il)- . ic-loaded dcs also enhanced production of il- and ifn-γ by co-cultured t cells. interestingly, the therapeutic efficacy of hbsag/anti-hbv ics has been tested in clinical trials (see below) in hbv-infected patients with encouraging results. [ ] [ ] [ ] more recently, in experiments aimed at visualizing immunopotentialization by hbsag/anti-hbv ics (see below), live-cell imaging revealed that ics were internalized via the fcγrs of apcs and were subsequently transported through early and late endosomes into lysosomes, where they co-localized with mhc-i and mhc-ii molecules. consistent with the latter observation, the administration of dcs loaded with hbsag/anti-hbv ics to mice increased the number of ifn-γ-and tumor necrosis factor-α-producing cd + and cd + t cells. similarly, in an siv infection setting, the incubation of apcs with ics made with a recombinant full-length gag p protein and an anti-p igg increased siv capsid cross-presentation. capsid cross-presentation was dependent on fcγr-mediated uptake of the immunocomplexed siv capsid protein, and required its proteasomal and endosomal degradation to generate stronger gag-specific cd + t-cell responses. from a mechanistic standpoint, these studies indicate that antiviral antibodies might enhance the priming and expansion of virus-specific cd + and cd + t cells by both promoting the secretion of key cytokines and facilitating the uptake and crosspresentation of viral ags by fcγr-expressing dcs. immune-complexed whole virions have also been shown to affect the functional activation of dcs. the stimulation of dcs with ics composed of siv virions and highly neutralizing siv-hyperimmune sera (svig) led to the increased virus-specific cd + t-cell responses in an fc-dependent manner. in contrast, dcs stimulated with ics made of hiv- and a polyclonal igg pool from hiv-infected subjects showed only weak hiv-specific ctl-stimulating activity. this suggested that opsonization of hiv- by iggs might be associated with decreased ctl-stimulatory dc activity. however, not all igg isotypes display equivalent effector functions. therefore, the undefined nature of the antibodies (both in terms of predominant isotypes and neutralization potential) used to form the hiv-ics in these experiments might explain these observations. whether hiv-ics made with highly neutralizing anti-hiv mabs of a specific igg isotype might induce stronger cd + t-cell responses is an important issue deserving further investigation (figure ) . moreover, the nature of the viral determinant present in ics might also be crucial in the stimulation of antiviral responses. interestingly, as mentioned above, in the mouse frcase infection model, ics made up of a neutralizing mab and infected cells, but not those made with virions, efficiently induce strong gag-specific cd + t-cell responses with high cytotoxic activity. this observation shows that the viral and cellular ics can trigger different immunologic outcomes. in the case of frcase, this is explained by the fact the frcase-gagl ctl immunodominant epitope is, at best, poorly incorporated into virions. taken together, these data indicate that the uptake of cellular ics might allow the presentation of a broader viral antigenic repertoire, leading to stronger effects on immunity ( figure ). ics formed with endogenous antibodies generated in virally infected mice have been shown to influence antiviral cellular immune responses in several models (table ) . notably, the highly neutralizing humoral response generated against the frcase retrovirus in mab-treated-infected mice was demonstrated to limit the viral propagation and to enhance memory cellular responses long after the disappearance of the therapeutic mab (which occurs within two weeks post administration, reflecting the natural igg lifespan in vivo). ic-mediated activation of dcs upon binding to fcγr was key for this effect. similarly, in an influenza virus infection model, ics formed with endogenous antiviral antibodies promoted more sustained antigen presentation by dcs, resulting in stronger cd + t-cell proliferation. interestingly, such prolonged antigen presentation by dcs was dependent on virus-specific, isotype-switched antibodies that facilitated the capture and cross-presentation of viral antigens by figure parameters to consider for achieving optimal ic-mediated modulation of antiviral immune responses. the optimization of vaccine-likeeffect-inducing protocols will require the consideration of several parameters such as the nature of the antigen (that is, purified viral proteins, whole virions and infected cells) and the antibody (that is, monoclonal vs polyclonal, nature of the isotype, engineered fc domain with improved effector functions and so on) used to form the immunogenic ics, as differences in these parameters might impact immunological outcomes. in addition, whether the optimized ics are used alone or in combination with immunostimulatory molecules might also be of paramount importance. several other parameters, including the ic dosage, the route of administration, the choice of adjuvant and the immunological status of patients, will also have to be considered. fcγr-expressing dcs. in addition, serum antibodies can affect the virus-specific cd + /cd + t-cell balance in an fc-dependent manner during rsv infection. an enhanced ratio of rsv-neutralizing to -non-neutralizing antibodies profoundly enhanced the cd + t-cell response. in a murine lymphocytic choriomeningitis virus (lcmv) infection model, endogenous virus-specific antibodies could stimulate innate immunity and thereby positively affect both the induction and the maintenance of the virus-specific cd + t-cell response. notably, anti-lcmv antibodies limited viral replication in peripheral organs, but allowed replication of the virus in the marginal zone of the spleen, promoting cd + t-cell priming. interestingly, anti-lcmv antibodies were also reported to be essential for long-term maintenance of the memory ctl response in infected mice. , these observations, together with the in vitro studies described above, demonstrate that virus-specific antibodies can promote the acquisition, processing and presentation of antigens that are subsequently instrumental for priming t-cell responses and programming functional cd + memory in an fc-dependent manner. they strengthen the concept that antiviral antibodies can regulate the quality and function of antiviral t-cell responses through the formation of ics. moreover, they also provide a rationale for developing novel ic-based therapeutic vaccination strategies. in , terres and wolins demonstrated the ability of ics to induce higher antibody titers than antigens alone. since then, the immunogenic potential of ics, alone or in combination with different types of adjuvants, has been tested in several viral infection systems, including animal models of human infections, for example, those involving hbv, hiv, rsv or flaviviruses. the immunostimulatory effects of ics are principally attributed to the ability of fc antibody fragments to recruit the host immune system. however, evidence also implicates fab fragments in modulation of the antiviral immune response, although the outcomes are less documented and were proposed to occur via alterations in antigen conformation and/or in the exposure of specific epitopes. we describe the enhancement in antiviral immune responses observed in ic-based vaccination experiments below (table ) . ics have been tested as vaccines to augment protective immune responses in different animal models of hbv infection. in ducks, ics made with duck hbsag and rabbit anti-duck hbsag (dhbsag/ anti-dhb) were used as immunogens in the form of solid matrixantibody-antigen complexes (smaa). such smaas contained killed staphylococcus aureus as a solid matrix and mab-opsonized viruses. they were initially shown to induce both humoral and ctl responses against the paramyxovirus simian virus in immunized mice. immunization of hbv-infected ducks with smaa-based dhbsag/anti-dhb ics led to the viral clearance in % of infected ducks. notably, the administration of dhbsag/anti-dhb ics lacking staphylococcus aureus showed decreased immunization efficiency, suggesting that the bacteria-based solid matrix functions as an adjuvant. ics have also been tested as a therapeutic vaccine against hbv infection in mice , , and woodchucks. balb/c mice immunized with hbsag/anti-hbv ics produced the increased levels of virusspecific antibodies. moreover, administering hbsag/anti-hbv ics to balb/c mice via intranasal inhalation induced both mucosal and systemic th -polarized immune responses, when administered with adjuvants such as cholera toxin or oligodeoxynucleotides containing modulation of antiviral immune responses by immune complexes j lambour et al immunostimulatory cpg motifs (cpg). this was not observed using hbsag alone. in addition, the administration of hbsag/anti-hbv ics to hbsag-positive transgenic mice decreased the serum hbsag levels and induced stronger ctl responses than hbsag alone. notably, the co-administration of ics and a plasmid coding for hbsag increased the antiviral immune response induced by ics, indicating the adjuvant effect of dna in this setting. a similar effect was also reported in a woodchuck model of hbv infection: immunization of woodchuck hepatitis virus (whv)-infected animals with whv surface antigen/anti-whv antibody ics combined with a dna vaccine resulted in a higher reduction of both viral load and antigenemia relative to ics alone. interestingly, the whv-infected animals were pretreated with lamivudine (a potent hbv antiviral drug able to enhance t-cell responses in chronically hbv-infected patients) before ic/dna immunization, suggesting that combination strategies should be considered in treating chronic hbv infections (figure ). the enhancement of antiviral immune responses by ics in vitro and in preclinical models of hbv infection paved the way for the development of ic-based therapeutic vaccination strategies against chronic viral hepatitis b infection. a therapeutic vaccine composed of yeast-derived recombinant hbsag/anti-hbv immunogenic complexes (yics) has been tested in a series of clinical trials. this vaccine approach was initially shown to be safe and to induce higher titers of hbsag antibodies, as well as to increase serum ifn-γ and il- levels in a phase i trial. importantly, a subpopulation of chronic viral hepatitis b patients showed a decrease in serum hbv viral load and hbsag levels together with an increase in anti-hbsag antibody titers in subsequent phase ii trials. , from a mechanistic standpoint, recent data showing that the administration of yic-loaded dcs to mice increased both cd + and cd + t-cell responses suggest that the improved immune responses induced by yics might account for the antiviral effect observed in a fraction of patients. in an attempt to enhance the immunogenic potential of yic-based vaccines, a phase iii trial tested the therapeutic effect of a higher number of ic doses. unfortunately, overstimulation with yic decreased the vaccine efficiency due to host immune fatigue. this suggests that vaccination protocols must be optimized and must take into account both the nature and the dose of ics, as well as other parameters such as the route of administration, the type of adjuvant and the immunological status of patients to achieve efficient protective immunization ( figure ). in , a study immunized healthy volunteers with hiv peptides. the authors found that compared with free antigen, recall immunization with ics induced stronger t-cell responses through uncharacterized mechanisms. other reports also describe alteration of the anti-hiv response by ics. [ ] [ ] [ ] in particular, the immunization of immunocompetent mice with ics containing a recombinant hiv- gp env glycoprotein and a mab ( -d mab) directed to the cd -binding site induced a higher virus-neutralizing antibody response relative to free antigen. as described above, humoral responses were further increased upon the co-administration of ics and monophosphoryl lipid-a/dimethyldioctadecylammonium adjuvants. notably, the interaction of the anti-cd -binding site mab with hiv- gp induced conformational changes in the latter, leading to the enhanced antigenicity and immunogenicity of neutralizing epitopes localized in the hiv- v loop. these observations highlight the ability of anti-hiv- antibodies to induce antigenic alterations in specific hiv- gp epitopes upon ic formation. interestingly, further improvement in the immunogenicity of the v loop was obtained in ics generated with gp mutants lacking site-specific n-linked glycans. , , taken together, these observations suggest that the ability of ics to stimulate the induction of neutralizing antibodies is dictated by the nature of the antigen, as well as the specificity and affinity of the mabs utilized. these results also indicate the potential contribution of fab-mediated activities in the enhancement of antiviral humoral responses by ics. tsouchnikas et al. investigated the influence of immunization with ics on the specificity of antibody responses using the e protein of the tick-borne encephalitis virus as an immunogen. mice were immunized with a dimeric soluble form of e (se) alone or in complex with mabs specific for each of the three domains of e. the antibody response induced by these ics was compared with that observed after immunization with se alone. unexpectedly, immunization with ics did not change the extent of the overall antibody response in immunized mice. however, substantially different antibody responses were observed between the different ics. these differences most likely reflected an epitope-shielding phenomenon and antibody-mediated structural changes that led to the dissociation of the se dimer. thus, such phenomena can profoundly influence the fine specificity of antibody responses to the same immunogen and must be considered in ic-based vaccination strategies. as mentioned above, serum anti-rsv antibodies can affect virusspecific t-cell responses. on the basis of this, kruijsen et al. tested whether ics made with the commercial rsv-neutralizing mab palivizumab could influence adaptive immune response priming after intranasal administration. substantial anti-rsv t-cell priming and b-cell responses were observed in mice receiving rsv-ics, resulting in predominant th -type cd + t-cell response and igg c antibody responses. importantly, the ics also primed anti-rsv cd + t cells. these data have important implications for the prophylaxis and treatment of pediatric rsv infections. nevertheless, interactions between ics and neonatal versus adult innate and adaptive immune systems still need to be investigated because mouse studies have revealed potential antibody-induced neonatal autoimmunity in certain settings. , ics and immune dysfunction in the course of viral infections, the formation of ics composed of viral determinants and the resulting host humoral responses can potentially produce deleterious effects. persistent ics are formed in a variety of chronic viral infections and may lead to unregulated and protracted fcγr signaling. this may lead to immune dysfunction instead of stimulating antiviral immune responses. in this regard, the high levels of ics formed during lcmv infection interfere with fcγr-mediated activities. , these endogenously formed ics were shown to outcompete the effector functions of exogenously administered therapeutic mabs, in particular binding to fcγrs expressed by immune cells. persistent endogenous ics are also linked to dysfunctional b-cell responses in hiv infection, including the suppression of antiviral iga responses and impaired production of neutralizing antibodies (reviewed in moir et al. ). the composition of ics might also negatively affect the efficiency of the antiviral immune response. for instance, the composition of ics has been shown to be dynamic throughout the course of hiv infections due to changes in both antibody specificities and virion levels. notably, circulating ics are initially comprised of antibodies that opsonize both infectious and non-infectious virions. this results in a decrease in the availability of antibodies able to blunt viral propagation. this phenomenon probably contributes to the reduced efficiency of the antibodies generated during acute infection. changes in circulating ics have also been reported in hcv infections. the level of circulating ics is low in acutely infected patients, whereas chronically infected individuals show a high proportion of immunocomplexed hcv, raising the possibility that ics may have a role in the pathogenesis of hcv, namely liver damage. moreover, the formation of ics with non-neutralizing antibodies may also lead to the antibody-dependent enhancement of viral infection of fcγr-expressing cells. this happens in several viral infections, including those by the dengue virus. [ ] [ ] [ ] [ ] along this line, the binding of ics to fcγrs on monocytes/macrophages can paradoxically suppress innate immunity, induce il- production and bias responses from th toward th . this in turn leads to the increased infectious outputs by infected cells via intrinsic antibodydependent enhancement. , finally, ics have also been reported to have a role in increasing viral loads in the context of gene transfer-based vaccination strategies. in the step hiv- vaccine trial, which evaluated a replication-defective adenovirus type vector vaccine, the ics formed with pre-existing anti-adv antibodies improved the environment for hiv- replication in t cells. this may have been due to the ic-driven activation of a dc-t-cell axis that induces the activation of cd + t cells and leads to a permissive environment for hiv- infection. this environment probably explains the increased propagation of hiv- infection among adenovirus type -seropositive vaccine recipients. several approaches can be considered to enhance the immunomodulatory potential of antiviral mabs, both alone and in the form of ics, in particular through combining neutralizing mabs and ic-based vaccination strategies with other therapies. a first possibility would consist of inhibiting immunosuppressive mechanisms in infected individuals by either depleting the treg response, as suggested by nasser et al. or targeting immune checkpoints, the latter strategy having already led to improved immune responses against both viral infections and cancer. [ ] [ ] [ ] [ ] in addition, the combination of antiviral mabs with different immunostimulatory agents can also be envisaged. because the primary structure and glycosylation pattern of the fc fragment are both essential for antibody effector functions due to their impact on the engagement of type i and type ii fcr family members, , fc engineering might also represent another approach, not only to improve direct antiviral effects, but also to induce stronger vaccine-like effects. in this regard, identification of the main fcrs and fcr-mediated mechanisms involved in enhancing the antiviral immune response will be of utmost importance. taking into account that the various igg isotypes display different effector functions and interact differently with fcγrs, the careful selection of antiviral mab subclasses is also crucial for enhancing antiviral immune responses. finally, as fcγr polymorphisms have already been associated with differences in viral disease progression and the therapeutic efficiency of anticancer mabs, it will be important to evaluate the extent to which such polymorphisms can affect the vaccine-like effects induced by mab-based antiviral immunotherapies. the therapeutic potential of antiviral mabs is now widely accepted, and their use as antiviral drugs is increasingly under consideration. the diverse biological activities of these mabs lead to the direct control of viral propagation and the modulation of antiviral immunity. this provides a novel rationale for their use in diverse prophylactic and therapeutic approaches. the improvement in both humoral and cellular responses achieved through the administration of mabs, either free or in the form of immunogenic ics, offers new therapeutic options. the challenge now is to improve our understanding of how ics convert mab-based immunotherapies from 'passive' to 'active' and to exploit the underlying mechanisms. this conversion will be crucial in reaching the goal of using antiviral mabs to induce longlasting protective immunity against life-threatening viral infections. molecular properties of human igg subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases trial watch: monoclonal antibodies in cancer therapy isolation of potent neutralizing antibodies from a survivor of the ebola virus outbreak protective monotherapy against lethal ebola virus infection by a potently neutralizing antibody prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus b -n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n / reversion of advanced ebola virus disease in nonhuman primates with zmapp therapeutic efficacy of potent neutralizing hiv- -specific monoclonal antibodies in shiv-infected rhesus monkeys antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology monoclonal antibodies for prophylactic and therapeutic use against viral infections broadly neutralizing antiviral antibodies broadly neutralizing antibodies abrogate established hepatitis c virus infection a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus clinical development of monoclonal antibody-based drugs in hiv and hcv diseases mechanism of human antibody-mediated neutralization of marburg virus cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection a single injection of anti-hiv- antibodies protects against repeated shiv challenges early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques overcoming drug-resistant herpes simplex virus (hsv) infection by a humanized antibody the growth and potential of human antiviral monoclonal antibody therapeutics a novel humanized antibody neutralizes h n influenza virus via two different mechanisms therapeutic efficacy of antibodies lacking fcgamma receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies viraemia suppressed in hiv- -infected humans by broadly neutralizing antibody bnc virologic effects of broadly neutralizing antibody vrc administration during chronic hiv- infection mouse and human fcr effector functions type i and type ii fc receptors regulate innate and adaptive immunity exploring the potential of monoclonal antibody therapeutics for hiv- eradication neutralizing antibodies and control of hiv: moves and countermoves which antibody functions are important for an hiv vaccine? fcgammar dependent mechanisms of cytotoxic, agonistic, and neutralizing antibody activities broadly neutralizing antibodies that inhibit hiv- cell to cell transmission properties of mouse and human igg receptors and their contribution to disease models how antibodies act as natural adjuvants antibodies as natural adjuvants antibody-mediated regulation of the immune response fcgamma receptors as regulators of immune responses intravenous immunoglobulin therapy: how does igg modulate the immune system? antibody-mediated immunomodulation: a strategy to improve host responses against microbial antigens antiviral monoclonal antibodies: can they be more than simple neutralizing agents? induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment endogenous cytotoxic t-cell response contributes to the long-term antiretroviral protection induced by a short period of antibody-based immunotherapy of neonatally infected mice efficient mother-to-child transfer of antiretroviral immunity in the context of preclinical monoclonal antibody-based immunotherapy a crucial role for infected-cell/antibody immune complexes in the enhancement of endogenous antiviral immunity by short passive immunotherapy long-lasting protective antiviral immunity induced by passive immunotherapies requires both neutralizing and effector functions of the administered monoclonal antibody control of regulatory t cells is necessary for vaccine-like effects of antiviral immunotherapy by monoclonal antibodies prophylaxis with a respiratory syncytial virus (rsv) anti-g protein monoclonal antibody shifts the adaptive immune response to rsv ra -line f infection from th to th in balb/c mice a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody neutralizing polyclonal igg present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus shivsf p -infected infant rhesus macaques passive neutralizing antibody controls shiv viremia and enhances b cell responses in infant macaques an anti-hiv- v loop antibody fully protects cross-clade and elicits t-cell immunity in macaques mucosally challenged with an r clade c shiv hiv- therapy with monoclonal antibody bnc elicits hoost immune responses against hiv- broadly neutralizing anti-hiv- antibodies require fc effector functions for in vivo activity broadly neutralizing antibodies and viral inducers decrease rebound from hiv- latent reservoirs in humanized mice hbsag-serum protein complexes stimulate immune t lymphocytes more efficiently than do pure hbsag selective functional deficit in dendritic cell-t cell interaction is a crucial mechanism in chronic hepatitis b virus infection evidence for antibody-mediated enhancement of simian immunodeficiency virus (siv) gag antigen processing and cross presentation in siv-infected rhesus macaques polyfunctional cd + t-cell induction in neutralizing antibody-triggered control of simian immunodeficiency virus infection antibodies attenuate the capacity of dendritic cells to stimulate hiv-specific cytotoxic t lymphocytes vaccination with recombinant hbsag-hbig complex in 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simultaneous injection of antigen and specific antiserum. i. effect of varying the amount of antigen used relative to the antiserum antigen-antibody complex as therapeutic vaccine for viral hepatitis b enhancement of the immune response to hepatitis b virus vaccine by antigen specific igm therapeutic efficacy of hepatitis b surface antigenantibodies-recombinant dna composite in hbsag transgenic mice combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model immunization against hepatitis b virus by mucosal administration of antigen-antibody complexes the use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on hiv- envelope gp improving immunogenicity of hiv- envelope gp by glycan removal and immune complex formation elicitation of broadly reactive antibodies against glycanmodulated neutralizing v epitopes of hiv- by immune complex vaccines intranasal administration of antibodybound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, simian virus solid matrix-antibody-antigen complexes induce antigenspecific cd + cells that clear a persistent paramyxovirus infection results of a phase iii clinical trial with an hbsag-hbig immunogenic complex therapeutic vaccine for chronic hepatitis b patients: experiences and findings antigenic peptides recognized by t lymphocytes from aids viral envelope-immune humans targeting a neutralizing epitope of hiv envelope gp by immune complex vaccine immunization with immune complexes modulates the fine specificity of antibody responses to a flavivirus antigen cutting edge: ly c/i(-) neonatal nk cells predispose newborns to autoimmune ovarian disease induced by maternal autoantibody the unique neonatal nk cells: a critical component required for neonatal autoimmune disease induction by maternal autoantibody antibody effector functions mediated by fcgamma-receptors are compromised during persistent viral infection suppression of fcgamma-receptor-mediated antibody effector function during persistent viral infection b cells in hiv infection and disease dynamic antibody specificities and virion concentrations in circulating immune complexes in acute to chronic hiv- infection immune complexed (ic) hepatitis c virus (hcv) in chronically and acutely hcv-infected patients paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development dengue antibody-dependent enhancement: knowns and unknowns intrinsic antibodydependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibodyenhanced viral infections antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of il- production in monocytes activation of a dendritic cell-t cell axis by ad immune complexes creates an improved environment for replication of hiv in t cells restoration of hbv-specific cd + t cell function by pd- blockade in inactive carrier patients is linked to t cell differentiation a randomized, double-blind, placebo-controlled assessment of bms- , a fully human monoclonal antibody to programmed death- (pd- ), in patients with chronic hepatitis c virus infection immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential augmentation of hepatitis b virusspecific cellular immunity with programmed death receptor- /programmed death receptor-l blockade in hepatitis b virus and hiv/hepatitis b virus coinfected patients treated with adefovir key: cord- -xzu faol authors: andreano, emanuele; nicastri, emanuele; paciello, ida; pileri, piero; manganaro, noemi; piccini, giulia; manenti, alessandro; pantano, elisa; kabanova, anna; troisi, marco; vacca, fabiola; cardamone, dario; de santi, concetta; benincasa, linda; agrati, chiara; capobianchi, maria rosaria; castilletti, concetta; emiliozzi, arianna; fabbiani, massimiliano; montagnani, francesca; depau, lorenzo; brunetti, jlenia; bracci, luisa; montomoli, emanuele; sala, claudia; ippolito, giuseppe; rappuoli, rino title: extremely potent human monoclonal antibodies from convalescent covid- patients date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: xzu faol human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the covid- pandemic. by single cell sorting sars-cov- spike protein specific memory b cells from covid- survivors, neutralizing antibodies were identified and of them were expressed as igg. up to , % of monoclonals neutralized the wild type virus at a concentration of > ng/ml, , % neutralized the virus in the range of - ng/ml and , % had a neutralization potency in the range of - ng/ml. only , % neutralized the authentic virus with a potency of - ng/ml. we found that the most potent neutralizing antibodies are extremely rare and recognize the rbd, followed in potency by antibodies that recognize the s domain, the s-protein trimeric structure and the s subunit. the three most potent monoclonal antibodies identified were able to neutralize the wild type and d g mutant viruses with less than ng/ml and are good candidates for the development of prophylactic and therapeutic tools against sars-cov- . one sentence summary extremely potent neutralizing human monoclonal antibodies isolated from covid- convalescent patients for prophylactic and therapeutic interventions. the impact of the sars-cov- pandemic, with more than million cases, over million deaths, trillion impact on the gross domestic product (gdp) and million people filing unemployment in the united states alone, is unprecedented (aratani, ) . in the absence of drugs or vaccines, non-pharmaceutical interventions such as social distancing and quarantine have been the only way to contain the spread of the virus. these interventions showed to be efficient when properly implemented but not all countries were able to do so showing the limits of these strategies. the urgency to develop vaccines and therapies is extremely high. the effort to develop vaccines is unprecedented and fortunately in october we already have several vaccines in advanced phase iii efficacy trials and many others in earlier phase of development. in spite of the big effort, it is predictable this wave of infection will continue to spread globally and it is likely to be followed by additional waves in the next few years until herd immunity, acquired by vaccination or by natural infection, will constrain the circulation of the virus. it is therefore imperative to develop in parallel both vaccines and therapeutic tools to face the next waves of sars-cov- infections as soon as possible. among the many therapeutic options available, human monoclonal antibodies (mabs) are the ones that can be developed in the shortest period of time. in fact, the extensive clinical experience with the safety of more than commercial mabs approved to treat cancer, inflammatory and autoimmune, disorders provides high confidence on their safety. these advantages, combined with the urgency of the sars-cov- pandemic, support and justify an accelerated regulatory pathway. in addition, the long industrial experience in developing and manufacturing mabs decreases the risks usually associated with the technical development of investigational products. finally, the incredible technical progress in the field allows to shorten the conventional timelines and go from discovery to proof of concept trials in - months (kelley, ) . indeed, in the case of ebola, mabs were the first therapeutic intervention recommended by the world health organization (who) and they were developed faster than vaccines or other drugs (kupferschmidt, ) . the sars-cov- spike glycoprotein (s-protein) has a pivotal role in viral pathogenesis and it is considered the main target to elicit potent neutralizing antibodies and the focus for the development of therapeutic and prophylactic tools against this virus , tay et al., . indeed, sars-cov- entry into host cells is mediated by the interaction between s-protein and the human angiotensin converting enzyme (ace ) (wang et al., b . the s-protein is a trimeric class i viral fusion protein which exists in a metastable prefusion conformation and in a stable postfusion state. each sprotein monomer is composed of two distinct regions, the s and s subunits. structural rearrangement occurs when the receptor binding domain (rbd) present in the s subunit binds to the host cell membrane. this interaction destabilizes the prefusion state of the sprotein triggering the transition into the postfusion conformation which in turn results in the ingress of the virus particle into the host cell (wrapp et al., ) . single-cell rnasequencing analysis revealed that ace expression was ubiquitous in different human organs have suggesting that sars-cov- , through the s-protein, can invade human cells in different major physiological systems including the respiratory, cardiovascular, digestive and urinary systems, thus enhancing the possibility of spreading and infection (zou et al., ) . during the first few months of this pandemic, several groups have been active in isolating and characterizing human monoclonal antibodies from covid- convalescent patients or from humanized mice and some of them have been progressing quickly to clinical trials for the prevention and cure of sars-cov- infection (shi et al., , hansen et al., , wang et al., a , pinto et al., , zost et al., , rogers et al., , andreano et al., . so far, most of the work on human monoclonal antibodies against sars-cov- started from one or few patients and it allowed to successfully isolate and characterize the first interesting antibodies with moderate neutralization potency. in many cases an hundreds of nanograms to micrograms of antibodies were required to neutralize the virus in vitro therefore grams of antibodies will be needed per single patient. in this scenario intravenous delivery results to be the only possible administration route for their therapeutic and prophylactic use. only recently very potent human antibodies have been isolated and these can be considered for intramuscular or subcutaneous administration. a striking example is a monoclonal antibody against rsv which delivered intramuscularly to premature babies has shown very promising results in clinical settings (griffin et al., ) . furthermore, giving the initial rush in isolating potential antibody candidates for clinical development, the whole picture on different types of neutralizing antibodies generated after infection is not yet clear. to identify and characterize potent mabs against sars-cov- , we isolated over , sprotein specific-memory b cells (mbcs) derived from covid- convalescent. from the screening of thousands of b cells, three extremely potent monoclonal antibodies were identified and are excellent candidates for further development. isolation and characterization of s-protein specific antibodies from sars-cov- convalescent patients to retrieve mabs specific for sars-cov- s-protein, peripheral blood mononuclear cells (pbmcs) from fourteen convalescent patients enrolled in this study were collected and stained with fluorescent labelled s-protein trimer to identify antigen specific memory b cells (mbcs). fig. summarizes the overall experimental strategy. the gating strategy described in fig. s was used to single cell sort into -well plates igg + and iga + mbcs binding to the sars-cov- s-protein trimer in its prefusion conformation. the sorting strategy aimed to specifically identify class-switched mbcs (cd + cd + igd -igm -) to identify only memory b lymphocytes that went through maturation processes. a total of , s-protein-binding mbcs were successfully retrieved with frequencies ranging from , % to , % (table ) . following the sorting procedure, s-protein + mbcs were incubated over a layer of t -cd l feeder cells in the presence of il- and il- stimuli for two weeks to allow natural production of immunoglobulins ( ). subsequently, mbc supernatants containing igg or iga were tested for their ability to bind either the sars-cov- s-protein trimer in its prefusion conformation or the s-protein s + s subunits ( fig. a -b) by enzyme linked immunosorbent assay (elisa). a panel of , mabs specific for the sars-cov- s-protein were identified showing a broad range of signal intensities (table ) . identification of s-protein specific mabs able to neutralize sars-cov- the , supernatants containing s-protein specific mabs, were screened in vitro for their ability to block the binding of the s-protein to vero e cell receptors. and for their ability to neutralize authentic sars-cov- virus by in vitro microneutralization assay. in the neutralization of binding (nob) assay, of the , tested ( , %) s-protein specific mabs were able to neutralize the antigen/receptor binding showing a broad array of neutralization potency ranging from % to over % (table and fig. c ). as for the authentic virus neutralization assay, supernatants containing naturally produced igg or iga were tested for their ability to protect the layer of vero e cells from the cytopathic effect triggered by sars-cov- infection (fig. s ) . to increase the throughput of our approach, supernatants were tested at a single point dilution and to increase the sensibility of our first screening a viral titer of tcid was used. for this first screening mabs were classified as neutralizing, partially neutralizing and not-neutralizing mabs based on their inability to protect vero e cells from infection, or to their ability to partially or completely prevent the cytopathic effect. out of , mabs tested in this study, a panel of ( , %) mabs neutralized the live virus and prevented infection of vero e cells (table ). the percentage of partially neutralizing mabs and neutralizing mabs (nabs) identified in each donor was extremely variable ranging from , - , % and , - , % respectively ( fig. a and table s ). the majority of nabs were able to specifically recognize the s-protein s domain ( , %; n= ) while , % (n= ) of nabs were specific for the s domain and , % (n= ) did not recognize single domains but only the s-protein in its trimeric conformation ( fig. b ; table s ). from the panel of nabs, we recovered the heavy and light chain amplicons of nabs which were expressed as full length igg using the transcriptionally active pcr (tap) approach to characterize their neutralization potency. the vast majority of nabs identified ( , %; n= ) had a low neutralizing potency and required more than ng/ml to achieve an ic . a smaller fraction of the antibodies had an intermediate neutralizing potency ( , %; n= ) requiring between and ng/ml to achieve the ic , while , % (n= ) required between and ng/ml. finally, only , % (n= ) of the expressed nabs were classified as extremely potently nabs, showing an ic lower than ng/ml ( fig. c -d; table s ). based on the first round of screening, nabs were selected for further characterization. all nabs were able to bind the sars-cov- s-protein in its trimeric conformation (fig. a ). the mabs named j , i , f , g , c , b , i , j and d were also able to specifically bind the s domain (fig. b ). the nabs named h , i , f and f were not able to bind single s or s domains but only the s-protein in its trimeric state, while the nab l bound only the s subunit ( fig. b -c) . among the group of s specific nabs only j , i , f , g , c and b were able to bind the s receptor binding domain (rbd) and to strongly inhibit the interaction between the s-protein and vero e receptors table ). on the other hand i , j and d , despite showing s binding specificity, did not show any binding to the rbd and nob activity ( fig s and table ). based on this description four different groups of nabs against sars-cov- were identified. the first group (group i) is composed by s -rbd specific nabs (j , i , f , g and c ) and showed extremely high neutralization potency against both the wt and d g live viruses ranging from , to , ng/ml ( fig. d -f; table ). the second group (group ii) is composed by s nabs that did not bind the rbd (b , i , j and d ). these antibodies also showed good neutralization potency ranging from , to ng/ml (fig. d -f; table ) but inferior to s -rbd directed nabs. antibodies belonging to group i and ii showed picomolar affinity to the s-protein with a kd ranging from . to . e - m (fig. s ). the third group (group iii) is composed by antibodies able to bind the s-protein only in its whole trimeric conformation (h , i , f and f ). antibodies belonging to this group showed lower affinity to the s-protein (kd . e - m - . e - m) compared to group i and ii nabs and medium neutralization potencies ranging from , to , ng/ml ( table ). this work describes a systematic screening of memory b cells from convalescent people to identify extremely potent human monoclonal antibodies against the spike protein of the sars-cov- virus, to be used for prevention and therapy of covid- . we found that approximately % of the total b cells against the spike protein produce neutralizing antibodies and these can be divided into different groups recognizing the s -rbd, s domain, s -domain and the s-protein trimer. we found that the most potent neutralizing antibodies are extremely rare and that they recognize the rbd, followed in potency by the antibodies recognizing the s domain, the trimeric structure and the s subunit. the l antibody against the s subunit, which had the lowest neutralizing potency, is representative of several other s specific antibodies identified in the preliminary screening. from these data we conclude that in convalescent patients most of the observed neutralization titers are mediated by the antibodies with medium-high neutralizing potency. indeed, the extremely potent antibodies and the antibodies against the s subunit are unlikely to contribute to the overall neutralizing titers because they are too rare and too poor neutralizers respectively to be able to make the difference. the observed antibody repertoire of convalescent patients may be a consequence of the loss of bcl- -expressing follicular helper t cells and the loss of germinal centers in covid- patients which may limit and constrain the b cell affinity maturation (kaneko et al., ) . it is therefore important to perform similar studies following vaccination as it is likely that the repertoire of neutralizing antibodies induced by vaccination may be different from the one described here. out of neutralizing antibodies that were tested and characterized three showed extremely high neutralization potency against both the initial sars-cov- strain isolated in wuhan and the d g variant currently spread worldwide. during the last few months several groups reported the isolation, structure and passive protection in animal models of neutralizing antibodies against sars-cov- . most of these studies, with few exceptions report antibodies which require from to several hundreds more ng/ml to neutralize % of the virus in vitro. these antibodies are potentially good for therapy. however, they will require a high dosage which will result in elevated cost of goods, low capacity to numbers large quantities of doses and intravenous infusion. the extremely potent candidates described in our study will allow to use small quantities of antibodies to reach the prophylactic and therapeutic dosage and as consequence decrease the cost of goods and implement sustainable development and manufacturability. this solution may increase the number of doses produced annually and therefore increase antibodies availability in high income countries as well as low-and middle-income countries (lmics). our work combined with institutions such as the elisa assay with s and s subunits of sars-cov- s-protein the presence of s -and s -binding antibodies in culture supernatants of monoclonal s- vector digestions were carried out with the respective restriction enzymes agei, sali and xho as previously described (tiller et al., , wardemann and busse, ) . briefly, ng of igh, igλ and igκ purified pcrii products were ligated by using the gibson assembly neb into ng of respective human igγ , igκ and igλ expression vectors. the reaction was performed into μl of total volume. ligation product was -fold diluted in nucleasefree water (depc) and used as template for transcriptionally active pcr (tap) reaction which allowed the direct use of linear dna fragments for in vitro expression. the entire process consists of one pcr amplification step, using primers to attach functional promoter (human cmv) and terminator sequences (sv ) onto the fragment pcrii products. tap reaction was performed in a total volume of μl using μl of q polymerase (neb), μl of gc enhancer (neb), μl of x buffer, mm dntps, , µl of forward/reverse primers and μl of ligation product. tap reaction was performed by using the following cycles: °/ ', cycles °/ '', °/ '', °/ ' and °/ ' as final extention step. tap products were purified under the same pcrii conditions, quantified by expi f cell line using manufacturing instructions. the sars-cov- virus was propagated in vero e cells cultured in dmem high glucose supplemented with % fbs, u/ml penicillin, µg/ml streptomycin. cells were seeded at a density of x cells/ml in t flasks and incubated at °c, % co for - hours. the sub-confluent cell monolayer was then washed twice with sterile dulbecco's phosphate buffered saline (dpbs). cells were inoculated with , ml of the virus properly diluted in dmem % fbs at a multiplicity of infection (moi) of . , and incubated for h at °c in a humidified environment with % co . at the end of the incubation, ml of dmem % fbs were added to the flasks. the infected cultures were incubated at °c, % co and monitored daily until approximately - % of the cells exhibited cytopathic effect (cpe). culture supernatants were then collected, centrifuged at °c at , rpm for minutes to allow removal of cell debris, aliquoted and stored at - °c as the harvested viral stock. viral titers were determined in confluent monolayers of vero e cells seeded in -well plates using a % tissue culture infectious dose assay (tcid ). cells were infected with serial : dilutions (from - to - ) of the virus and incubated at °c, in a humidified atmosphere with % co . plates were monitored daily for the presence of sars-cov- induced cpe for days using an inverted optical microscope. the virus titer was estimated according to spearman-karber formula (kundi, ) and defined as the reciprocal of the highest viral dilution leading to at least % cpe in inoculated wells. the neutralization activity of culture supernatants from monoclonal was evaluated using a cpe-based assay as previously described negative for sars-cov- in elisa and neutralization assays). following expression as full-length igg recombinant antibodies were quantitatively tested for their neutralization potency against both the wild type and d g strains. the assay was performed as previously described but using a viral titer of tcid . antibodies were prepared at a starting concentration of µg/ml and diluted step : . technical triplicates were performed for each experiment. characterization of sars-cov- rbd-antibodies binding by flow cytometry containing the captured mab for sec at a flow rate of µl/min. dissociation was followed for sec, regeneration was achieved with a pulse ( sec) of glycine ph . . kinetic rates and affinity constant of spike protein binding to each mab were calculated applying a : binding as fitting model using the bia t evaluation software . . ng/ml). in all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the sars-cov- s -rbd, s -domain, s-protein trimer only and s -domain respectively. table s . identification of neutralizing antibodies. the table shows numbers and percentages of neutralizing, partially neutralizing and not-neutralizing antibodies identified for each donor assessed in this study. table s . s-protein binding distribution of neutralizing antibodies. the table summarizes the binding distribution of neutralizing antibodies against the s -domain, s -domain and s-protein trimer. table s . potency distribution of sars-cov- s-protein specific nabs. the table reports the number of recombinant nabs expressed per each subject and their distribution based on the neutralization potency. jobless america: the coronavirus unemployment crisis in figures. the guardian single-dose nirsevimab for prevention of rsv in preterm infants studies in humanized mice and convalescent humans yield a sars-cov- antibody cocktail. science isolation of human monoclonal antibodies from peripheral blood b cells developing therapeutic monoclonal antibodies at pandemic pace one-hit models for virus inactivation studies successful ebola treatments promise to tame outbreak evaluation of sars-cov- neutralizing antibodies using a cpe-based colorimetric live virus micro-neutralization assay in human serum samples cross-neutralization of sars-cov- by a human monoclonal sars-cov antibody isolation of potent sars-cov- neutralizing antibodies and protection from disease in a small animal model a human neutralizing antibody targets the receptor-binding site of sars-cov- the trinity of covid- : immunity, inflammation and intervention efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning structure, function, and antigenicity of the sars-cov- spike glycoprotein a human monoclonal antibody blocking sars-cov- infection structural and functional basis of sars-cov- entry by using human ace . cell expression cloning of antibodies from single human b cells cryo-em structure of the -ncov spike in the prefusion conformation single-cell rna-seq data analysis on the receptor ace expression reveals the potential risk of different human organs vulnerable to -ncov infection key: cord- - z yfj s authors: ho, mei-shang; chen, wei-ju; chen, hour-young; lin, szu-fong; wang, min-chin; di, jiali; lu, yen-ta; liu, ching-lung; chang, shan-chwen; chao, chung-liang; king, chwan-chuen; chiou, jeng-min; su, ih-jen; yang, jyh-yuan title: neutralizing antibody response and sars severity date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: z yfj s using the taiwan nationwide laboratory-confirmed severe acute respiratory syndrome (sars) database, we analyzed neutralizing antibody in relation to clinical outcomes. with a linear mixed model, neutralizing antibody titer was shown to peak between week and week after onset and to decline thereafter, with a half-life of . weeks. patients with a longer illness showed a lower neutralizing antibody response than patients with a shorter illness duration (p = . ). when early responders were compared with most patients, who seroconverted on and after week of illness, the small proportion ( . %) of early responders (antibody detectable within weeks) had a higher death rate ( . % vs. . %) (fisher exact test, p = . ), had a shorter survival time of < weeks (fisher exact test, p = . ), and were more likely to be > years of age (fisher exact test, p = . ). our findings have implications for understanding the pathogenesis of sars and for sars vaccine research and development. using the taiwan nationwide laboratory-confirmed severe acute respiratory syndrome (sars) database, we analyzed neutralizing antibody in relation to clinical outcomes. with a linear mixed model, neutralizing antibody titer was shown to peak between week and week after onset and to decline thereafter, with a half-life of . weeks. patients with a longer illness showed a lower neutralizing antibody response than patients with a shorter illness duration (p = . ). when early responders were compared with most patients, who seroconverted on and after week of illness, the small proportion ( . %) of early responders (antibody detectable within weeks) had a higher death rate ( . % vs. . %) (fisher exact test, p = . ), had a shorter survival time of < weeks (fisher exact test, p = . ), and were more likely to be > years of age (fisher exact test, p = . ). our findings have implications for understanding the pathogenesis of sars and for sars vaccine research and development. s evere acute respiratory syndrome (sars) is a newly emerged infectious disease. its etiologic agent is a novel coronavirus (sars-cov) ( , ) , which can readily infect a variety of wild and laboratory animals without causing apparent clinical symptoms ( , ) , making the existence of an animal reservoir possible. in humans, sars appears with a wide clinical spectrum, ranging from selflimited pneumonia to acute respiratory distress syndrome (ards) and death ( , ) . anecdotally, asymptomatic infection has also been reported ( ) . autopsies of sars patients have found the virus to be widespread throughout a variety of tissues and organs ( ) . during the acute phase, the virus is found in the excreta of infected persons ( , ) and is thought to be transmitted by direct contact, droplets, or contaminated environmental surfaces. infection can be prevented largely by good hand hygiene, although some healthcare settings and communities may be prone to the aerosolization of contaminated human excreta, and in these cases, precautionary measures should be instigated accordingly ( , ) . the chain of human transmission has been successfully interrupted by public health measures, but potential reintroduction of the virus from an unidentified natural reservoir remains a concern. a wealth of clinical and epidemiologic observations have emerged and contributed to the successful control of the sars epidemic (see peiris et al. [ ] for a review). however, information on immunity and pathogenesis is insufficient to provide a comprehensive basis for specific drug or vaccine design. nor have animal pathogenic models been established that adequately resemble the pathogenesis of sars in humans. without a good experimental model to study the biologic basis for human disease, the observational data collected from reported sars casepatients, along with the associated laboratory diagnostic tests, will continue to provide essential leads in controlling a possible reemergence of sars. to gain a better insight into the humoral responses in the context of epidemiologic and clinical settings, we analyzed the neutralizing antibody data, along with a variety of epidemiologic elements in the database. the epidemiologic database contains basic demographic information (age, sex, city/county of residence); symptoms at onset; date of onset of first symptoms; date of diagnosis; dates of hospitalization, discharge, or death; results of all epidemic investigations on contact tracing; travel history; and results of laboratory tests of reverse transcription-polymerase chain reaction (rt-pcr) on sars-cov and other pathogens in the differential diagnosis of atypical pneumonia. the analysis of epidemiologic data has been reported previously ( , ) . the detailed laboratory data taken from molecular and serologic tests of sars-cov infection were compiled in a separate file that could be linked to the epidemiologic data. the concordance and discordance between various serologic tests and molecular diagnostic methods of sars have also been reported previously ( ) . the serum neutralizing antibody was measured by microtiter assay and by enzyme-linked immunosorbent assay (elisa) (centers for disease control and prevention, atlanta, ga, usa) as described ( ) . hospitalization served the dual purposes of isolating patients and providing health care; therefore, criteria for discharging patients, i.e., being afebrile for days and clinical improvement, were stringently adhered to by the clinicians as a part of public health practice. since no antiviral drug was known to effectively shorten the clinical course of sars, the duration of illness, defined as the number of days between onset of fever and time of discharge from the hospital, can be assumed to reflect the clinical severity of sars manifested by the patient. to validate the consistency of the interhospital practices in patient care in relation to the severity of patients, we collected and analyzed anonymous and computerized clinical data, focusing on oxygen supplementation and respiratory therapy, on a sample of sars patients from hospitals that represented healthcare accreditation levels in taiwan: a major medical center (national taiwan university hospital), a regional teaching hospital (taipei mackay memorial hospital), and a district hospital (taipei hospital). regardless of hospital, duration of illness correlated highly with the supplementation of oxygen, which is a good surrogate for the level of pulmonary dysfunction (p for trend < . ) ( table ) . thus, in our analysis, duration of illness was used as a surrogate for clinical severity among the surviving sars patients, and death rate was also used as severity index. for the convenience of discussion, a duration of illness < weeks was considered mild, - weeks as intermediate, and > weeks as severe. a fatal case, regardless of the length of survival, was considered severe. our data corroborate the report that sars patients with a severe clinical course mainly had a slower and prolonged recovery ( ) . data were analyzed with sas software (version , sas institute inc, cary, nc, usa). differences in frequencies or proportions were tested using a χ test and by risk ratios. the continuous variables, i.e., age distribution or titers of neutralizing antibody, were compared by using the wilcoxon rank-sum nonparametric method. multivariate logistic regression ( ) was used to analyze factors that can affect seropositivity, including demographic information, source of infection, and duration of illness. to adjust the time effects and other covariates of interest, the relationship between antibody titer, based on logarithmic transformation of base (serum dilution) and other potential factors, i.e., age, sex, infection source, and duration of illness, was quantified by linear mixed models ( ) , which took into account the correlation between repeated measurements of each study participants. specimens from all patients with probable sars in taiwan were serologically tested for case confirmation, with a particular focus on the convalescent-phase serum specimens, as previously reported ( ) . positive neutralizing antibody results, which correlated well with those of elisa (table ) , were used as the standard assay for case confirmation. thus, of reported probable sars cases were confirmed. these included cases in patients whose diagnoses were based on serologic testing alone, whose results were positive by both tests, and patients whose diagnoses were based on the rt-pcr alone. of these diagnoses based on rt-pcr alone, had convalescent-phase serum specimens that tested negative, and did not have appropriate convalescent-phase serum specimens for antibody testing because of death, loss to follow-up, or inappropriate timing of serum collection (table ) . we examined whether the seronegative results of the patients were false-positive instances of virus detection by rt-pcr, most commonly caused by laboratory error or contamination. cross-contamination in the laboratory should occur without any correlation with the patients' demographic or clinical parameters. the seronegative rate ( . %, / ) was significantly higher in men than in women ( . %, / ) (mantel-haenszel test, p = . ), but the effect of age was not statistically significant (p = . in men, χ test) ( figure ). based on the transmission risk of known or unknown sources, patients whose sources could not be ascertained, i.e., had no apparent history of having contact with sars patients, were significantly more likely to be seronegative ( . %, / ) than those with known sources of infection ( . %, / ) (χ , p< - ) ( table ). patients with a shorter duration of illness were more likely to be seronegative; ( . %) of patients with a duration of illness < days, ( . %) of patients with illness durations of and days, ( . %) of patients for those with and days, and none of those who survived for > days (χ for trend = . , p = . ). a logistic regression model confirmed that patients with a known source of infection (odds ratio [or] = . , p< . ) and a longer duration of illness (or = . for each additional day of illness, p = . ) were more likely to possess a detectable level of neutralizing antibody than those with no discernible infection source and shorter duration of illness (table ). the total number of serum specimens collected from each patient ranged from to , including > convalescent-phase serum sample collected after week . of the seropositive sars patients, ( . %) of the patients were seropositive by week ; ( . %) of patients who were seropositive within the first weeks of illness and are called early responders hereafter. on rare occasions ( . %, / ), seroconversion occurred after week of symptoms onset. because of the differences in the distribution of age and sex among patient groups and the differences in the number and timing of specimens collected for antibody measurement, we used regression-based modeling approach to examine these factors simultaneously and tried to analyze the relationship between their potential interactions and antibody titers (table , figure a ). this model was based on the neutralizing antibody titer of the convalescentphase serum assays, representing patients who had had convalescent-phase serum sample collected between weeks to after onset of fever, patients who had convalescent-phase samples, and patients who had . the number of serum specimens collected ranged from to per week from week of illness through week . the model suggested that neutralizing antibody rose and diminished during the follow-up period between weeks and after onset of illness (p = . for linear term and p = . for quadratic term); the estimated half life was ≈ . weeks. patients with a more protracted clinical course tended to have a higher antibody titer than patients with a shorter clinical course (p = . ). antibody in patients with more severe clinical courses tended to decay at a faster rate than in patients with shorter clinical course (the interaction between duration of illness and time of serum collection, p = . ). this pattern of decay followed a half-life of ≈ . weeks after reaching the peak, which occurred between weeks and after infection ( figure b ). the time that the blood was collected for each patient was examined, and an equally dispersed pattern of blood collection was found in all clinical groups (figure ). of the patients who had a second convalescentphase specimen collected after weeks, ( . %) of showed a -fold ( dilution) drop by week postinfection. three patients had a negative seroconversion during the same period. conversely, a measurable neutralizing antibody persisted in ( . %) of patients who had been followed up between weeks and . to better examine the dynamics of the antibody profile, cross-sectional views of figure a were extracted ( figure b) . the model suggested that antibody response was higher and occurred earlier in patients with a more severe clinical course than in those with a shorter clinical course. in the model, patients with a more severe clinical course had earlier and higher antibody responses; we then examined the death rate of the early responders (table ). these early responders had a significantly higher mortality rate ( . % vs. . %) than others who did not undergo seroconversion until week of illness or later (p = . , χ test). these early responders also tended to die early during the acute phase: of died during the first weeks of illness, and the other died on days and of illness, respectively (fisher exact test, p = . ). of the patients who died and who seroconverted after the second week of symptom onset, only one died during the first weeks of illness (wilcoxon rank sum, p = . ). among the early seroresponders, the antibody titer of those who died (n = ) (median titer = ) was not significantly higher than that of those who survived (median titer = ) (wilcoxon rank sum, p = . ). however, the early seroresponders were significantly older (mean age . years) than the case-patients who seroconverted after week of illness (mean age . years) (wilcoxon rank sum, p = . ); i.e., older patients were more likely to be early responders, % of patients > years of age versus . % of patients < years (fisher exact test, p = . ). neutralizing antibody plays an integral role in immunoprotection from viral diseases, and serologic tests are important to their diagnosis. this report relates sars neutralizing antibody profiles to clinical outcomes. the lack of a readily available, well-characterized diagnostic assay that could be used as a standard and the additional lack of a well-established typical clinical description that encompasses all clinical syndromes are intrinsic difficulties of working with a new infectious disease. therefore, analysis of the interrelation between clinical, epidemiologic, and laboratory data might provide further insight into these elements. results of our analysis, although they passed a certain level of statistical scrutiny, should be interpreted with caution. the seronegative sars patients whose diagnoses were based on positive rt-pcr results of nasopharyngeal swab specimens warrant further discussion concerning whether they were indeed sars patients or were merely misdiagnosed by the false-positive rt-pcr of sars-cov. the false-positive rt-pcr is most commonly due to cross-contamination, which pertains to the nature and quality of a laboratory procedure and should be independent of patient's profile. however, we found that seronegative patients were more likely to have a short duration of illness and no clear source of infection. lack of specificity of the test is another reason for having a false-positive rt-pcr, but none of the commercial tests we used have been reported to have nonspecific cross-reactivity with other known pathogens. furthermore, after may , sars diagnosis required positive results of > specimens collected at different time or from different sites, or tested by if only specimen was available. therefore, a specimen that yields false-positive results with different test methods is deemed unlikely. alternatively, these patients were indeed sars patients, but the negative neutralizing antibody reading was due to patent's low antibody level in combination with the low sensitivity of the antibody test. the sensitivity of our neutralizing assay is comparable to that of elisa ( ), but the possibility that our assay had a low sensitivity remains because the neutralizing antibody test is based on the reading of a complete inhibition of cytopathic effect. thus the absolute titer is expected to be lower than the results, based on reading of % inhibition. the neutralizing antibody of sars patients has been reported in only other study, in which a pseudovirus containing the s protein of sars-cov was used; antibody titers were found to be low ( ) . a low antibody response may be associated with a primary infection of sars-cov, as seen with primary infection of respiratory syncytial virus (rsv) ( ) , and infection through the respiratory tract was shown to stimulate a less vigorous immune response than infection by an invasive intravenous rejection of rsv ( ) . furthermore, a robust humoral immune response requires antigen in sufficient doses through a proper route; this fact has been demonstrated in vaccine studies, including research on several live vaccines ( − ) . the lack of detectable antibody among patients without history of contact with a known sars a b patient might be associated with a low inoculum of the virus because of incidental exposures, in contrast to patients who acquired sars in hospitals under circumstances assumed to have a high virus density. when systematically screened during the sars outbreak, some healthcare workers and public health personnel who had a history of direct contact with patients were shown to harbor nasopharyngeal sars-cov. subsequently, however, they did not show seroconversion (y.-t. lu et al., unpub. data), which raises the possibility of asymptomatic mucosal epithelial colonization by sars-cov. our seronegative patients with mild symptoms might fit into the spectrum between the seronegative asymptomatic colonizers and the severe sars patients with high neutralizing antibody response. all considerations appear to favor the possibility that seronegative patients indeed had acquired sars. since the natural reservoir of sars-cov has not been clearly identified, and reintroduction of sars-cov to humans is possible, the short duration of having detectable antibody should be considered when a vaccine against sars-cov is developed. the clinical course of sars patients with severe infection is described as follows: pulmonary functions worsen during week of illness ( , − ) , while the virus load in the airway decreases ( ) , and patients with mild disease would begin to stabilize clinically. those in whom ards later develops usually show pulmonary decompensation during week . severity was intensified by a slower and prolonged recovery with complications of pulmonary fibrosis occurring in week in some patients ( ) . results of a high-resolution computed tomographic scan in followup of sars patients corroborates this observation by showing a high correlation between bilateral fibrotic lung changes and clinical severity ( ) . findings of these studies, in conjunction with clinical study on cytokines during the acute phase ( − ) , suggest that activation of th cell-mediated immunity and a hyperinnate inflammatory response, rather than direct damages from uncontrolled virus growth, are responsible for the pathogenic process in severe infection ( ) . in previous vaccine studies, immunization conditions that could induce a stronger activation of th response would concurrently result in a higher antibody response ( , ) . thus, the high antibody response and a strong cell-mediated th response may reasonably be understood as concurrent events, and the latter may be causally related to a severe clinical course of sars. neutralizing antibody is unlikely to be causally related to the pathogenesis of sars because treating sars patients with convalescent-phase serum collected from patients who had recovered from sars showed no adverse effect and probably had beneficial effects ( ) . thus, for all the reasons stated above, our finding that high neutralizing antibody correlating with clinical severity should not be interpreted to mean that neutralizing antibody is harmful. having a detectable neutralizing antibody during the first weeks of illness, in our analysis, coincides with a high and an early sars mortality rate. the basis for early antibody response is not apparent, but possibility is the priming effect of a previous non-sars-cov infection. indeed, antibody against sars-cov has been shown to cross-react with human coronavirus e ( ) . the finding that early responders are older than other sars patients is figure . scatterplot of antibody titers of the seropositive study participants (titers of the same participant measured at different times are connected); superimposed is the fitted mean curve (in red) of log (antibody titer) between weeks and postinfection based on the linear mixed model by severity (duration of illness) and sex at the median age of years. each dot represents > titer; no distinction is made between single values and those with > value. in agreement with the priming effect since cumulative infection rate increases with increasing age. the priming effect of a previous viral infection can induce cross-reactive but nonneutralizing antibody, as well as neutralizing antibody to sars. furthermore, the nonneutralizing antibodies are known to facilitate viral infection, termed antibodydependent enhancement (ade), which is the pathogenic basis of feline infectious peritonitis virus (fipv, a type ii coronavirus), dengue hemorrhagic fever, and other viruses ( ) ( ) ( ) ) . in the case of fipv, ade can occur even with neutralizing antibody ( ) . however, this type of ade resulted directly from neutralizing antibody is unlikely to occur with sars-cov infection because a number of sars patients have been treated with convalescent-phase serum of sars patients and show no adverse effect ( ) . the hypothesis that the early responders may have experienced a priming effect could be verified by demonstrating that a significantly higher proportion of early responders than other sars patients possess antibody against non-sars coronavirus during the acute phase. early death occurring within the first weeks of illness is also associated with high nasopharyngeal virus load among a subset of sars patients with information on nasopharyngeal virus load (j.-y. yang et al., unpub. data). unfortunately, the number of early responders for whom information on virus load was available was too few to yield a meaningful statistical analysis on whether high virus load is correlated with an early humoral response. while antibody induced by a variety of sars-cov antigen preparations protects against sars-cov infection in mice and ferrets ( , ) , these animals do not develop clinical symptoms resembling that of sars-cov infection in humans and thus are not models of pathogenesis. since ade can occur through a number of mechanisms and is not completely understood, 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prophylaxis for sars coronavirus infection in ferrets we are grateful to all public health nurses who helped collect epidemiologic data; the staff in the division of surveillance in the taiwan-cdc, who compiled the database, tom ksiazek for the generous supply of elisa reagents, and eli chang and kathleen ahrens for assistance in preparing this article. this study was supported by an academia sinica institutional grant.dr ho is a physician epidemiologist at the institute of biomedical sciences, academia sinica, taipei, taiwan. she also serves as an advisor to taiwan-cdc. her research activities focus on various aspects of host-virus interaction, including genetic susceptibility to severe viral infection. key: cord- -o eelqcw authors: stadlbauer, daniel; baine, ian; amanat, fatima; jiang, kaijun; lally, kimberly; krammer, florian; jhang, jeffrey s.; arinsburg, suzanne a. title: anti‐sars‐cov‐ spike antibodies are stable in convalescent plasma when stored at ° celsius for at least weeks date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: o eelqcw nan as nations around the world continue to address the public health crisis of coronavirus disease (covid ), the global medical and scientific communities continue to search for and develop therapeutic strategies for patients. to date, remdesivir presents one such option and can shorten the duration of hospitalization in patients with severe disease . convalescent plasma (cp) has presented another option, with its use and safety supported by numerous case series and retrospective studies [ ] [ ] [ ] . while its degree of effectiveness has been variable, there is general agreement that it acts as a supportive adjunctive therapy in patients with moderate to severe disease , . current cp protocols such as the mayo clinic early access program (eap) specify that once thawed, cp may be stored for up to days at ˚ celsius. presumably, this is intended to mimic current guidelines which apply to thawed plasma (tpl), which specify a similar shelf-life. however, the rationale for this guideline stems from the fact that under normal circumstances, donor plasma is transfused or exchanged therapeutically for the purposes of correcting or preventing a coagulation factor deficiency, to replace deficient factors necessary for hemostatic balance such as adamts , or to restore complement-regulatory factors in the setting of atypical hemolytic uremic syndrome. for these purposes, tpl is largely considered equivalent to fresh frozen plasma, with the exception of decreases in factors v and viii activity levels, and several other components , . however, the therapeutic goal of cp transfusion is to deliver passive immunity to the patient via antibodies. studies demonstrating antibody stability under refrigerated conditions have largely focused on peripheral blood samples stored for several days , . however, studies describing the stability of antibodies in refrigerated donor plasma over longer periods are lacking. here, we demonstrate the long-term stability of anti-sars-cov- spike antibodies in donor cp samples collected at a local blood donor center for transfusion. after thawing of fifteen cp units, segments were sampled and anti-spike antibody titers were determined via enzyme-linked immunosorbent assays (elisas) . segments from five non-cp units were sampled as a negative control. all samples were then stored at ˚c, and plasma endpoint titers were reevaluated at , and days. we detected no reduction in antibody titers for any of the samples ( table ). the anti-spike elisa assay demonstrates a between-run %cv ranging from . % at an antibody concentration of . mcg/ml, to % at a concentration of . mcg/ml. therefore, the perceived decrease in titer for most samples between days and , and the perceived increase between days and reflects the serial dilution cutoff values of the elisa assay itself and run-to-run variation, and are not reflective of a true change. this is supported by the results obtained when the area under the curve (auc) values for raw absorbance data are calculated, and show no significant variation over time ( figure accepted article ). while our study does not address the neutralization capacity of these antibodies, previous studies demonstrate significant correlation between spike antibody titers and neutralization capacity of plasma and serum samples . we do note that our findings are limited by several factors. first, while limited case series have identified that cp may be effective in reducing morbidity and overall mortality, its efficacy has not been formally evaluated in large randomized clinical trials. similarly, while there is correlation between spike antibody titers and neutralization capacity, as well as historical basis for the antibodies themselves as this would need to be formally determined by a randomized trial comparing cp to standard frozen plasma. there may also be a slight increase in risk for septic reactions, though we believe this risk remains low, given the estimated incidence of septic reactions from red cell units of in , - , , a product which in some cases is also stored for up to days at ˚c. accepted article this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. accepted article remdesivir in adults with severe covid- : a randomised, doubleblind, placebo-controlled, multicentre trial treatment of covid- patients with convalescent plasma early safety indicators of covid- convalescent plasma in , patients convalescent plasma treatment of severe covid- : a matched control study evaluation and comparison of coagulation factor activity in fresh-frozen plasma and -hour plasma at thaw and after hours of to degrees c storage comparison and stability of adamts activity in therapeutic plasma products long-term stability of trastuzumab in plasma and whole blood samples stored under different conditions one-month stability study of a biosimilar of infliximab (remsima((r))) after dilution and storage at degrees c and degrees c a serological assay to detect sars-cov- seroconversion in humans covid- and its implications for thrombosis and anticoagulation bacterial contamination of blood components: risks, strategies, and regulation: joint ash and aabb educational session in transfusion medicine new york, ny *these authors contributed equally to this work key: cord- - hqhty o authors: yu, meng; stevens, vicky; berry, jody d.; crameri, gary; mceachern, jennifer; tu, changchun; shi, zhengli; liang, guodong; weingartl, hana; cardosa, jane; eaton, bryan t.; wang, lin-fa title: determination and application of immunodominant regions of sars coronavirus spike and nucleocapsid proteins recognized by sera from different animal species date: - - journal: journal of immunological methods doi: . /j.jim. . . sha: doc_id: cord_uid: hqhty o abstract knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. although there have been many reports of such work done for sars–cov, these were mainly focused on the immune responses of humans and mice. in this study, we aim to search for and compare immunodominant regions of the spike (s) and nucleocapsid (n) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. twelve overlapping recombinant protein fragments were produced in escherichia coli, six each for the s and n proteins, which covered the entire coding region of the two proteins. using a membrane-strip based western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. the s fragment (aa – ) and the n fragment (aa – ) were the most immunodominant among the six s and n fragments, respectively. antibodies raised against the s fragment were able to block the binding of a panel of s-specific monoclonal antibodies (mab) to sars–cov in elisa, further demonstrating the immunodominance of this region. based on these findings, one-step competition elisas were established which were able to detect sars–cov antibodies from human and at least seven different animal species. considering that a large number of animal species are known to be susceptible to sars–cov, these assays will be a useful tool to trace the origin and transmission of sars–cov and to minimise the risk of animal-to-human transmission. in late to middle , outbreaks of severe acute respiratory syndrome (sars), also known as atypical pneumonia, spread through more than countries and caused more than cases with close to human deaths . the aetiological agent was a previously unknown coronavirus, now named sars associated coronavirus or sars-cov, which seems to be evolutionally distant to any of the existing coronaviruses known at the time (rota et al., ; marra et al., ) . initial epidemiology studies indicated an animal origin , which was further confirmed by the detection and isolation of almost identical viruses from wild life animals in live animal markets (guan et al., ) . more recently, sars-like-covs were identified among different species of horseshoe bats lau et al., ) , implicating bats as the potential reservoir of these novel coronaviruses . coronaviruses are enveloped positive-stranded rna viruses containing several structural proteins including the spike glycoprotein (s), nucleocapsid protein (n), membrane protein (m), and envelope protein (e). receptor-binding and membrane fusion between the virus and host cell is mediated by the s protein, which is also the main target for neutralizing antibodies (lai et al., ) . the s protein is a large class i transmembrane glycoprotein consisting of two domains: the s domain at the n terminus responsible for receptorbinding and the c-terminal s domain functioning in fusion. for sars-cov, the minimal receptor-binding domain has been defined as a -aa fragment (aa - ) which binds to the receptor molecule, ace , in vitro wong et al., ) . the n protein is thought to participate in the replication and transcription of viral rna and interfere with cell cycle processes of host cells. in addition, the n protein of many coronaviruses is highly immunogenic and abundantly expressed during infection, making it an attractive target for detection of virus-specific antibodies (lai et al., ) . there are several diagnostic approaches developed for the detection of sars-cov infection, including pcr-based assays, antigen detection and antibody detection (shi et al., ; he et al., ; houng et al., ; manopo et al., ; wang et al., b; chou et al., ) . most, if not all, of these detection methods were developed for human applications. there is a lack of attention to the study of immune responses in different animal species and a lack of proper diagnostic tools for wide range surveillance of animal populations. yet, it is well documented that sars-covs and sarslike-covs can infect a wide range of animal species . in most cases, there is no obvious clinical sign associated with the infection, making early detection and diagnosis even more difficult. knowing that sars-cov originated from an animal reservoir and animals played a key role in the adaptation and transmission to the human population (holmes ; li et al., ) , accurate diagnosis of sars-cov infection among different animals species should be an important and integral part of an effective prevention strategy for reducing animal-to-human transmission. in this study we identified and characterized the major immunodominant domains of the sars-cov n and s proteins recognized by different animal species, and then developed competition elisas based on these findings. by conjugating the detection antibodies with horseradish peroxidase, we developed one-step-competition elisas (or oscelisa) which are simple to use and can detect sars-specific antibodies from different species. with further improvement and validation, we are hopeful that this will become a powerful tool for early detection of sars-cov infections in animals, which will in turn help to reduce the risk of future sars outbreaks in humans. the hku- strain of sars-cov was used in this study and all live virus work was carried out in the biosafety level (bsl- ) laboratory under protocol approved by the institutional biosafety committee (ibc). culturing of virus in vero e cells was conducted as previously reported (tu et al., ) . for use outside the bsl- laboratory, virus preparation was inactivated by gamma-irradiation at a dose of kgy. anti-sars-cov sera from rabbits, rats and mice were generated in the bsl- animal facility at the australian animal health laboratory (aahl). to test the susceptibility and to generate antibodies from live virus, , tcid sars-cov was administered oronasally to rabbits (new zealand white, n = ), mice (balb/c, n = ) and rats (sprague-dawley, n = ). daily monitoring for clinical signs did not detect disease and after three weeks the animals were euthanized and blood samples were taken for serology. anti-sars-cov antibody in serum was detected using immunofluorescence antibody test (ifat) and western blot assays, and confirmed by virus neutralization test (vnt). in addition, a collection of human and animal antisera was also used in this study. they included human sera from sars patients , hyperimmune horse, pig and rabbit sera raised against formalin inactivated sars-cov (c. tu, unpublished results) , pig sera from experimental infection studies , sera from naturally infected civets (tu et al., ) and bats , and a panel of mouse monoclonal antibodies (berry et al., ) . for cdna preparation, sars-cov rna was extracted from the inactivated virus pellet using the qiaamp viral rna mini kit (qiagen), followed by cdna synthesis using the omniscript rt kit (qiagen) and random hexamer primers. pcr primers were designed using published sequence to amplify fragments coding overlapping regions of the s and n proteins, respectively. all forward primers contain the mlui site and the reverse primers contain the noti site (primer sequences will be provided on request) to facilitate cloning into the modified expression vectors phan and pban, which were modified, respectively, from the prset vector (invitrogen) for expression of his -tagged fusion proteins and the pdw vector (tsao et al., ) for expression of fusion proteins with a biotin tag at the n terminus. both vectors have inframe asci and noti sites in their multiple cloning sites. e. coli bl (de ) (novagen) harbouring appropriate recombinant plasmid was grown in ml of lb broth containing ampicillin ( μg/ml) at °c with shaking overnight. the culture was diluted : and incubated at °c with shaking until od reached . . protein expression was induced by addition of isopropyl-beta-dthiogalactoside (iptg) at a final concentration of mm. after h, bacterial cells were harvested by centrifugation. the cell pellet was resuspended in lysis buffer ( mm nacl, mm phosphate buffer, ph . ) and lysed by sonication. the insoluble protein fraction was collected by centrifugation and solubilised in m urea (in the same lysis buffer). the recombinant proteins were then purified by preparative sodium dodecyl sulphatepolyacrylamide gel electrophoresis (sds-page) as previously described (tachedjian et al., ) . four -week-old chickens were immunized with recombinant proteins for antibody production, two with his -s and two with biotin-nc (see fig. ). for primary immunization, μg of recombinant protein was mixed with the csiro triple adjuvant (tachedjian et al., ) before injection. three weeks later, the same injection was repeated for boost immunization. the final boost was carried out by injection of μg protein without adjuvant at three weeks after the second injection. chickens were bled before each injection and sars-cov reactive antibodies were determined by elisa using inactivated sars-cov antigen (see below). eggs were collected for a period of five months and egg yolk was pooled for purification of igy antibodies. the igy extraction and purification kit (pierce) was used for purification as follows: one part of egg yolk (approximately ml) was mixed with parts of phosphate buffered saline (pbs), ph . , followed by the addition of peg to a final concentration of . % (w/v). the mixture was stirred for min at room temperature and centrifuged for min at ×g. the supernatant was then filtered through cotton wool in a funnel, and peg was added to the clear supernatant to a final concentration of % (w/v). the mixture was stirred for min at room temperature and centrifuged as above. after dissolving the precipitate in ml of pbs, the same procedure was repeated once by addition of peg to %, mixing and centrifugation. the final precipitate was slowly dissolved in . ml of pbs and dialysed against pbs overnight at °c. aliquots of igy antibodies were stored at − °c for future use. conjugation of igy antibodies to horseradish peroxidase (hrp) was carried out following the protocol provided by the ez-link plus activated peroxidase kit (pierce). briefly, ml of igy antibodies at approximately mg/ml in conjugation buffer was added to the lyophilized ez-link plus activated peroxidase. the mixture was incubated for h at room temperature, followed by the addition of μl reductant solution and a further incubation of min. the reaction was stopped by adding μl of quench buffer, followed by incubation at room temperature for min. the conjugate was dialysed against pbs buffer overnight at °c and aliquots were stored at − °c in the presence of % glycerol. all immunofluorescent antibody tests were carried out following the standard protocols previously described by our group (tu et al., ; zhang et al., ) . western blot analysis was carried out as described (wang et al., ) . to produce membrane strips for testing reactivity of the same antigen with a panel of different sera, μg of purified protein was loaded into a wide comb, separated by sds-page and transferred to nitrocellulose membrane. the membrane was then sliced into mm-wide strips and each strip tested with a different serum sample. alkaline phosphatase (ap)-conjugated secondary antibodies and apstreptavidin were obtained from chemicon australia. for virus neutralization, serial two-fold dilutions of test sera and control sera were prepared in duplicate in a well tissue culture plate in μl cell media (minimal essential medium containing earle's salts and supplemented with mm glutamine, µg/ml fungizone, units/ml penicillin, µg/ml streptomycin and % fetal calf serum). under bsl conditions, an equal volume of sars-cov working stock (hku- ) containing tcid was added and the virus-sera mix incubated for min at °c in a humidified % co incubator. μl of vero cell suspension containing × cells/ml was added and the plate incubated at °c in a humidified % co incubator. after three days, the plate was observed for cpe. all elisa assays were carried out using standard procedures established in our group zhang et al., ) . all incubations were conducted at °c for h with gentle shaking, and all washes (five times between each step) were carried out at room temperature using pbs containing . % tween (pbst). antigens were coated in . m carbonate buffer (ph . ) and antibody dilutions were done in blocking solution ( % skim milk in pbst). pvc -well plate (dynatech) was used for all elisa analysis. for indirect elisas, viral or recombinant antigens were coated at a concentration of - ng/well, and bound antibodies were detected using hrp-conjugated species-specific secondary antibodies (chemicon) or hrpconjugated protein a/g (zymed). color development was conducted using the substrate , , , , tetramethylbenzidine (tmb) and the absorbance at nm was determined. for competition elisa, detecting antibody (mab or chicken mono-specific antibody) was added directly after the antigen-coated plate was pre-incubated with test sera without wash steps. the percentage inhibition of detecting antibody binding was calculated using the following formula: ¼ od obtained with buffer only: to test for susceptibility and to generate antisera in experimental small animals, rabbits, rats and mice were inoculated with , tcid sars-covoronasally and monitored daily for clinical signs. none of the animals showed any signs of disease. three weeks postinfection, all animals were euthanized and serum samples were taken for analysis using three different assays, i.e., immunofluorescent antibody test (ifat), western blot and virus neutralization test (vnt). the results are shown in table . seroconversion was observed in all three species although not in all animals. from the limited number of animals used in this study, it appeared that rats were the most susceptible and displayed the highest vnt titres in their sera varying from : (n = ) to : (n = ). four of the six mice produced antibodies detected by ifat and western blot, but only two of them were positive in vnt. one of the two rabbits also seroconverted, with a low vnt titre of : . recombinant proteins, full-length or serially overlapping fragments, were produced in e. coli for determination of immunodominant regions in the s and n proteins. pcr fragments were cloned into two expression systems, the prset-derived phan vector for production of his -tagged proteins and the pdw -derived pban vector for biotin-tagged proteins. all of the recombinant proteins generated in this study are summarised in the diagram in fig. with their aa positions indicated. for each construct, two positive clones were confirmed by dna sequencing for correct insert sequence and in-frame fusion with the vectorencoded tagging sequences. as expected, the level of expression varied with the different constructs, and some expressed better in the his -tagging system and other vice versa (data not shown). overall, the expression level varied from . - mg/l to approximately - mg/l. most of the expressed proteins were present largely in the insoluble fractions when analysed by western blot using anti-his antibody (for his tagged proteins) or ap-conjugated streptavidin (for biotin-tagged proteins). this observation prompted us to purify the recombinant proteins using preparative sds-page under denaturing conditions. a purity of % or greater was achieved for all recombinant proteins when analysed by direct staining with commassie blue. for mapping of immunodominant regions of the s and n proteins, western blot using membrane strips was conducted. each of the recombinant protein fragments, his -tagged s to s and biotin-tagged n to n , was tested against a panel of animal sera from six different animal species. the results are compiled in table together with the reactivity of each serum sample in vnt. the hyperimmune horse serum was known to contain a very high titre of antibodies and was b immunofluorescent antibody test was done using a serum dilution of : and the intensity of staining in sars-cov-infected cells, but not in mock infected cells, was indicated by the sign + with ++++ representing the strongest signal observed. c western blot was carried out using a serum dilution of : and the intensity of the reactivity against a recombinant full-length n protein was indicated by the sign + with ++++ representing the strongest reactivity. used as a positive control in this study. as shown in table , the horse serum reacted with all of the s and n protein fragments tested. overall, the hyperimmune sera (especially those from horse and rabbit) displayed more reactivity towards different fragments than sera obtained from experimental or natural infection (civet, rabbit, mouse and rat). however, the results in table clearly indicate that s and n represented the most crossreactive regions of the s and n proteins, respectively. in addition, the data also reveal that the c-terminal half of the n protein is more immunogenic than the n-terminal half, at least for the epitopes which were recognizable under denaturing conditions, i.e., in western blot. based on the results obtained above, mono-specific antibodies were raised in chickens against the s and nc fragments. two chickens each were used for production of anti-s and anti-nc antibodies. after three immunizations, the peak antibody titres were determined by elisa using whole virus antigen. for both s and nc, a final titre of : , to , was obtained (data not shown). the specificity of the antibody was further confirmed using western blot against viral antigen (data not shown) and ifat using sars-cov infected vero cells. as shown in fig. , both antibodies reacted with sars-cov infected vero cells whereas the pre-bleed chicken sera did not. when tested in vnt, neither of the anti-s or anti-nc antibodies showed any evidence of neutralization activity (data not shown). for conjugation of hrp to the chicken antibodies, crude igy antibodies were purified from egg yolks using the peg precipitation method and protein concentration and purity were determined by sds-page in comparison with serially diluted bsa standards run on the same gel. the protein concentration of anti-s and anti-nc igy was at mg/ml and mg/ml, respectively. the purity for both antibodies was approximately - %. the antibody titres of the partially purified anti-s and anti-nc igy were : and : , respectively, tested using the same elisa as for the serum antibodies above. an aliquot (approximately mg) of each igy antibody was then conjugated to hrp using the pierce ez-link plus activated peroxidase kit. the final titres of the hrp-anti-s and hrp-anti-nc antibodies were very similar, : and : , respectively. among a panel of mabs generated using inactivated sars-cov, five were directly shown to be s-specific by western blot analysis (berry et al., ) . in addition, four other mabs were shown to neutralize sars-cov although failed to react with the s protein in western blot. in this study, competition elisa (celisa) was carried out between chicken anti-s antibodies and these mabs, using chicken anti-nc as a control. the data shown in fig. a indicated that the binding of three known sspecific mabs and four neutralizing mabs to sars-cov antigen was blocked by the anti-s antibodies, but not by the anti-nc antibodies. among these mabs, f g displayed the most efficient blocking by the chicken anti-s antibodies. western blot analysis of the six s-protein fragments indicated that f g was able to bind both the s and s fragments (fig. b) , suggesting that its epitope is located in the overlapping region (aa - ) of the two protein fragments. one of the main aims of this study was to assess the feasibility of developing a competition elisa for detection of sars-cov antibodies from different animal species. based on the results above, it was evident to us that a celisa based on either mab f g or the chicken mono-specific antibodies might be ideal for such an application. to confirm this, three separate celisas were developed and tested against a panel of negative and positive sars-cov sera from different species. firstly, a celisa based on mab f g was tested against a panel of known positive and negative sera from different species, and the results in fig. fig. . mapping of mab binding regions by celisa and western blot. a. competition elisa using whole virus as antigen. the igy antibodies were used at : for both nc and s . the percentage inhibition was calculated using the formula given in materials and methods. b. western blot analysis of all six s fragments using mabs rgs-his and f g . suggested that each serum was able to inhibit f g binding, with the civet sera displaying the strongest inhibition. we then tested the feasibility of using the hrpconjugated chicken mono-specific antibodies as detecting antibody in a celisa. since the elisa only requires one-step incubation with both detection and testing antibodies, we termed the assay one-stepcompetition elisa or oscelisa. as shown in fig. , although different sera showed slightly different inhibition patterns against the hrp-igy/s or hrp-igy/nc antibodies, both assays, especially the oscelisa using hrp-igy/s antibody, were able to detect positive sera from a wide range of animal sera. it is worth noting that the sera from bats infected with sars-like-covs inhibited hrp-igy/nc antibodies more effectively than hrp-igy/s antibodies (see more in discussion). compared with the great success in rapid identification of the sars causative agent involved in and the control of the sars outbreaks, our understanding of the origin and mechanism of animal-to-animal and animal-to-human transmission of sars-cov is less satisfactory. with recent identification of sars-like-covs in bats lau et al., ) and the key role played by civets in direct transmission of sars-cov to humans wang et al., a wang et al., , , it is evident that further research is required before we are able to get a fig. . inhibition of f g binding to sars-cov by sera from different species. all animal sera were used at a dilution of : . the percentage inhibition was calculated using the formula given in materials and methods. abbreviations: if, infected sera; im, immunized sera; neg, negative control sera. fig. . inhibition of binding of mono-specific chicken antibodies to sars-cov by sera from different species. all animal sera were used at : for this study. since animal sera of different species gave rise to different level of background inhibition, the percentage inhibition was calculated using the following formula:% inhibition = − (od n − od t / od b ) × , in which od n = od obtained in the presence of known negative sera from the same species; od t = od obtained in the presence of test sera; and od b = od obtained with buffer only. abbreviations: if, infected sera; im, immunized sera; neg, negative control sera. better understanding of what was the true origin of the sars-covs responsible for the - outbreaks and whether such events can happen again in the future. there are two major difficulties associated with the study of roles played by animals leading to potential sars outbreaks. first, there are more than a dozen animal species known to be susceptible to sars-cov by either natural or experimental infection , and it is likely that more animal species could be susceptible too. it is important to differentiate between those which play a key role in the origin and transmission of sars-cov and those that are infected, but play no significant role in disease spread and spill over to the human population. secondly, there is a lack of an ideal detection approach to monitor infection status of different animal species. the most commonly used tool is direct pcr analysis for the presence of viral material in animal specimens (guan et al., ; poon et al., ; kan et al., ; woo et al., ; tang et al., ) . while this is effective to detect active infection, it is not very sensitive to monitor prior exposure in a given animal population. we believe that a serological test which detects sars-cov antibodies will be a more powerful tool for investigation of sars-cov prevalence among different animal populations. to develop an antibody test which can be applied to different animal species, it is essential to first have a panel of different animal sera which are known to be positive to sars-cov. this was achieved by a broad international collaboration among groups who have access to such sera from naturally infected wild life animals, experimentally infected animals or animals immunized with inactivated virus. for production of sars-cov antisera in small animals at aahl, we chose to use live virus for two reasons. firstly, this allowed us to test the susceptibility of these animals to sars-cov (the work began in , before any experimental animal infection studies were published). secondly, if the animals were susceptible, this would provide us better quality reagent than that obtained from immunization with inactivated virus and adjuvant. the seroconversion data in this study indicate that rats, mice and rabbits are susceptible to infection of sars-cov by the oronasal route. when the same amount of virus was administered subcutaneously and intra-peritoneally, none of the animals seroconverted (data not shown), suggesting that the seroconversion is due to the replication of sars-cov rather than immunization and that the route of administration is important for the infection to occur. to determine the immunodominant regions of the n and s proteins recognized by different animal species, a series of overlapping truncated recombinant fragments were produced in e. coli. reactivity of different animal sera against each of the protein fragments was conducted by western blot. while it is known that western blot is not ideal for detecting conformational epitopes, it is the most feasible and effective way to unequivocally locate at least the linear or continuous immunodominant epitopes. the two most immunodominant regions, s and n , were identified for the s and n proteins, respectively. for the n protein, it seems that the c-terminal region is more immunogenic than the nterminal region. for the s protein, the location of s (aa - ) is consistent with previous studies conducted with sars patient sera, which revealed that the immunodominant regions are located in the following regions of the s protein, aa - , - and - . combined with our data, these results suggest that the common immunodominant regions are shared among humans and animals. based on the results above, mono-specific antibodies targeting the s and nc regions were produced in chickens. the chicken was chosen for several reasons. it was known that avian species were not susceptible to sars-cov swayne et al., ) , and antibodies produced in chicken can be used as detecting antibodies in a competition elisa for sars-cov sera from any mammalian species without the concern of potential cross reactivity of anti-species secondary antibodies. collection and purification of igy antibodies from egg yolk have proven to be a very effective and economical way of producing large quantities of antibodies (tachedjian et al., ) . by collecting the lymphocytes (bursa or spleen) of antigensensitized chickens, the immune gene repertoire can be stored for later production of recombinant chicken single chain antibodies, which is technically less challenging than the production of rabbit or mouse single chain antibodies (foord et al., ) . the successful production of s -and nc-specific antibodies was confirmed by several assays, including ifat, elisa and western blot. the results presented in fig. demonstrated not only the specific staining of viral antigens in sars-cov infected cells, but also the different staining patterns for the two antibodies. this was expected considering that s antibodies should react to the extracellular domain of the s proteins whereas the n proteins are known to be located intracellularly (lai et al., ) . in addition to the main application of these antibodies in the development of celisa, these mono-specific antibodies also proved useful in determining the specificity of several mabs produced in a previous study (berry et al., ) . the data presented in fig. indicated that all four conformational neutralizing mabs (f g , f g , f g and f g ) are s-specific and bind to a domain overlapping with the s region. in comparison with an indirect elisa, the celisa has the advantage in that it can be used for detection of antibodies from different species without having to use species-specific secondary antibodies. most celisas employ mabs as the detecting antibodies to achieve maximal specificity. however, for viruses like sars-cov, which are known to have many variants and are rapidly mutating, a mab-based celisa may potentially give false negative diagnosis due to subtle changes of protein sequence close to or in the epitope region. for this reason, we have developed in parallel celisas using mono-specific antibodies as detecting antibodies. also, by directly conjugating the chicken antibodies with hrp, we were able to develop oscelisas for detection of sars-cov antibodies from any species, from human, civets, to bats. it is important to point out that due to the limited availability of positive sera from different animal species, the celisas developed in this study need more optimization and validation before they can be used as robust diagnostic assays. it is also important to note that depending on the purpose of application, one may wish to use different assays under different circumstances. although sequence analysis would suggest low probability of interference by cross-reactive antibodies to other animal coronaviruses, this is yet to be confirmed experimentally for the hrp-igy/nc oscelisa. however, we are certain that this will not be an issue in the sprotein-based celisas. this was deduced from the preliminary data obtained with bat sl-cov antisera in this study. it was clear that not all bat sl-cov sera were detectable in the hrp-igy/s oscelisa. the failure to detect some positive bat sera using the hrp-igy/s oscelisa can be explained by the significant sequence difference present in the n-terminal half of the s proteins of sars-covs and sars-like-covs . the development of three celisas was intended to provide choices for different applications. if the most specific antibody test is needed to detect sars-cov infection, the mab-based celisa may be the best choice. on the other hand, if one wishes to detect antibodies to all of the related sars-covs and sarslike-covs, the anti-nc-based celisa will be more appropriate. the anti-s -based celisa will be most useful to detect antibodies for sars-covs only, but allow for 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identification of dominant epitopes of synthetic immunocontraceptive that induce antibodies in dogs characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus e and e rns using phage-displayed random peptide library the authors would like to thank d. middleton, j. bingham, k. halpin, and r. fogarty for help with animal infection experiments; m. tachidjian and s. wilson for assistance in production of chicken antibodies; k. selleck, and j. brangwyn for technical support; d. magoffin and k. halpin for critical reading of the manuscript; and t. pye and e. hansson for dna sequencing service. the major component of the study described in this paper was funded by a grant (project . r) from the australian biosecurity cooperative research centre for emerging infectious diseases to l.-f.w. key: cord- -otwps u authors: parray, hilal ahmed; shukla, shivangi; samal, sweety; shrivastava, tripti; ahmed, shubbir; sharma, chandresh; kumar, rajesh title: hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: otwps u the advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. there are more than antibody-based molecules that are in different stages of phase i/ii/ iii clinical trials targeting new unique targets. to date, approximately more than monoclonal antibodies (mabs) have been approved. a total of novel antibody therapeutics had been granted the first approval either in the united states or european union in the year , representing approximately % of the total number of approved drugs. most of these licenced mabs or their derivatives are either of hybridoma origin or their improvised engineered versions. even with the recent development of high throughput mab generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. the recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. this has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. in this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. this review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mabs. antibodies are the glycoproteins produced by the b-cells also known as immunoglobulins, which are present in higher eukaryotes. immunoglobulins are present in either as a soluble form (blood or plasma) or as membrane-bound form (b cell receptors). antibodies are the major component of the humoral immune system that provides protection against the invading pathogens i.e. viruses and bacteria [ ] . an antibody is made up of two structural unit's i.e. heavy and light chain. generally, each heavy chain has one variable and three constant regions whereas the light chain has one variable and one constant region. the variable region of antibodies is mainly responsible for its interactions with the invading pathogen and antigen recognition. the antigen-antibody recognition mechanism works like a lock and key fashion. each antibody has a particular paratope (i.e. lock) that binds to a particular antigen (i.e. key). one type of b cell produces one type of antibody against a particular antigen. there are five different types of heavy chains based on the structure of crystallizable fragments (fc) that is attached to the antigen-binding fragments. on the basis of different fc region, antibodies are grouped into five different isotypes i.e; igm, igg, iga, igd, and ige. among all the isotypes igg is the smallest and the most common isotype with the highest therapeutic potential. it makes - % of the total antibodies. iggs have a longer half-life and are permeable to extravascular spaces [ , ] . https://doi.org/ . /j.intimp. . received april ; received in revised form may ; accepted may antibodies are potentially used for various applications as extraordinary tools in biomedical research for many years. high specificity and selective binding have expanded the scope of antibodies to various applications such as flow cytometry, magnetic cell sorting, immunoassays, therapeutic approaches etc. [ ] . antibodies have developed about years ago and have expanded the scope of antibodies to various applications due to their specificity and selective binding ability. these antibodies are classified into two primary subtypes, monoclonal and polyclonal on the means they are basis of their origin from the lymphocytes [ , ] . both polyclonal and mabs have their advantages and limitations which make them equally suitable for different applications. polyclonal antibodies (pabs) are a pool of immunoglobulin molecules that are secreted by different b cell lineages and react against multiple epitopes of a specific antigen. the pabs are generated by injecting an immunogen into an animal using a prime-boost immunization strategy to produce high titres of antibodies against the particular antigen. after immunization, pabs can be used directly or in the purified form (through affinity column chromatography to remove other serum protein components). polyclonal sera display multiple epitope binding properties which make them an attractive reagent for various purposes, like its use as research or therapeutic reagent either directly or in purified form. the polyclonal serum is widely used for several decades for the treatment of toxin-mediated bacterial and viral diseases [ ] . emil adolf von behring was awarded nobel prize in physiology and medicine in for his work on serum therapy, especially its application against diphtheria, through which he opened doors for new ways of treatment in medical sciences [ , ] . animal serum-derived therapy has been successfully applied for different medical aspects like overdosing of medication, viral disease (like rabies) and as antitoxins in snakebite envenoming [ ] . the beneficial effects of pabs come from its polyclonal nature and biophysical diversity. the poly clonality nature allows targeting multiple sites in a single window of the application and biophysical diversity provides greater stability in environmental changes [ , ] . despite having beneficial effects of serumderived pabs therapy has several limitations, which need to be evaluated before introducing new interventions. as blood-derived products, intravenous polyclonal immunoglobulins (ivig) have limited availability, batch-to-batch variability; carry the risk of blood-borne disease transmission and only a small fraction of antibodies from the pool of antibodies binds to the target of interest to exert the desired effect. this sometimes results in low specific activity and relatively needs high doses to observe a desired beneficial clinical effect [ , ] . the other limitation of polyclonal serum is that it cannot be used for the treatment of chronic diseases [ ] . due to some of these potential drawbacks of pabs, the way for mab isolation, need, and urgency comes into the light. however, mabs are most suitable and frequently used because of their high sensitivity, specificity, affinity and homogenous nature [ ] . monoclonal antibodies are monospecific in nature and produced by identical b cells having high affinity and specificity towards a single epitope of an antigen. in hybridoma based technology was used to generate the mabs showing very minimal and acceptable batch to batch variation, produced in an indefinite amount continuously. the mabs can be produced against any given epitope present on an antigen or immunogen. moreover, they can be used to detect, purify and characterize the substance of interest. since the development of mab, the scope of antibodies has expanded to various further applications due to their target specificity [ ] . this has made mabs a powerful tool in the fields of biochemistry, molecular biology, and medicine. antibody-based biologics are one of the best-selling classes of biomolecules in today's market. advancement in mab generation technologies in recent years has made ease in identification of new target antigens to be explored in diagnostic and therapeutic approaches [ , ] . several mab generation technologies had been developed over the years, to isolate mabs from immune and non-immune sources using hybridoma, display methods, and more advanced novel mab development technologies like single b cell amplification and culturing methods. unlike the hybridoma method, other methods rely mostly on recombinant production of mabs. each of these technology platforms has their respective advantage, limitations, and applicability [ , , ] . hybridoma technology is the primitive, most fundamental and successful methodology in the field of mab isolation [ ] . this technology is quite robust and useful in discovering thousands of antibodies for different applications [ ] . the basic practical advantage associated with hybridoma technology is, once the hybridoma clones are established, the production of mab becomes simple and efficient. the antibodies isolated through hybridoma methodology preserves the native pairing of variable and constant regions gene combination, which further supports studies on both direct and indirect functions of a mab. hybridoma technology relies on b cells that are matured in secondary lymphatic organs in response to an antigen. these b cells undergo natural antibody maturation process where the variable region of antibodies diversified by accumulating somatic hypermutations which further results in the selection of high-affinity tight binders [ , ] . resulting antibodies possess the natural pairing of variable heavy and light chain genes with naturally class-switched matured constant region gene through class switch recombination (csr). such freedom of natural csr is not possible in other mab isolation method that makes hybridoma a unique way to produce naturally matured in vivo antibodies in the laboratory [ ] . nevertheless, hybridoma technology is the most preferred technology for mab discovery for in vivo applications. presently there are more than % of the antibodies approved by the united states food and drug administration (us fda) are generated by traditional hybridoma technology and are used either directly or in chimeric or humanized versions [ , , ] . a list of these fda approved antibodies has been listed in table . the dominance of this technology has been continued with the recent development of transgenic humanized animals that has opened new avenues for more effective generation of high-quality human antibodies in the modern biotherapeutic era. in this review, we have discussed in detail about hybridoma technology, how it has been expanded to different animal species and their associated biological applications which determine their usage for certain intends. we have also discussed the advancement and challenges associated with hybridoma development, and how it has been overcome in years. georges kohler and cesar milstein in invented the hybridoma technology, for which they received the nobel prize in in physiology and medicine [ ] . during the same period, herve bazin in louvain-la-nerve belgium created a rat myeloma cell line ir , allowing the generation of first rat mabs (https://www.synabs.be/ / / /hybridoma-vs-display-the-fight-of-the-century/). hybridoma cells are generated via fusion between a short-lived antibody-producing b cell and an immortal myeloma cell. each hybridoma cell constitutively expresses a large amount of one purely specific mab, and favoured hybridoma clones can be cryopreserved for continuous mab production for a long period. hybridoma generation process takes advantage of a host animal's natural ability to generate functional, highly specific and high-affinity mabs [ ] . to date, several mabs are developed using this technology and are presently used for diagnosis, prevention, and treatment of different diseases tables , and . initially, hybridoma technology was limited to murine antigens but with advancement in this field, it is a well-established technology to develop mabs against a vast range of antigens and from different species like rabbits [ ] , humans [ , ] , chickens [ ] , goats, sheep [ ] , cows [ ] , mice [ ] , guinea pigs, and rats [ ] . choice of animal species especially for mab isolation depends on several factors such as the presence of a homologous protein in the immunized species, table a list of fda approved rabbit mabs that are used in diagnostics. device name availability of suitable fusion partner, the amount of protein or antigen available for immunization, the time required to obtain an antibody response and finally, a purpose for which these mabs are needed. most commonly used hosts for mab production are the mice followed by rabbits. the inbred balb/c strain of mice is usually the right choice and preferably suited for mab isolation [ ] . chickens are also considered as a preferred host of choice due to its distinct phylogenetic relationship between the antigen donor and the antibody producer [ , ] . hybridoma technology has not gained much attention because its success mainly depends on the availability of a suitable fusion partner. in the initial years of hybridoma discovery, technology was limited to mice but in progressive years researchers used this platform for human and rabbit hybridoma development. however major limitation associated with the production of mabs from other species is the instability of hybridoma clones produced with fusion partners from heterologousspecies [ ] . this instability resulted in the hybridoma clones are due to the fusion of two cells from different species which leads to chromosomal instability. to overcome this, in the past few years many different strategies have been used to increase the fusion efficiency. generally, there are two types of hybridomas one is homo-hybridomas and second is hetero-hybridomas. in homo-hybridomas both, the igg secreting b cells and fusion partners are from the same species. in hetero-hybridomas the antibody-secreting b cells and fusion partners are from two different species. homo hybridomas are genetically more stable and secrete stable igg as compared to hetero-hybridomas as it gradually lost the chromosomal recombinants during the clonal selection step due to their genetic instability. . animal species used for hybridoma development over the years: mouse polyclonal and mabs held the largest market in as they are more specific and easier to produce in nature. the structural similarities between human and mice antibodies are the prime reason for their high acceptability rate. upgradation and simplicity of mice hybridoma process have made it a more prime reason for their high adoption rate in research and therapeutics [ ] . the mice hybridoma technology is a multi-step process that takes advantage of a host animal's natural ability to produce highly specific, high-affinity and fully functional mabs. it involves the development and optimization of specific immunogenic antigen (ag). following the optimization, a host animal is immunized with the ag along with adjuvant for several weeks. the sera from immunized animals are tested for their reactivity and specificity to the immunizing antigen while the animals with high titres of binding antibodies are selected further for splenocytes isolation [ ] . the spleen cells are fused with the immortalised myeloma cells in the presence of fusogenic agents like viruses, chemicals and electric pulses. the most common myeloma fusion cell lines are x -ag . [ ] and sp / -ag [ ] , with the origin from balb/c mouse. the fused cells are then selected on hypoxanthine-aminopterin-thymidine (hat) medium. the myeloma cells are sensitive to hat medium as they lack hypoxanthine-guanine phosphoribosyltransferase (hgprt) gene required for nucleotide synthesis by the de novo or salvage pathways while the unfused b cells die as of short life span. in this process, only the hybrid (b cell-myeloma) survives, as they harbour the functional hgprt gene from the b cells. however, hybrid cells retain the dual properties, antibody secreting property of b cells and continuously growing property (immortality) from myeloma cells. fused or hybrid cells are then screened by "limited dilution cloning" method or with semi solid selective medium to select only those hybridoma that produce antibodies of appropriate specificity. a detailed schematic representation of steps involved in hybridoma production is shown in fig. . production of antibodies by mouse hybridoma technology is quite robust and has been useful to discover thousands of antibodies for different applications. academic and industrial research groups with expertise in the field of antibody isolation, hybridoma methodology continues being the methodology of the first choice, particularly if the goal is to obtain antibodies for analytical purposes. the main associated advantage is that, once the hybridoma clone is isolated, mab production in mouse ascites is simple, efficient and reproducible. these hybridomas can be stored in liquid nitrogen for several years making them virtually immortal [ ] . the mouse mabs can be potentially used for diagnostic, therapeutic as well as for other research applications. the first therapeutic antibody that was approved by the food and drug administration (fda) in , developed by murine hybridoma technology was potentially used to reduce the graft rejection in transplant patients [ ] . okt , the first mab to be used in organ transplantation and during the past one decade there has been an extensive experience of its use both for prevention and treatment of organ transplantation rejection. okt blocks t cell function by modulating cd and the t cell receptor from the t cell surface, functions as an immunosuppressant [ ] . since its discovery, okt was further improvised in progressive years from chimeric to humanized version, to further reduce adverse effects and to increase immunologic efficiency [ ] . the use of murine derived mabs has less therapeutic value as they are entirely from mouse origin and can cause immunogenic reactions in the target host. such limitations have been addressed by chimerization and humanization of antibodies, potentially removing the mouse immunogenic content [ ] . several murine mabs has been approved by the fda for use in diagnostic and therapeutic purposes over the years as listed in table . since the discovery of mouse mabs, rabbit hybridoma has been potentially used as a dominant tool in the field of research, diagnostics and therapeutics from several decades [ ] . however, a new technology for generating mabs with improved affinity, specificity and having the ability to recognize non-immunogenic rodent's epitopes, are in need as an alternative for the scientific community. the rabbit immune system has been documented as a vehicle for developing antibodies with higher affinity and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic in mouse [ ] . antibodies produced in rabbits usually have about to fold greater affinity than those produced by mice. rabbits generate more diverse and complex immune response towards target antigen as compared to human and mice because of gene conversion and somatic hypermutations phenomenon leads towards more mutations in rabbit antibody repertoire [ , ] . the gene conversion is responsible for introducing mutations and affinity maturation of variable antibody fragments which takes place in double-stranded rearranged v(d)j dna segment of antibody gene via homologous recombination [ , ] . the rabbit iggs are somewhat simpler than the mouse and human antibodies. rabbit igg has only one subclass i.e. cγ gene and the majority ( - %) of light chains are derived from isotype cκ . only % to % of the total igg light chains are isotype l. fig. . several efforts were made to generate rabbit mabs after the development of mouse hybridoma technology in the s. due to the favourable properties of rabbit antibodies, many scientific groups tried to develop methods for the generation of rabbit hybridomas. this endeavour was significantly complicated by the absence of rabbit myeloma cell lines. viral transformation of rabbit b cells to generate myeloma-like cell lines also proved to be difficult and rather inefficient [ ] . for these reasons, substantial efforts are focused on generating rabbit-mouse hetero-hybridomas. unfortunately, all hetero-hybridomas generated in the early days of hybridoma technology revealed poor fusion efficiency, genetic instability and impaired functional rabbit heavy-and light-chain pairings. in , raybould et al. generated the first stable rabbit-mouse hetero-hybridoma by polyethylene glycol-mediated fusion of rabbit spleen b cells with the mouse myeloma cell line sp / -ag . even though they observed stable rabbit igg expression for several months, other groups observed genetic instability and concomitant decrease of mab secretion [ ] . these shortcomings could be partially addressed by extensive efforts to regularly subclone the rabbit-mouse hetero-hybridoma in , weimin zhu and robert pytela, at the university of california, developed an improved rabbit hybridoma fusion partner by repeatedly subcloning. after multiple rounds of subcloning, they selected high fusion efficiency clones based on characteristics like robust growth, morphological characteristics and named it as a new cell line e-w, with better fusion efficiency and stability. since then this cell line e-w has been further developed and optimized to eliminate endogenous igg and has been used for the production of rabbit mabs for research and commercial applications [ ] . the cell line e-w was further modified to a superior version e-w . abcam patented this technology to develop highly specific mabs, under the name of ranmab, which has been potentially explored for the production of diagnostic and research antibodies. a large number of rabbit mabs are used in basic laboratory research. rabbit mabs are preferred over the mouse and human mabs in diagnostics and pharmaceuticals applications because of their high specificity and affinity. various rabbit mabs have been approved by the fda to use as in vitro tumour diagnostic tool [ ] . a list of fda approved rabbit mabs for diagnostics are listed in table . recently, wei et al. developed an ultrasensitive test for ebola virus diagnostics using carbon nanoparticle-labelled pad with rabbit anti-ebola virus (ebov)-vp igg for rapid detection lateral flow test strip for ebola virus [ ] . limited success has been gained in the development of rabbit mabs as therapeutic agents but the potential use of rabbit pabs for the prophylaxis and treatment of acute rejection against t cells in organ transplant opened venture to explore the use of humanized rabbit mabs as a therapeutic agent [ ] . a company name epitomics has developed a humanized rabbit monoclonal drug candidate and demonstrated in vitro and in vivo efficacies [ ] . recent fda approval of humanized rabbit monoclonal single-chain antibody fragment, brolucizumab in for the treatment of wet age-related macular degeneration (amd) has increased hope and venture of other humanized rabbit monoclonal for therapeutics in near future [ ] . a detailed description of these antibodies has been reviewed by mage et al. [ ] . human hybridoma technology which allows the direct generation of human antibodies in a native form, is the most direct effective approach for the production of natural therapeutic and diagnostic antibodies with no additional modifications require [ ] . it is believed to be the most promising and convenient technological platform for the isolation of therapeutic mabs. however, success of human hybridoma technology for the therapeutic purposes has been limited since years due to several technical challenges like unavailability of human fusion partners, as most of the fusion partners available are from rodent origin or heteromyelomas. a fusion of human b cells with different fusion partners limits the use of these mabs for therapeutic applications. several hetero-myelomas fusion has been successfully employed for the generation of mabs of human origin for different diseases like hiv [ , ] , chikungunya [ , ] , dengue [ ] etc. however, such hybridomas are unstable, leads to a loss in the ability of antibody secretion hence there is a challenge in achieving desired pharmacokinetic characteristics of natural human antibodies in terms of distribution, metabolism and excretion [ ] . several scientific groups have attempted to develop natural human fusion partner cell lines but limited success stories are reported using these fully human fusion partners. one such example of fusion cell is human karyochi cells which were successfully used to develop complete human stable hybridomas with igg secreting properties for several months. the fusion efficiency of these human karyochi cells was in the range of - to - with no reports on the endogenous generation of immunoglobulin or chains that can interfere with subsequent synthesis, assembly and purification of mabs. the other major limiting step in the development of human hybridomas is low fusion efficiency of human b cells with the myeloma partner ( . %) and secondly, the low percentage of circulating antigen-specific b cells in the peripheral blood ( . %) also limits the use of this technology [ ] . frequency of antigen-specific b cells is very rare in circulation therefore, selection of a proper blood donor is critical for the success of this method. acutely infected patients have a higher number of circulating b cells as compared to the convalescent patients. donors showing a high titre of serum binding/neutralizing antibodies may have a higher frequency of peripheral b cells, and an indication of the greater chance of successful production of hybridomas. to overcome these challenges different groups have tried ebv(epstein-barr virus) transformation approach to enrich the population of b cells [ ] . the most commonly used cell line b - is a continuous cell line releases high titres of transforming ebv in supernatants [ ] . the b - cell line was initiated by exposing marmoset blood leukocytes to ebv fig. . schematic drawing of natural rabbit, mouse, chicken and human igg. generally -kda igg comprises of two identical κ or λ light chains paired with two identical heavy chains. the light chain consists of an n-terminal variable domain (vl), followed by one constant domain (cl). the heavy chain consists of an nterminal variable domain (vh), followed by three constant domains (ch , ch and ch ) generally, however, the heavy chain of avian igy contains four constant regions (ch , ch , ch and ch ). schematic drawing of natural rabbit antibodies in igg format. the~ -kda rabbit igg molecule contains two identical κ (white) or λ (light grey) light chains paired with two identical heavy chains (dark grey). the light chain consists of an n-terminal variable domain (vl), shown with its three cdrs, followed by one constant domain (cl). the heavy chain consists of an n-terminal variable domain (vh), also shown with its three cdrs, followed by three constant domains (ch , ch and ch ). ch and ch are linked through a flexible hinge region that has the amino-acid sequence apstcskptcp (or apstcskpmcp in an allotypic variant) and anchors three disulphide bridges (orange) of the igg molecule, one for each of the two light-and heavy-chain pairs, and one for the heavychain pair. notably, rabbits have two κ light chains, k and k . the more frequent κ light chain, k , contains an additional disulfide bridge that links vl and cl. rabbits of the commonly used new zealand white strain have~ % igg-κ (k ),~ % igg-κ (k ) and < % igg-λ antibodies. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) extracted from a human leukocyte cell line. b - provides a source for ebv to establish continuous lymphocytic cell lines from human donors. ebv mainly binds with the cells in peripheral blood that contains cd receptor and b cells in peripheral blood express these antigens on their surface and activates latent membrane protein (lmp) a and lmp [ , ] . due to low antibody-secreting and chromosomal instability characteristics transformed b cells cannot be cultured for a long period. the transformation efficiency of ebv to b cells is low, ranges from . to %, however, the transformation efficiency of b cells in presence of ebv can be increased by the addition of cpg, which acts as toll-like receptor (tlr) agonist [ ] . cpg oligo-deoxynucleotides (or cpg odn) are defined as short single-stranded synthetic dna molecules where c refers for a cytosine triphosphate deoxynucleotide ("c") followed by a guanine triphosphate deoxynucleotide ("g"). the "p" denotes to the phosphodiester bond between consecutive nucleotides [ ] . in unmethylated form, these cpg motifs act as immune stimulants and are recognized by the pattern recognition receptor (prr) toll-like receptor (tlr ), which is constitutively expressed on immune system cells like b cells. these b cells undergo polyclonal response to cpg dna stimulation by proliferation and differentiation to antibody-producing cells [ ] . these transformed b cells are grown for a specific time for the emergence of immortalized cells, which further fused with the fusion partner to establish hybridomas. this cpg activation method has been successfully used to produce mabs against severe acute respiratory syndrome coronavirus (sars-cov) [ ] and hiv [ ] . the main concern of developing hybridoma for therapeutic purpose is that the final hybrid cells should be free of ebv and other human viruses [ ] . the two most common cell lines used for fusion are shm-d and hmma . . the shm-d ; produced by fusing the human myeloma cell line fu- , clone e- (hat sensitive, -azaguanine resistant and resistant to g- -an antibiotic similar to gentamicin) with the murine myeloma p x ag . [ ] . this cell line has been used as a fusion partner to stabilize the lymphoblastoid cell lines (lcl's) secreting immunoglobulins to produce mabs against envelope proteins of hiv- and parvovirus b [ , ] . hmma . is a human × mouse cell line that was generated by fusing mouse myeloma cell line p x ag . with bone marrow mononuclear cells of a patient with iga myeloma [ ] . several mabs have been produced using this cell line as a myeloma fusion partner [ ] . the other limiting steps in human hybridoma development are fusion efficiency. fusion is mainly performed by three methods (i) chemical agents like polyethylene glycol (peg) (ii) viral agent mediated methods and (iii) electric fusion methods. peg mediated fusion is the most common and traditional method of fusion due to its simplicity and most commonly convenient fusing agent of choice for hybridoma production. peg fuses the plasma membranes of two adjacent mammalian cells by dehydrating the lipid head groups, leading to the asymmetry of the membrane bilayer, favouring fusion of two cells leading to a single cell with two or more nuclei. one major drawback of peg mediated fusion is a generation of non-specific fusion between different kinds of cells [ ] . over the year's viral agents have also been used as a successful agent to perform fusion among two different cells. sendai and vesicular stomatitis virus are the two most commonly viruses used as fusion partners. the most efficient and innovative method for cell fusion is electrical cytofusion. this mainly works on the principle of fusion of cells in the presence of high-intensity electric field pulse that causes transient membrane permeabilization. this method has higher fusion efficiency over chemical or viral-based fusion methods. however, electrofusion yields in low fusion efficiency when fusion partner cells are different in size [ ] . hmma . myeloma cell line is found to be the most preferred cell line for electrofusion showing maximum fusion efficiency as compared to other myeloma cells [ ] . rems et al., developed a numerical calculation method to fuse cells with shorter, nanosecond (ns) pulses. the performance of this method works on the principle of contact areas between two fusion cells, regardless of their cell size [ ] . use of cephalin as fugenic reagent along with emetine and actinomycin d, golestani as selection method dramatically increased the fusion efficiency and recovery ( - %) [ ] . the major advantage of human hybridoma technology is that antibodies produced by this technology are derived from human origin have more therapeutic applications than rodent derived counterparts due to attributable differences between the human and rodent immune systems. due to evolutionary differences between mammalian (e.g. human, mice) and avian species (chickens), the avian/chicken immune system recognizes more epitopes on mammalian proteins as foreign and generates a more vigorous and diverse immune antibody repertoire [ ] . phytogenic differences among different species have been illustrated in fig. . as the phylogenetic difference between the immunizing antigen and the immunized animal increases, the immune response increases accordingly. it is, therefore, possible to produce antibodies in chicken that are difficult or impossible to produce in mammals such as g-protein-coupled receptors (gpcrs) against which producing antibodies in rodents is a challenge as of high sequence conservation (> %) at the protein level. chickens are emerging as valuable immunization hosts specifically for therapeutic antibody discovery for difficult targets having sequence conservation in mammals. the use of chicken egg yolk for antibody production represents a reduction in animal use (ethical issues) as chicken produces a larger amount of antibodies than laboratory rodents [ ] . the advantages of chicken (gallus domesticus) antibodies as diagnostic and therapeutic biomolecules are less characterized than their mammalian counterparts [ ] . initially, in a successful attempt was made to generate chicken hybridomas against newcastle disease virus (ndv) by fusing the peripheral blood lymphocytes (pbl) and thymidine-kinase deficient (tk-) chicken myeloma cells [ ] . surprisingly, the secreted antibody hybridomas were initially obtained, but they lose the ability to produce antibody in the culture immediately [ ] . to overcome this issue nishinaka s et al., in developed a new improved fusion cell line r h , for the production of chicken mabs [ ] . the new cell line was efficient in the development of antibody-producing hybridomas with highly reactive igg secretion ability of months. these cell lines were further improved and several chicken hybridomas were successfully developed [ , ] . however, chicken hybridoma technology has been explored in limits and most of the investigators prefer to use phage display method over hybridoma for the development of chicken mabs [ ] . the main avian antibody isotype igy shares structural and functional homology to mammalian igg and ige isotype counterparts but the difference in a constant region of antibodies, igy has constant regions whereas igg has constant regions [ ] [ ] [ ] fig. . igy present in chicken sera gets passed to the embryo through the egg yolk [ ] . egg igy antibodies have been used previously against bacterial and viral infections [ , ] . humanization of these antibodies can have great potential for biopharmaceutical development [ , ] . the recent development of transgenic chicken with human immunoglobulin loci has expedited the use of transgenic chicken derived mabs directly for human therapeutic use [ , ] . these engineered transgenic chickens express antibodies from immunoglobulin heavy and light chain loci containing human variable regions and exhibit normal b cell development raising immune responses to conserved human targets that are non-immunogenic in mice [ ] . these transgenic chickens can be potentially used for the development of hybridoma secreting antibodies of human origin. however, like others, the unavailability of robust hybridoma fusion partner limits its potential utility [ ] . in recent years different research groups have extensively explored the display method to overcome the limitations of chicken hybridoma. antibodies isolated through hybridoma methodologies have the advantage of being used directly and could be cryopreserved for future uses till an indefinite time, as the fusion partners are myelomas possessing remodelled transcriptional machinery to secrete a continuously large amount of antibodies [ ] . advancement in recombinant technology has overcome the challenges by cloning of variable heavy (vh) and variable light (vl) from unstable hybridoma; cloning in transient transfection vectors to produce antibodies in mammalian expression system [ ] . currently the development of stable cell lines has advanced the antibody production system by developing stable cell lines from unstable hybridomas to produce consistent antibody production. chinese hamster ovary cell line (cho) is the most preferred cells used for large scale production of mabs. however, the development of stable cell lines is a tedious process that sometimes takes a month to year time where the success of this process depends on random genome integration of transgenes [ ] . development of crispr-cas in recent years has overcome by immunogenomics reprogramming using plug and play technology, where crispr-cas was used to engineer immunogenomics by homology-directed repair to replace endogenous immunoglobulin region by exogenous donor counterpart with the help of guide rna. this technology platform has enabled the rapid generation of full-length antibody-secreting cell lines [ ] . a major limitation of hybridoma technology is the lack of suitable fusion partners which limits the use of this technology and limits its applicability to other species. to overcome the problem of suitable fusion partners, the transgenic mice model h- k b -tsa has been developed. h- k b -tsa transgenic mice express the simian virus (sv ) antigens (tag) under the control of mouse major histocompatibility complex h- k b promotor. this promotor allows differential expression of sv antigen in different tissues at various levels. expression of this antigen can be increased by simply increasing the levels of interferons (ifns) [ ] . the higher expression level of this antigen is responsible for tumorigenesis and aberrant development. in this approach transgenic mice h- k b -tsa were specifically used to isolate monoclonal against the filamentous phage. transgenic mice were immunized with filamentous phage suspension. splenocytes were recovered from the immunized animals and spleen cells were limited diluted from , -, -well plates. cells from the positive wells were selected to develop monoclonal lines. the main advantage of this technology is that it eliminates the use of fusion partners and can parallelly be used for the development of monoclonal and polyclonal based therapies [ ] . the other major challenge associated with hybridoma development is the requirement of purified antigen to generate a specific immune response. in some of the cases, it's a challenging task to purify the antigen. the lack of specific immune reagents for characterization and monitoring of these numerous proteins limits the overall time process for the production of hybridoma. expression and purification of recombinant protein are time-consuming and sometimes not cost-effective. additionally, immunization of animals with these purified recombinant proteins in formulation with adjuvants sometimes leads to alteration of native conformation of these proteins which finally leads to an undesired immune response in animals. in recent years, different approaches have been successfully implemented to overcome these challenges; a novel strategy has been developed to isolate mabs against the native proteins [ ] . in this strategy, animals were directly immunized with transiently transfected hek cells that express desire protein on the surface of these cells in a proper folded and glycosylated form. this modified technology is mainly useful for the isolation of mabs against native antigens. the advantage of this technology is that expressed protein on transfected cells closely mimics as it expresses in naturally infected cells. a similar type of strategy was developed by hazen m et al; where they had attempted to isolate monoclonal against the native conformation of extracellular loops & multi transmembrane proteins using dna based immunization strategy in combination with immunomodulatory agents [ ] . use of immunomodulators in dna immunization has positively enhanced the immune response and proved to be a successful approach to isolate desired specificity binding mabs to native conformations. kato et al., in developed a cell-based immunization and screening (cbis) method for isolation of mabs for podoplanin (pdpn) [ ] . in this strategy, they immunized the mice with transfected cells overexpressing pdpn and screened the fused hybridoma cells using flow cytometry. these cell-based immunization methods are useful in the isolation of mabs against various membrane proteins which is still difficult to achieve otherwise [ , ] . the other challenge in hybridoma technology is animal immunization. the overall efficiency of hybridoma depends upon the efficiency of immunization. some factors that affect the efficiency of immunization are; route of administration of antigen, dose, choice of adjuvant, number of boosts and immunization protocol. dna based immunizations are generally more preferred where it is difficult to express fulllength proteins and immune response are mainly targeted towards native or conformational epitopes because the structural integrity of protein is critical for induction of functional mabs. the major routes of dna based delivery are intramuscular, intravenous and intrasplenic. intrasplenic routes are considered to be the most efficient route. a single dose of dna delivery is sufficient to induce antibody responses. antibodies against different proteins can be produced at the same time by immunizing with several nucleic acids encoding for different proteins or single plasmid encoding different subunits of the protein. in such cases multiple booster doses are usually avoided, to reduce immune-dominance amongst antigens. another major challenge in the field of human hybridomas is the requirement of lymphocytes from actively infected patients or who have been exposed to antigen. if the active immune response is absent or not sufficient then the probability of circulating b cells in such cases is very poor or negligible. due to ethical issues and considerations, it is generally not possible to immunize humans. to overcome these limitations li et al., in have developed a novel and rapid combined ex-vivo immunization strategy with morphogenics platform process for the isolation of therapeutic human mabs. in this strategy, the group has purified b cells (cd +) and cd positive t cells from healthy volunteer blood samples using magnetic bead-based sorting. these b/t cells were cultured in the presence of growth factor and antigen to activate b cells. the pool of these b and t cells were then fused with myeloma fusion partner using electric cyto plus. the fused cells were screened for antigen-specific antibody secreting properties by limited dilution method. the isolated mabs had shown high specificity, biological activity and high affinity. this method avoids the collection and screening of a large number of patient samples which are normally the basic requirement for the generation of therapeutic human hybridomas. it also avoids the risk of potential viral transmission associated with conventional methods where pbmcs are sometimes used from viral infected patients. screening of volunteers and blood cells can be performed before ex vivo immunization and after hybridoma development. this platform technology offers a rapid and cost-effective way for therapeutic human mabs having natural pairing of immunoglobulin genes [ ] . after cell fusions between b cells and myeloma cells, protocols of hybridoma technology include multistep screening and cloning processes to identify antigen-specific hybridomas, which is labour-intensive and time-consuming. however, recent advances in robotic screening methods have alleviated this to some extent [ ] . the screening process on semi-solid selective medium has made it easy and reduces the overall time in hybridoma production by repeated selection and cloning steps. this screening technology has been used by companies to sale ready to use kit based systems for the development of murine hybridoma. it has also facilitated the use of murine hybridoma technology in a less cumbersome and user friendly. methylcellulosebased semi-solid selective medium is preferentially used in hybridoma selection [ ] . technically it avoids the loss of rare clones from an overgrowth of faster-growing cells, which can occur during selection in a liquid medium. the selected clones are further dispersed into a liquid growth medium for screening and expansion. similarly, paul et al recently developed microarray-based screening technology for direct identification of high-affinity clones which avoids the loss of slowgrowing clones. additionally, this approach eliminates the enrichment, isolation, and purification of igg for the characterization process. the crude culture supernatants can be directly used and thus avoids expensive and lengthy screening steps [ ] . the screening process has advanced through flow cytometry-based methodology where single cells could be sorted from a bulk mixture of fused hybridoma cells. it has advanced by saving time and labour instead of the traditional multimicro well plate seeding and limiting dilution sub-cloning [ ] . in recent years tedious hybridoma screening and cloning processes are replaced with flow cytometry-based sorting methods. these methods avoid effort and time of tedious repeated screening processes. this method could also differentiate igm and igg secreting hybridoma. facs based screening methodology can be applied in any laboratory easily setup as it doesn't require any special reagents [ ] [ ] [ ] . the other alternative approach that some groups had used is the screening of hybridoma supernatant directly by bio-layer interferometry based on disassociation rates to select clones containing high-affinity antibodies for further expansion and subsequent characterization. the main drawback of the elisa based screening method is that clones that express high levels of a low-affinity antibody can give an equivalent signal to clones that express low levels of a high-affinity antibody. as a consequence, superior clones can be overshadowed by inferior clones because elisa method score antibodies based on the binding signal strength and do not provide accurate affinities or dissociation rate constants [ , ] . current drug approval rates underline the revolutionary effect of fully human mab therapeutics on drug development. antibodies isolated for therapeutic applications from different species excluding human needs a multistep process of humanization and developability through rational sequence optimization [ ] . mouse is the most common and preferred progenitor used in mab isolation. to avoid the multistep process of humanization, the concept of transgenic mice harbouring the human antibody repertoire has gain attention, where large human immunoglobulin loci are transferred into the mice germline using yeast artificial chromosome approach [ ] . the xenomouse and humab mouse are the first engineered transgenic mice that carry the majority of human vh & vl antibody repertoire [ ] . xenomouse transgenic technology was developed by cell-genesya a biotech company (now a part of bristol myers squibb, new york, usa). the first therapeutic antibody developed by this transgenic technology was approved by the fda in for the treatment of advanced colorectal cancer. the strength of transgenic technology can be evaluated by the recent data on approved mabs. more than fully human mabs developed by transgenic animal-based technology are used for human therapy. a list of fda approved mabs developed by transgenic technology is listed in table . initially, this technology was limited to mice but over the years this technology has been established for other animal models like rabbits, rats, and cows. the success of this technology table a list of fda approved mabs derived from transgenic technology. treatment of colorectal cancer mainly depends on the proper representation of the target antigen to the immune system. in addition, designing a proper immunogen is very critical for success. to avoid the ambiguity of immunogen design in soluble form, genetic immunizations are more preferred over the traditional methods. in contrast to human, mouse possesses very less antibody diversity represents the main limiting step in xenomouse transgenic technology. human antibody repertoire shows diversity more than , however, the number of b cells in a mouse is~ only. a single mouse can harbour the only fraction of the antibody repertoire from human antibody [ , ] . moreover, immunization of a large cohort of mice to increase the diverse response against antigens could be used to overcome the limited antibody diversity. the other transgenic mice technologies, where kymouse & trianni mouse models were developed to represent a more diverse human antibody repertoire that allows the selection of diverse human antibodies and overcomes the limitations of xenomouse [ ] . fig. represents an illustration of transgenic antibody technology showing the antibody production route. besides, the other limitation of this technology platform is immune tolerance when attempting to raise an immune response against human targets. a large number of human targets possesses a very high degree of sequence and structural homology, because of this homology the transgenic immune system recognizes these antigens as self-antigen. different groups have tried to overcome the immune tolerance mechanism by adding t cell epitopes to the antigen [ , ] . the other similar approach tried by different groups to abolish the expression of murine orthologues gene [ ] . but the major limitation with these defected mice models is that sometimes these mice suffer from health issues and some of these knockdown genes are necessary for the development of a foetus. recent developments of transgenic rat and fig. . illustration of transgenic antibody technology shows the antibody production route: mouse immunoglobulin gene loci were functionally inactivated in embryonic stem (es) cells by targeted gene deletion used to generate mice homozygous for the necessary deletions. crossbreeding between the transgenic mice (containing both human and mouse antibodies) with mice incapable of producing mouse immunoglobin, resulting in the xenomouse strain which expresses human antibodies but not the mouse antibodies. b cells, isolated from immunized xenomouse, are fused with myeloma cells to produce hybridomas producing human mabs. chicken (omnichicken) models have partially overcome these limitations. the transgenic model-based technology harbours some advantages over the phage display derived antibodies. the antibody developed by transgenic technology requires less development or optimization and thus require a shorter time to reach the product development stage. production of stereo-specific mabs is still a very big challenge. there are hardly any practical technologies available for the generation of stereospecific antibodies, because of hurdles like how to immunize a mouse maintaining the antigen structure intact in the presence of adjuvant. in addition, adjuvants allow more effective sensitization, disrupting the native structure of proteins. however, if an adjuvant is not used, immunization efficiency could be very low. another difficulty is strict selection of stereospecific mab producing sensitized b lymphocytes. although, if the immunization is successful with a native intact antigen, the number of desired sensitized b lymphocytes is extremely small, accounting for very less population of total spleen cells after repeated immunization [ ] . most of the established therapeutic mabs have specificity for the antigen targets having primary structures. generally mabs can recognize two types of antigen epitopes which includes linear in the primary structures of proteins and the conformational, dependent on secondary and tertiary structures [ ] . stereo-specific mabs recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. besides primary, secondary and tertiary structures, proteins may also exhibit quarterly structures. which are formed by hetero or homo-subunits, providing unique interfacial geometric structures on their complexes. native or conformation-specific mabs are quite reasonable and attractive for future therapeutic purpose. there is need to replace the conventional mabs with stereospecific mabs in the near future for therapeutic medicine as they recognize the dimensional conformation, which intrinsically determine their fundamental biological functions [ ] . stereospecific mabs that recognize d molecular configuration has advantages over linear epitope-specific mabs that consider only d configuration. in general, mabs against primary structures, in target therapeutic antigens with the dominant secondary and tertiary folding are having affinity only with full antigens in restricted areas, as their linear epitope may be masked due to conformational folding. alternatively, target antigens in the immune system can be identified by assuring indigenous structures via immunizing dna of the target antigens expressed on the surface of the cell, allowing healthy and intact structural conformation. this can be linked to membranes, which mimics membrane protein, with a soluble protein harbouring sufficient signal peptides and membrane-penetrating areas that are genetically linked to the ′ and/or ′ terminals of genes of the desired soluble proteins as nucleic acid sequences to express as a fusion protein [ ] . in the emerging diseases like human immunodeficiency viruses- (hiv- ), coronavirus - (sars-cov ), dengue, and chikungunya, the immunogenic proteins of pathogens harbour complex structural glycoproteins, needs an urgent high-quality stereospecific mabs to control their spread. development of therapeutic antibodies against these pathogenic diseases is aimed to the structural antigens. the complex native structures of envelope proteins on viral surface facilitate the attachment of virus to the host cell, and subsequently entry inside the cell [ ] . these native structural proteins induce predominantly cross reactive neutralizing antibodies. the neutralizing antibodies have shown promising results in the protection against pathogens however nonneutralizing antibodies help the virus in evading immune system [ ] . in hiv- , researchers are working to develop broadly neutralizing antibodies targeting conformational epitopes [ ] . similar approaches have been taken for other viral antigenic targets [ ] . in many of these viral infections "conventional abs" are generated in response to virus infection, but the virus adopts numerous evasion strategies like conformational masking of antigenic targets by glycosylation, high mutation rate etc. [ ] . generation of stereospecific mabs are required to display impressive breadth and potency against the conformational proteins. these stereospecific mab productions with promising therapeutic potential can be achieved by inclusion of critical steps like i. dna or soluble protein immunization with native like targets or structural proteins ii. selection of antigen specific myeloma cells and iii. selective fusion of myeloma and b cells to generate hybridoma cells secreting stereospecific mabs. different novel approaches and attempts are being taken to produce the soluble trimers which can display native-like conformational stable structure mimicking with virus surface. these conformational protein structures are promising targets for protein or dna immunization and could subsequently require for the production of stereospecific mabs. one major challenge associated with the use of native like antigens for the development of stereospecific antibodies is use of adjuvants in immunization process. most of the adjuvants usually disrupts the original protein native structure and hence impede its ability to present relevant epitopes or occludes the trimer conformational epitope [ ] . in recent years' studies using iscom class of adjuvants in animal preceded by in vitro analyses showed that it has no adverse effect on native trimer conformation or antigenicity [ ] . the other way to generate stereospecific antibodies is the direct immunization of mammalian cells expressing cell surface antigens in its native conformation [ ] . a number of stereospecific antibodies has been generated against several targets like receptors [ ] , ligands [ ] , antagonist and chemical compounds [ ] . a detailed schematic representation of different approaches used for isolation of stereospecific mab is shown in fig. . the production of stereo-specific mabs could be achieved by tweaking the conventional hybridoma fusion through stereo-specific targeting (sst) technique invented for the first time by tsumoto et al [ ] . crucially, it involves a strict selection of the required sensitized b lymphocytes by intact antigens, expressed on myeloma cells, through b-cell receptors (bcrs) and their selective electrofusion (only attached cells can be fused among themselves) to generate a specific hybridoma [ ] . sst technology offers selective production, against different protein types, of monoclonal stereospecific antibodies not only for membranous but also for soluble non-membranous antigens. morshed et al recently showed efficiency in activation of the g-protein coupled receptor which holds seven-transmembrane domains by a stereo-specific mab [ ] . furthermore, the new promising class of therapeutic mabs, catalytic antibodies are capable of identifying and degrading antigens, has fundamentally demonstrated. hifumi et al. have developed a catalytic antibody to degrade the active site for urease o helicobacter pylori and eliminates the bacterial infection in the mouse [ ] . moreover, the catalytic antibodies have proven their utility in suppressing infection of the rabies virus [ ] and the influenza virus [ ] in vitro and in vivo using human antibody light chains. in addition, they have recently been noted in their ability to effectively reduce the accumulated ß-amyloid in the mice's brain [ , ] . medi is a mab that inhibits cd (ecto- ′nucleotidase) activity on a non-competitive basis and is considered a promising immuno-oncology target [ ] . this mab is antagonistic to cd employing dual inter-cd dimer cross-linking and/or steric blockage mechanisms that prevent the adoption of the cd catalytic active conformation [ ] . bispecific mabs with stereo-detection may be especially effective for cancer cell detection of membranous antigens which have not been easily detected by conventional linear mabs. in a nutshell, as proteins retain their native conformation in nature, development of stereospecific, alternate forms of bispecific and catalytic mabs, for selective therapy is the founding factor in therapeutic future drugs. the mab has come a long way since the days when unmodified murine mab was explored against the cancer-causing agents. from the last two decades, mabs have been a standard element of cancer therapy, however, with still much room for further improvement in future [ ] . the mabs are always preferred over the chemical compound based therapies because of their high specific reactivity and affinity towards the target antigen recognition. they show minimal side effects with favourable pharmacotoxicity and pharmacokinetics properties. the high specificity of mabs towards their targets presents an attractive and successful option for the development of medical treatment and molecular drug targets. [ ] . early clinical attempts exploring mab-based therapeutics were very primitive and disappointing years ago, some clinical experts considered the antibody-based therapy treatment for cancer as a failed hypothesis [ ] . the first mab which was clinically evaluated against cancer was the murine mab. although there were some fascinating hints that mab therapy could be successful, however, the problems related to the administration of murine mab to humans limited their clinical utility and applications [ ] . rise in immune response against the therapeutic mab, very rapid clearance of the mab from the system and suboptimal ability of the murine mab to interact with the human immune system in a manner that led to immune destruction were the challenging tasks. however, some investigators tried continuously to explore the use of mab as a possible cancer treatment. they also evaluated other strategies such as using; igg to target cancer directly, alter the host immune response to cancer, provide cytotoxic substances to cancer, and retarget the cellular immune response towards cancer [ ] . over the last twenty years, the effectiveness of antibodies in the treatment of patients with cancer and other deadly diseases has been increasingly recognized, as mentioned in tables and . many of these antibodies are specific for antigens expressed by the disease-causing agents itself [ ] . in the case of viral targets, mab-based therapeutics have shown limited success. however neutralizing antibodies play an essential part in antiviral immunity and human protection against viral diseases is primarily mediated by the humoral immune response [ , ] . it is well documented that early administration of mabs in the treatment regimen reduces mortality rate significantly up to % [ ] . to date, only two mab is licensed for viral infection i.e. respiratory syncytial virus (rsv) and the other one is ibalizumab, which has been recently approved in for the treatment of hiv positive people with multidrug resistance towards anti-retroviral therapy (art) [ , ] . several other candidates are at the different stages of clinical trials e.g. leronlimab, an anti-ccr igg for hiv infection and regn-eb : a mixture of igg mabs for ebola virus infection (https://www. antibodysociety.org/antibodies-to-watch-in- -at-pegs-europe/). lack of vaccines against various deadly viral diseases necessitates the development of antibody therapeutics to save the loss of lives and control deadly diseases [ ] . however, it is always easy to generate monoclonal to protect the population at the time of the outbreak in a much shorter time as compared to vaccine production. though vaccines are one of the most cost-effective ways to manage infections, vaccines also require time to elicit protective immunity and depend on the host's ability to mount an immune response. a number of a prophylactic vaccine against pathogens such as; herpes simplex virus type- (hsv- ) and human immunodeficiency type- (hiv- ) have shown protection in animal immunization studies, but, so far, no effective human vaccine against these diseases are available [ , ] . antigenic drift and high diversity among the emerging pathogens; such as influenza virus and hiv have been reported [ , ] which further add to the complexity and may lead to vaccine mismatch drop in vaccine effectiveness against circulating serotypes and strains. in developing countries, it is not economically feasible to make a vaccine of every disease because of a lack of awareness of disease burden [ ] . the mabs are widely used in the fields of diagnostic, therapeutic and biological applications due to their high specificity and affinity. at present, the majority of mabs approved for therapeutics are humanized or the chimeric versions of mouse mabs and were generated using hybridoma technology. in recent years, these engineered humanize and chimeric antibodies are potentially used to generate different forms of antibody fragments such as scfvs [ , ] , diabodies [ ] , tandom abs [ ] , and domain antibodies [ ] , pegylated fabs [ ] to target novel antigenic sites. technical advancement in the applicability of hybridoma technology to other animal species (other than mice) of different phytogenic origin has led to the development of novel mabs to conserve human antigens. it has opened a new path for therapeutic and diagnostic mabs with high specificity and affinity to poorly immunogenic targets. with the recent development of high throughput mab generation technologies, hybridoma technology is the most favoured method due to its indigenous nature to preserve natural cognate pairing information of antibodies that is lost in other methodologies, reduces the specific diversity of antibodies [ ] . advancement in recombinant dna technology methods like chimerization and humanization has increased the potential of hybridoma technology to a great extent. an antibody that undergoes the process of humanization preserves the natural specificity and limits risk of cdrs causing an immune response. the unnatural pairing of antibodies in terms of affinity maturation and recombination pairing in display methods sometimes results in high immunogenic response [ ] . all these features have make hybridoma platform as first and most preferred mab isolation technology. recent advances in development of hybridoma cells secreting stereo-specific mabs have opened new avenues of future therapeutics. in comparison with other anti-viral drug treatments, a stereospecific antibody-based therapy could offer potent anti-viral actions by more comprehensive target and potent neutralizing effect. the mab market has shown tremendous increase in the last five years as a diagnostic and therapeutic reagent. the commercial development of therapeutic mabs commenced in the early s, and by the first therapeutic mab was fda approved for the prevention of kidney transplant rejection. over the years mab market has changed rapidly as a major class of therapeutic agents for the treatment of many human diseases, in terms of global sales revenue for all mab products was~$ . billion in . a graphical representation of the global antibody-based therapeutic market trend is represented in figs. and . the global mab therapeutics market is expected to grow at a compound annual growth rate (cagr) of . % and is expected to reach market revenue of around usd . billion by the end of [ ] . humanized mab accounts for the largest revenue-generating share among antibody-based therapeutics, showing to its widespread acceptance for numerous diseases including cancer, autoimmune diseases, inflammatory diseases, infectious diseases, haematological diseases, and others. the other potential area where mab has shown great success is diagnostics. the mab -based diagnostic reagents potentially identify abnormal cell targets, infectious agents, or elements of the body's this article does not contain any studies with animals performed by any of the authors. 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and therapeutic applications specific amyloid beta clearance by a catalytic antibody construct anti-cd in cancer immunotherapy: awakening new opportunities inhibition of cd amp hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action beyond peptides and mabs-current status and future perspectives for biotherapeutics with novel constructs overview of monoclonal antibodies in the diagnosis and therapy of cancer a clinical trial of anti-idiotype therapy for b cell malignancy congenital prostato-urethral fistula caused by persistence of the mesonephric (wolffian) duct antibody therapies for the prevention and treatment of viral infections broadly neutralizing anti-hiv- monoclonal antibodies in the clinic the contribution of vaccination to global health: past, present and future single-chain antigen-binding proteins protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli diabodies: small bispecific antibody fragments bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics binding activities of a repertoire of single immunoglobulin variable domains secreted from escherichia coli efficacy of a novel pegylated humanized anti-tnf fragment (cdp ) in patients with rheumatoid arthritis: a phase ii doubleblinded, randomized, dose-escalating trial veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas storyid= &title= global-monoclonal-antibody-therapeutics-market-to-be-worth-usd- -billion-by- : global monoclonal antibody therapeutics market to be worth usd . billion by supplementary data to this article can be found online at https:// doi.org/ . /j.intimp. . . key: cord- -ep ay authors: herron, j.b.t.; dennis, j.; brennan, p.a. title: coronavirus antibody positive tests and continued use of personal protective equipment throughout the pandemic date: - - journal: br j oral maxillofac surg doi: . /j.bjoms. . . sha: doc_id: cord_uid: ep ay the covid- pandemic has thrust not only a novel virus onto the world, but new challenges resulting in novel approaches. governments have reduced regulation in order to facilitate timely advances to combat the disease. antibody testing has rapidly been deployed but it is creating challenges for staff and patients. mask use has come to the forefront and human factor (hf) strategies must be examined to reduce risk associated with lack of engagement from both healthcare staff and patients. in this we explore these issues and suggest some solutions. globe from december creating an unparalleled situation, particularly in the western world renowned to have excellent primary and tertiary medicine facilities ( ) . this unprecedented sociopolitical and economic upheaval will have long reaching ramifications. as our understanding of this pandemic evolves, so too has the approach to combat the disease. we have seen retractions of published literature in high impact factor journals ( ) and spread of misinformation from all sources including senior political figures ( ) . despite mapping of its genome, the virus is still not understood ( ) . key aspects such as long term immunity remains unknown with much of the current knowledge applied from mers-cov and sars-cov( ). while there is evidence of antibody response, studies are low in participant numbers and the follow-up testing has been done over a relatively short period of time( ). a worrying study showed % of patients tested had very low neutralizing one area of concern is reliable testing as the covid- rt-pcr swab has a % false negative rate( ) and a delayed virus clearance can mimic re-infection due to the presence of dead rna ( ) . these outcomes can result in patients believing they have never had covid- or that they continue to have the disease. the nhs has recently introduced serum antibody testing ( ) with the theory that with development of an immunity passport, individuals could to go about their business secure in the knowledge that they are "immune" to the virus ( ) . suggested plans have included developing a cohort of immune staff to care for covid- patients allowing for a relaxation of overstretched personal protection equipment (ppe) resources. there are a number of serological tests available of dubious provenance (fig. ) . the most reliable are those being developed by pharmaceutical giants such as abbot and roche ( ) . as mentioned in a recent publication current covid- antibody tests are similar to the first-generation hiv tests ( ) . if current covid- antibody testing was similar to the equivalent hiv tests with a specificity of . %, public and healthcare confidence in them would be much higher ( ) . the % confidence intervals for roche antibody testing kits are between % to % whilst abbot is between % to %. this variation shows that up to % could be given an incorrect result with the roche test ( ) . public health measures such as good hand hygiene, the use of mucous membrane protection with goggles and masks, social distancing, isolation and contact tracing are the mainstay of prevention of this disease( ). masks reduce nosocomial spread and are important, particularly for healthcare staff ( ) . on the june , the uk secretary of state for health and social care announced that from june all healthcare workers and visitors will need to wear masks in hospital ( ) . with increasing antibody testing in medical staff it is pertinent that those with positive antibody tests ( ), but offers relatively little protection to the wearer ( ) . with healthcare staff often requiring to be within one metre of a patient for examination and treatment, social distancing is impossible in these circumstances. it is imperative that both patient and staff wear a surgical mask as a minimum to help mitigate this risk( ). it is important that even if staff are immune, they continue to use masks as others passive followers may follow the leader ( ) . this is also critical for patient's as it is known the four key elements to a good patient-doctor relationship are trust, knowledge, loyalty and regard ( ) . if patients observe a doctor without a mask, they may lose trust, assume that the doctor has dismissed the risk and not wear a mask themselves despite being highly infectious. this could reduce patient concordance with future medical professionals and increase spread of the disease. as learnt from military medicine team briefings can be an excellent opportunity to remind staff of a unity of effort ( ) and their obligation to wear a mask and to encourage a more assertive followership style ( ) . covid- has assisted the flattening of team hierarchy and reduced barriers between those working in health and social care. however, some team members will emulate the behaviour of their leaders and this is particularly prevalent between healthcare professionals and those less experienced or non-qualified staff. it is therefore important that those in positions of authority provide adequate j o u r n a l p r e -p r o o f role modelling. for the antibody test, even after the exact nature of protection is determined, basic public health measures are not forgotten and that staff feel able to challenge those in more authoritative positions regarding ppe. it is even more important than ever for leaders to conduct team briefings reminding others of the importance of facemask use, continue to flatten hierarchy and encourage assertive followership. physical interventions to interrupt or reduce the spread of respiratory viruses. part -face masks, eye protection and person distancing: systematic review and meta-analysis high-profile coronavirus retractions raise concerns about data oversight covid- infodemic: more retweets for science-based information on coronavirus than for false information a pneumonia outbreak associated with a new cov- through the postpandemic period reducing medical error during a pandemic generational and occupational differences in human factors knowledge and awareness: a comparison study mission command: applying principles of military leadership to the sars-cov- (covid- ) crisis laboratory diagnosis and monitoring the viral shedding of -ncov infections persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients covid- : antibody tests will not be rolled out in uk until at least may, mps hear covid- immunity passports and vaccination certificates: scientific, equitable, and legal challenges expert reaction to phe laboratory evaluations of roche and abbott antibody tests. science media centre waiting for certainty on covid- antibody tests -at what cost? human immunodeficiency virus diagnostic testing: years of evolution personal protective equipment and covid -a risk to healthcare staff? covid- : the case for health-care worker screening to prevent hospital transmission. the lancet do theatre staff use face masks in accordance with the manufacturers' guidelines of use? respiratory virus shedding in exhaled breath and efficacy of face masks assertive followership: how to make a team safer impact of the doctor-patient relationship key: cord- -ut gczm authors: nan title: education day monday: plenary session monday: parallel sessions date: - - journal: vox sang doi: . /j. - . . .x sha: doc_id: cord_uid: ut gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases ed- - standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be g (fda criteria). however, actual measurements have found a mean hemoglobin content of . ± . g per unit, with variability between manufacturers. percent of units may contain less than . g of hemoglobin while percent may contain more than . g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be . to percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of . g/dl. for pc transfusion, about percent of transfusions achieve the targeted dose of - ¥ platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of g/dl rather than g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of % yielded the same outcomes as one of %. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below % were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of g/dl and unlikely to be necessary at a hb above g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: ( ) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; ( ) the quality of retrieved studies is assessed systematically; ( ) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and ( ) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. ed- - biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf- /cxcr pathway has been emphasized, either by down regulation of cxcr , changes in sdf- gradients, or by disruption of sdf- /cxcr signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf- /cxcr , disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla /vcam- pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt- l, il- ) or chemokines (i.e. il- , gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf- molecules (i.e. ctce ), or cxcr antagonists (i.e. amd ) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. ed- - the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by - fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by %. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. ( ) ann med : - ). determination of the coding sequence of all the genes giving rise to antigens within the blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl ( ) human blood groups, nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. ( ) transfusion : - ), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. ( ) transfusion : - ). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. ed- - the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd + t cells and class ii molecules present exogenous antigens to cd + t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 's and 's -protect oncology patients from clinically relevant bleeding in more than % of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below per microliter (or in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol ; : - . additional data on lumbar puncture are reported in ann hematol ; : - . another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about % to about % of patients. ed- - ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over % in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: . studies of interventions comparing ffp with no ffp; . studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; . studies of interventions comparing ffp with a different blood product; . studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range - patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication ) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in . first in the commission agenda was a recommendation on donor selection criteria, given in . then came the european blood directive / /ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over donations per inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of nd august which came into force as of january st . this act was then updated in november according to eu directive / /ec and came into force as of january th . this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of nd august , in force since january st , has been supplemented by decrees: . procedures for external bts audits; . requirements for donor selection; . requirements and procedures for organization and safe management of blood transfusion in hospitals; . requirements for implementing of national and regional donor registers; . employment criteria for bts personnel; . training requirements for hospital personnel involved in blood and blood product administration; . national, uniform price list for blood and blood products; . organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age years, range - ; median weight kg, range . - . kg) with solid tumors at onset or on relapse underwent collections with the p y kit of the fresenius hemocare com.tec blood cell separator. our cd + cells target was ¥ e /kg. collections were started if a peak of at least . e /l cd + cells ( per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio : - : was combined with heparin u/kg. in children with < kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in % albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of ml (range . - . ml) was processed in a separation time of min (range - min). the median product weight was g (range - g) and yield of cd + cells was . ¥ e /kg body weight (range . - . ¥ e /kg body weight). three poor mobilizing patients (peripheral blood cd + peak of - cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose ( . and ). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under kg. m-pa- alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in , aubuchon and birkmeyer published a paper (jama ; ( ) : - ) where they concluded that the cost was usd per qaly, which is far above the 'acceptable limit' of usd . this estimate was adjusted to usd . mill. per qaly in a letter to jama (jackson, jama ). in , riedler et al. published (vox sang : - ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp for neonates and gbp for patients aged . the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = ) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas ) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination ( . mmol/l), maximum storage time of whole blood before separation ( °c, h), maximum storage time of mb plasma before freezing ( h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+ . %), fibrinogen (clauss) (- . %), factor v (- . %), factor viii (- . %), factor xi (- . %) and protein c (- . %). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha- -antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment + , thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at °c for months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of < . mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within hours (usually - hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but : plasma-containing transfusions is the most commonly cited figure. in % of patients, recovery is well underway within hours, and leads to complete resolution. death occurs in - %. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: ( ) antibody-mediated and ( ) -hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than % of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in % these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since . since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past years, cases of trali and cases of possible trali have been confirmed by critical review of each questionnaire. of cases of definite trali, donor specimens were obtained in cases. of cases, anti-leukocyte antibodies were detected in cases ( %) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (< %). of cases of antibody positive donors, anti hla antibodies were detected in cases, anti hna antibodies were detected in cases, and both were detected in cases. of cases of positive anti hla antibodies, class i antibodies were detected in cases, class ii antibodies were detected in cases, and both were detected in cases. on the other hand, the anti-leukocyte antibodies were detected in % of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion ( %). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of adverse events analysed by the serious hazards of transfusion (shot) scheme ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , ( %) were haemolytic transfusion reactions (htrs). were due to incorrect blood component transfused (ibct): / were abo incompatible and / caused by other red cell antibodies. a further cases were reported as acute htrs (ahtrs; i.e. occurring within hours of transfusion) whilst were recognised more than hours after transfusion and reported as delayed htrs (dhtr). / ( %) patients died and suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. / ahtrs occurred in group a ( / ) or group b ( / ) patients given group o platelets. of the ahtrs related to red cell transfusion, were due to errors, were in patients with auto-antibodies, in only of whom alloantibodies had been adequately excluded/identified. at least / dhtrs were potentially avoidable; in cases the antibody was detectable retrospectively in the pre-transfusion sample; in cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. % htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in of patients ( %) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations - allogeneic from matched sibling healthy donors and autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf ( - g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was - ¥ e /l (autologous setting) or on the th day - hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was . - . l, and . l for one pbsc transplantation in average. results: using a minimal target dose of cd + cell count ( ¥ e /kgbm), performing one mnc-apheresis procedure for % recipients sufficient number of pbscs were obtained. the mnc yield was . ¥ e /kgbm in allogeneic and . ¥ e /kgbm in autologous setting in average. the mean cd + yields for allogeneic and autologous transplantations were . ¥ e /kgbm and . ¥ e /kgbm, respectively. hematopoietic reconstitution was achieved on the . th day for leukocytes and the . th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd + cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late s but it was only in the s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive / /ec off. j. european union. . . :l / -l / ) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa- years of shot data - : a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to deaths and cases of major morbidity in the uk between and . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that % of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for % of all pre-transfusion testing but % of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. % of hospitals reported that they participated in shot in , but only % of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa- passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b ) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds ( depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa- evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for days. red cell quality was determined by measuring the in vivo red blood cell recovery hours after re-infusion of the -day stored red cells. storage hemolysis and atp were also determined. methods: units of blood ( ml) were collected from normal volunteers into the leukotrap wb system containing cp d/as- anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within hours at room temperature (rt units), or after hours at - °c (cold units). the prion filter set was sterilely connected to the units on day , and the units were filtered and stored for days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with -cr radioisotope, re-infusion, and determination of the -hour in vivo rbc recovery. a donor sample was also labeled with m-tc to allow for red cell mass determination. thus, both single-and double-label -hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on units. the mean single-label -hour recoveries were . % and . % for the rt and cold units, respectively. the mean double-label recoveries were . % and . % for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the -hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than % of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp d/as- (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~ ¥ e platelets in ml of % plasma and % intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria ( , , cfu) were added to the double units. each double unit was divided into two identical products containing , , or cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process ( mm amotosalen + j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days , and of storage. samples ( ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at and cfu per product, strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and strains (e. coli, c. perfringens) tested negative after days of platelet storage, respectively. k. pneumoniae tested positive after - hours of culture when sampled on day of platelet storage for both and cfu per product. at cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after days of platelet storage. the anaerobic cultures of p. acnes became positive after hours of culture when sampled on day of platelet storage. of the strains studied, only s. aureus consistently tested positive after - hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire -day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv- and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from to showed a significant decrease from / and / before nat implementation to / and / after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is / , five times higher than hiv one and times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples / , / , and / , in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of days, ultrio assay is projected to close the wp by an average of days on undiluted samples, days in minipools of samples, days in minipools of samples and only days in minipools of samples. the projected yield calculated on the basis of . million donations collected per year in france, would be . unit per year for minipool-nat and to units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: of clinical samples ( . %) typed identically with this system and cat, and of the discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average genome equivalents(geq)/ml early in pregnancy to about geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t / being only minutes. in we have shown that this cell free fetal dna could be used for rhd genotyping. in the last years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over %, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only - % of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. ( ) transfusion ( ), ]. this system utilized low-speed centrifugation of -well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for . % of samples, compared to . % for cat (p > . ). when samples were tested for unexpected alloantibodies, fc determined the correct result for . % of samples, as compared to . % for cat (p > . ). this novel method was better than cat at detecting weak anti-a (p < . ) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (< bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = . ) but a better correlation was observed with the langmuir equation (mean r † = . ). in maternal sera, the mean ka of anti-d was . ¥ to the m- (from . to ¥ to the m- ). in the sera from immunized donors, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ) and in lots of anti-d ig, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ). the comparison of anti-d affinity measured in cases of hdn in which infants presented a fetal anemia and in cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was . ¥ to the m- whereas in the cases of postnatal anemia the mean value of ka was . ¥ to the m- . conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa- alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa- alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, ; anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, and anti-hpa- b, ). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges . - . ). interestingly, we used lyophilized platelet with a high expression of cd bearing the hpa- system and we have detected anti hpa antibodies among sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa- system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between and g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between and g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for cases of cardiac bypass surgery. in addition, exclusive use of fuwb for burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa- transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention . the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from centres ( patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < . ) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < . ). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults ( ml/kg in premature; ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < ¥ cells/l if bleeding is observed; (b) a plt count < ¥ cells/l without bleeding; (c) a plt count < ¥ cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in children ( premature infants and at term newborns). the weight of premature infants ranged from - g and at term newborn from - g. gestation age of premature infants ranged from - weeks and of at term ones, of course, from - weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of - ml/kg, with a plt count < ¥ cells/l. results : in the study period, plt transfusions have been performed: children have been only transfused one time, while multiple plt transfusions (ranged - ) needed for children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome ( cases) , haemorrhagic syndrome without sepsis ( cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome ( cases). after hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all little patients. the highest increase in plt count was ¥ cells/l, while the lowest ¥ cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. % of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies fusion society of turkey (bbtst) in with contribution of blood transfusion centers. according to these figures % of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the blood banks performing apheresis, were university hospital blood banks. another blood banks were producing both productive and therapeutic, produce only productive and produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only centers reported to prepare random platelets as well. each center had apheresis machines between - . a total of centers was carrying out around < procedures, around - , at centers about - , at a further centers around - procedures a year (one center was excluded). of the responders to the survey , all procedure were done at blood banks, whereas at of them all were carried out by the hematology clinics. at other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: centers < , centers - , centers - and centers > units have donations a year. around % of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are % lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the hospital based transfusion services, which are financed within the budget of the whole hospital. the hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to % ( blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population ( in the north, in the central and in the south region). each sub region will have one standard mobile blood collection team to collect blood units daily, i.e. annually. the blood units per teams provide the blood units ( %) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p- years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in . since and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in , / . codex of voluntary blood donation and health care staff has also been established; blood donors donates blood annually. in the past years, there was over million blood donations, performed in accordance with who regulations. over transfusion medicine specialists and technicians specially trained for the work in blood transfusion service (n = ), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with . million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from . now, they have been regulated by tender. in , test to lues was introduced, to hbsag in , to a-hiv in and to a-hcv in . information system was introduced in . nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso implementation. in the current reform of transfusiology system we are aiming for percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde . the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in in stations of blood transfusion and in blood transfusion departments at big hospitals. amount of donors in was equal to , voluntary donors being . % of them. the average number of whole blood donations in relation to the general population is per inhabitants, and on average percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is . ml per inhabitant and ml per one bed. an average volume of one blood donation is ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about . % of collected blood is used for procurement of blood components and preparations, . % of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv- / , hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is . %. % of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: % albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first . cross-matches last year (in months) were found incompatible due to dat positivity of blood donors' red cells ( . %). dat positive [( +)-( +)] samples were only igg positive in cases, only c d positive in and igm positive and c d positive in case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c c, -c d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the donors that participated in the survey ( . %) answered the questionnaire, ( . %) men and ( . %) women. that the survey was useful thought % and % that it was not. opinions were elaborated by . %. that the questionnaire should be completed before each blood donation was the opinion of . %, % thought it should be filled out only the first time blood is donated and . % that the questionnaire should not be completed at all. the answers given were sincere in . % of blood donors, % were not and . % were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years ( ) ( ) ( ) ( ) ( ) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from . . till . . . statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total donors in nitm and ( . %) deferrals. . % of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in ( . %) blood donors, use of drugs - ( . %), low blood pressure - ( . %), high blood pressure - ( . %), infections - ( . %), cardiovascular diseases - ( . %) and others. relation voluntary/family donors is almost equal ( . : . ). in the last two years the number of voluntary blood donors is increasing ( : ), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~ %, which is result of insufficient data and it is increasing in the last two years (> %). reasons for deferral are predominantly from temporary character ( . %). permanent deferrals are only ( . %), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of % macedonians, % albanians and % others (serbs, gypsies, turks). over % of blood donors are voluntary non-remunerated and ~ % are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from till . results: there were blood donations for the mentioned period. predominant blood donors are employed and high school students in %. family blood donors are ~ %; between them % are from albanian population. the ratio between blood donors macedonians vs. albanians is : . woman blood donors are presented with %. first time blood donors are %, and regular donors arẽ %. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than %. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donat- conclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype b of hepatitis c virus is the most frequent in blood donors d, from a to f, from a to k, a and a. these are differently distributed in the world: types and are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from . to % of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may to december , blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: blood donors have resulted hcv-rna positive with identification of the following genotypes: a = cases ( . %); b = ( . %); a + b = ( . %); a/ c = ( . %); = ( . %); = ( . %); none was a or a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv- a has showed a double prevalence in men ( cases, . %) respect in women ( cases, . %), while genotype b is more frequent in women ( cases, . %) than in men ( cases, . %), moreover genotypes and do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt > iu, our results show that . : donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype b has resulted the most frequent in blood donors' population, followed by type , while the others have showed a very low prevalence. the high frequency of genotype in blood donors is explained by the observation that hcv is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype is principally linked to a not transfusion transmission way; genotype is linked to old age, to female sex and to post-transfusion transmission; genotypes and are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes and are still little known because extremely rare in europe. p- kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel . the kel (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family - ( . %) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger - ( . %). motives for their donation are the following: their wish to help the ones that need blood - ( . %), concern that some day everyone can be a potential recipient of blood - ( . %), because of offered benefits - ( . %), for a friend or relative - ( . %), care for their health - ( . %), because of citizen duty - ( . %), because the others donate - ( . %), curiosity - ( . %). they want to be invited every months - (%) students, every months - ( . %), every months - ( . %) and ( the mean age of case group was / ± / and the mean weight of them was / ± / , / % was male and the mean number of blood donation was / ± / . the mean age of control group was / ± / and the mean weight of them was / ± / . / % of them was male and the mean number of blood donation was / ± / . the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < . ). the rate of vasovegal rx was higher in blood donor (p < . ) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < . ) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than - and - whole blood units per year, respectively. we present studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. mg and mg daily combined with mg ascorbic acid over months (males) or months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (> mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period - . methods: the donation of whole blood and aphaeresis platelets from donors of age from to (max. years for each class) were count for the period of five years ( ) ( ) ( ) ( ) ( ) . the percentage of the man´s donation was calculated for each target class ( ) ( ) ( ) ( ) ( ) . results please see tables and . in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from to decreased during objected years. unfavourable state of total number of births in the czech republic ( birth in republic ( birth in , birth in ) and its decreasing tendency ( birth in !) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of women from the district of limassol (the second largest urban center of cyprus) between the ages of and . using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. % of women do not donate blood and attribute their reluctance to do so to health-related problems, while about % of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to % of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively and beat per minutes and blood pressures (bp) were both in the normal range ( / and / mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to / and / mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to / mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the - data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period ( ) ( ) , . percent of persons who presented for donation were deferred; . % were temporary deferrals ( % due to laboratory test results, among others low hemoglobin, . % due to risk of acquiring a transfusiontransmissible infection) and . % were permanent ( % due to the infectious diseases markers, . % due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption : percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure ( . % of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact - % of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia ( . % of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future ( - % of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn , abbott laboratories, usa), in a blood donor setting. methods: the nbm- device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of subjects ( females, males) aged - . subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm- device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device ( donors). hb levels were considered normal when readings were equal to or > . g/dl. results: venous hb measurements ranged from . - . g/dl. the mean nbm- hb level was . ± . g/dl, only . g/dl lower than the mean hb result obtained by venous sampling, which reached . ± . g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ± . g/dl. the mean absolute error (mae) of their difference was . g/dl. when checked against the cell-dyn in the usa center, where subjects had hb of . g/dl or lower, the nbm- and hematastat devices showed comparable sensitivity results. the nbm- using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the december to march were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: donors -control group: donors). computer records were evaluated during the next years ( march to march to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure ( %), deferred for medication ( %) and colds and/or sore throats ( . ). non deferred donors were a little more likely than donors with sttds to return over the next years ( . % vs. . % pv = . ) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers - ( %), while all other ethnic groups are present with only %. the most prevalent is the group of adults aged - ( - . %), with high school education - ( . %) and mostly those who donated blood - times ( - %). conclusion: having in mind that % of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with % in relation to the total number of blood donors surveyed in the period - . this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta -microglobulin (b -mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta -microglobulin (b -mg) is a low molecular weight protein ( daltons) and found in all biological fluids it is light chain of histocompatibility class -human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class -hla antigen plays a role in the elimination of the virus. method & samples: beta -micro globulin was measured in serum drawn from hbs ag positive blood donors include ( . %) female and ( . %) male in age between - years, and healthy ( %) female and ( %) male in the same age we detected serumic b -mg by enzyme immunoassay (ela). results: our studies showed b mg level increased in ( . %) hbs ag positive donor that was significant differences with healthy control (p = . ). conclusions: it seems that serum b mg is a good marker for hbs ag replication. the role of b mg in monitoring of response therapy needs to be more evaluated. and ( . %) were contributed by vd, rd and dd respectively. over the last / years, voluntary donations have shown a rising trend from . % to . %, where as rd ( . % to . %) and dd ( . % to . %) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd ( . % vs. . % vs. . %) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable ( . % vs. . % and . % vs. . %). hiv antibodies was found more frequently in vd as compared to dd [ . % vs. . % (p < . )] whereas, vdrl reactivity was lower in formal as compared to latter [ . % vs. . % (p < . )]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between and . results: during this period people volunteered to donate blood, out of which were allowed to donate blood, while were declined. out of the total number of blood donors were male and female donors. the reasons for declining potential donors were the following: . % had low levels of hb, . % were taking antibiotics, . % were ill, . % had low blood pressure, . % had high blood pressure, . % for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in and , but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. . the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. . the lack of motivation of the studied lot is another cause. the questionnaire was applied on a lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to , which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vÖrÖs*, c bercsÉnyi † , o petrÓ † , r jÁger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the . . in hungary. as the consequence of reorganization till . . , blood banks had been established instead of existing before, under direction of the hhnbts. the working profile of the regional blood centers and local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about inhabitants and hospital beds). local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually donors donate blood in this region. this donation activity covers about the % of all inhabitants. the acceptance ratio of the donors is good ( - % of the donors were deferred). there are hospitals in our region. the regional demand on rbcc is - . u/year, on ffp is . - . u/year and on pc is - . u/year. the poster shows the donor data and the details of blood application of this region since . p- implementation of rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pÉlissier and jl beaumont efs ile de france, paris, france background: single donor rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for rbc collection (weight ≥ kg, height ≥ cm, hb ≥ . g/dl, ferritin ≥ ng/ml for repeat rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the recruited donors, all donors were male, % were regular whole blood donors, % were regular whole blood or apheresis donors and % were new donors. the medical staff was trained on rbc collection with the sdr protocol and disposable set ln pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as to months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table . six percent of the donors had a ferritin level below ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: hematomas and citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of min). the results of the survey were very favorable as more than % of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: ( ) possible risk of infectious disease in seronegative study; ( ) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than % blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in (c = . ; p < . ) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs + apparatus were used for platelet apheresis. apheresis were performed between / / and / / . including criteria of donors are that estimated process time is smaller than minute and estimated postapheresis platelet count is higher than ¥ /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls ( procedures on patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count > ¥ e /l or blast count > ¥ e /l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was . %. tp-treatments ( procedures on patients with symptomatic thrombocythemia and/or platelet count higher than ¥ e /l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction ( . % in average) and with clearly noted clinical improvements, subsequently. tes ( procedures on patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from . % till . %. rbcx treatment ( procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with % albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of collections (range - ). the inlet blood flow ranged between . and . ml/min (median . ml/min). the median blood volume processed was ml (range - ). leukapheresis lasted a median of min (range - ). the median total nucleated cell yield was . ¥ e /kg (range . - . ), mononuclear cell (mnc) yield was . ¥ e /kg (range . - . ) and cd + cell yield was . ¥ e /kg (range . - . ). the median of mnc collection efficiencies was . % (range . - . ). in ( . %) patients, in only one apheresis procedure more than ¥ e cd + cell/kg were collected. during ( . %) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than ¥ e /l in cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately / pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a -year-old pregnant woman, alloimmunized to the hpa- a (p a , zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt . /mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was . ml in a short time procedure ( minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected ( . ¥ e ) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing . ¥ e each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from . /mmc to . /mmc and after a week the child had plt . /mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to - minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm ( - seconds), thrombin time ( - seconds), the dilute fibrin monomer complex ( - mg%), coagulation time ( - min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was - % of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of - procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: pts ( females, male), median age . years ( - ), affected with agvhd grade ii ( ) and extensive cgvhd ( ) gtx with median total granulocyte doses of ( - ) ¥ per gtx corresponding to . ¥ granulocytes/kg in children and . ¥ granulocytes/kg in adults. the wbc counts increased from baseline values of . ( - . ¥ ) g/l for both pediatric and adult patients to peak values of . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at one hour after gtx and to . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at hours after gtx. in out of patients ( %), the crp levels significantly declined ( ( - )%; p£ . ) during the granulocyte transfusion period; in almost all cases ( / ; %) after the initial or nd transfusion. thirty-eight patients ( %) were alive at day + after termination of neutropenia and gtx. patients without crp response to gtx ( / , %) and patients with severe viral infections / ( %) were not among the day + survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: of them with mcs p and with mcsplus (haemonetics), according to automatic standard protocols a p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was : ). most of the donors ( % men and % women) were patient´s relatives. half-hour before the initiation of the procedure, mg of calcium ( tablet cal-c-vita) were administered to each donor. the mean platelet yield was . e /unit. the overall rate of the donor related adverse events was . %. feeling faint was the most frequent event, which was occurred in . % of donations. hypotension and citrate related rates were . % and . %, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was . %. other adverse events were venipuncture related ( . %), machine related ( . %) and miscellaneous complications ( . %). ( ) plateletpheresis using the mcs p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. ( ) with the used acd-a-to-blood ratio ( : ) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. ( ) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis . from the donors immediately with the initiation of the procedure (citrated whole blood) and . from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap- c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs p device (p = . ). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a year old pregnant woman in the th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially mg/kg bw prednisolone for weeks and subsequently mg/kg bw for weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table ) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was . ml per apheresis. after day plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. again lead to a recovery of the patient which held on until day . conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: procedures were carried out with mnc program and procedures with autopbsc on patients with cgvhd. both hemoglobin increased from . ± . to ± mg/tu, k+ from . ± . to . ± . mmol/l, level of glucose decreased from . ± . to . ± . to . ± . mmol/l, ldh from . ± . to . ± . ukat/, lactate from . ± . to . ± . mmol/l, ph from . ± . to . . . the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was . ± . ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage and , in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/ - . background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the . to -fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in cases with bone marrow volume over ml, and cases with bone marrow volume under ml. fifteen of these cases were allogeneic, and were autologous procedures; a software uploaded with cobe pbsc coll vers . and (catalog no: - - ) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: - - ). results: the mean volume reduction was . % ( . - . ) for volumes over ml, and . % ( . - . ) for volumes less than ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd + cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of donors who donated platelets by apheresis procedure from january till april . procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than ¥ /l and the volume of the processed blood was not less than ml. according to histogram, the most frequent donors' initial platelet value ranged from ¥ /l to ¥ /l ( %). final concentration of the samples of tested donors ranged from . ¥ to . ¥ in the average volume of ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < . . leukocyte contamination of the final concentrate prepared without the filter ranged from . ¥ /l to . ¥ /l. presence of red blood cells in the final concentrate ranged from . ¥ /l to . ¥ /l. p- therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of diseases into categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in croatian hospitals in the period - , respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in patients suffering from various diseases. in ( %) patients ta was performed by membrane filtration, while in ( %) separation by centrifugation was used (table ) . according to the ctg, s of the aabb/asfa guidelines, ta was performed in ( %) diseases from ctg i, ( %) ctg ii, ( %) ctg iii, and ( %) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis ( %), collection of pbpcs ( %), sy. guillain-barré ( %), and plasmacytoma ( %). in ctg ii frequent indications were: poisonings ( %), systemic lupus erythematodes ( %), and rapidly progressing glomerulonephritis ( %), and in ctgs iii and iv: cytoreduction-polycythaemia ( %), thyroid storm ( %), gvhd ( %), and reumatoid arthritis ( %). ( ) the time spent for resolving h / , ( ) mtp / , ( ) discarded blood units / . iii group: wrong data input / , donor replacement / , marking errors / , error in determining blood group at the first blood taking / , errors in input medical consulting / and disregard of prohibitions / . the consequences are: ( ) the time spent for resolving h / , ( ) mtp . / . , ( ) discarded blood units / . conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso parameters (soon to be updated with iso ). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p- software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from to more than . indeed, bacterial detection is performed for pools of to platelet samples and to red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt . in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of different reports can be generated on the website as a pdf file. for this study, reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or , -dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) -phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis nd. edition. academic press, new york ; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from ml to a total volume of ml and tests were performed in microtiterplates. results: samples were analysed in hplc and modificated commercial testkits, another samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range - mm atp) and statistical analysis demonstrated a given linearity (r = . ). variability has been calculated as . % (intraassay; n = ) and . % (inter-assay; n = ). the hand-on-time calculated for samples has been decreased from . hours to minutes. at least the costs of atp-determination have been reduced from € . to € . per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: ( ) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. ( ) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about whole blood units were collected and processed by mda national blood services in [ ] [ ] [ ] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some different types of dm were defined. out of reports sent to the corresponding manufacturers for investigation, responses ( %) were received and analyzed. about % of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly % of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table analysis and characteristics of t ( ) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. ( ) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. ( ) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. ( ) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in / / and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso : standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of . million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of %. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of %. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of %, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min . %-max %). but because with vista any procedure brings between . and . products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between € and € (mean value €) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx- filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of units of red cells, measurements of volume, platelet concentration and platelet count/pool of ld platelet pools produced by a team of technologists and of ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by . % of participants. the remaining . % of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in . % and . % of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. % of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso -quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on / iso -quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into levels; -most severe, potentially harmful to patient. -severe, damage to process and result. -moderatly severe, damage to process only. -benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years - , events were detected and recorded in the blood bank, they comprised . % of all tests performed ( ). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level & , whereas technical events were mainly of severity levels & . analysis of event recurrence in the quality system revealed that % of events were resolved, whereas only %- % of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of . %. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: . administrative for defining the it security organization, the information security policy, and information security awareness and training. . physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. . technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: < . ¥ /l, leukocytes: < . ¥ /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv- group m arna, hiv- group o arna, hiv- arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv- group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing % of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives / /ec and / /ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since . blood samples are classified according to adr in division . under un as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. . of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso : standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso : standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and , dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on erythrocyte suspension (es) samples during days of storage at oc on days , , , and . for whole blood (wb) samples during days of storage, same parameters were measured on days , , and . the measurement of the complex impedance of blood samples were performed in the frequency range from khz to mhz. by using the a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and , dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and , dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and , dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that , dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of plateletpheresis procedures performed using two cell separators (cs baxter, usa, mcs p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs was . ± . ¥ and in mcs p, it was . ± . ¥ per unit, however, only - % of sdps showed wbc levels < ¥ . six of donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in , the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in , centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = . ), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured times in order to determine within-run imprecision. coefficient of variation (cv) calculated from consecutive measurements was . %. to investigate between-day imprecision of the hemocue method one sample was divided in aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was . %. during the period of validation measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were . % (low), . % (medium) and . % (high). in order to investigate possibility of batch testing, supernatants of different rbc products were divided in aliquots and measured periodically during -month period. cvs calculated from measurements of each sample were in range . - . %. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a . mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer ( wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the -year period ( - ) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w ' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in . purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is years, of which at least are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: . organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. . collection, processing, storage and transport of blood and blood components . laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) . clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is -year specialization in transfusion medicine, established years ago (over specialists finished this specialization till now). we have postgraduate studies in tm started years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of theoretical and practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory -month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished -mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at - % of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of - %). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than time in - years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost % from the general number of patients of hospitals with positive results at % from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to , whole blood was comprising the majority of transfused units (more then %) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized symposiums, national courses, national and international congresses since . due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to %. in most of the university hospitals and training hospitals this is around %. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was ± and postoperative ± mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived ( ± ) and survived patients ( ± ) was not significant (p = . ). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il- and anti-inflammatory cytokine il- in relation to clinical outcome and complications after cardiac surgery. methods: in participating patients blood samples were taken before and after surgery. using elisa il- and il- were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il- and il- were low. mean postoperative level for il- was ± and for il- ± . compared with patients without mods and without infections and survived patients only the il- was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il- were higher in patients receiving more then units blood transfusions compared to transfusions. conclusion: there is an association between mods and mortality and il- , not with il- . ld has no influence on mean levels of il- and il- . there is a correlation between transfusion of more then units and il- , not with il- . these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july to december . patients were enrolled within h of icu admission. follow-up time was days, hospital discharge or death, whichever occurred first. results: a total of patients ( male, female, mean age . ± . years, range - ) were included in the study. the mean hemoglobin (hb) level at baseline was . ± . , which level was descending during the study. overall . % ( / ) of the patients received one or more rbc units while in the icu stay (mean . ± . units per patients). the mean pretransfusion hb was . ± . g/dl and the mean time to first icu transfusion was . ± . days. more rbc transfusions were given in the first week of the icu stay ( units vs , , units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = . , p < . and r = . , p < . respectively). baseline hb level was significantly related to the number of rbc transfusion (r = . , p < . ), but was not an independent predictor risk factor of length of stay or mortality (r = . , p > . and r = . , p > . respectively). the mean baseline apache ii and saps scores were . ± . and . ± . respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of < g/dl ( . ± . vs . ± . and . ± . vs . ± . respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < . ). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population ( . mill) the trauma team performed initial assessment and resuscitation of patients, % males, during the first six months of . the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: units of erythrocytes (er), of thrombocytes (thr) (buffy coat preparations from donors) and of s/d-treated whole plasma (op) (octaplasÒ) were transfused in uuh during this period. . % of er were given to surgical, . % to medical and . % to gynaecological and obstetric patients. % of er were given to patients above years. er units were given to patients (mean . units/patient; range - ). mean age of trauma patients was ± , median , range - years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb < g/dl, irrespective of age and sex. eighty-eight patients ( . %) received er, of them as massive transfusion ( er units in < hours) ( . %), of these died ( %). altogether trauma patients received units of er ( . % of total uuh consumption), . thr ( . % of total) and units of op ( . % of total). massive transfusion consumed er ( %), . thr ( . %) and op ( . %) units. fourteen adult trauma patients ( . % of those transfused) received or er units only. lowest pre-transfusion hgb was . - . g/dl, median . , mean . ± ; highest post-transfusion hgb . - . g/dl, median . , mean . ± . five patients received er transfusion at higher pretransfusion hgb level than g/dl, but in er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for > days, while only % were issued before days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year . in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of - mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved health institutions across the country. as many as deliveries and abortions were reported in . the institutions reported the usage of doses of mg rhd gamma globulin or doses/ deliveries + abortions. we compared our data with those obtained from similar surveys conducted in , i.e. before the issuance of the guidelines. in that year deliveries and abortions were reported, and doses of rhd gamma globulin or doses/ deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of - beds, general hospitals with the capacity of - beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/ deliveries + abortions in the first category was - with the median being ; in the second category there were - doses with the median being , in the third category - doses with the median being , while in the fourth category the range was - doses with the median of doses. the number of pregnancies which should have been protected with rhd immunization in year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = . ¥ . = . . if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in , doses of rhd gamma globulin or mg/ deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in , the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in a study was carried out regarding the usage of plasma derivates. as many as health institutions took part having the capacity of acute beds out of a total of . the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of - beds and general hospitals with the capacity of - beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in . the consumption of albumin was . kg or kg/million inhabitants in and consumption per bed for the first category institutions was in range . - . kg with the median being . , for the second category the range was . - . kg with the median being . , while for the third category the range was . - . kg with the median being . . in in croatia the consumption of albumin was . kg or kg/million inhabitants. the consumption of i.v. gamma globulin was . kg or . kg/million inhabitants in and the consumption per bed for the first category institutions was in range . g- . g. with the median being . , for the second category was in range . - . g. with the median of . and for the third category was in range . - . with the median . g. in in croatia the consumption of i.v. gamma globulin was . kg or . /million inhabitants. the consumption of the concentrate of factor viii was . iu or . iu per inhabitant in . . iu, i.e. . %, was a recombinant factor viii. in the consumption of factor viii was . iu or . iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased . times and the use of the concentrate of factor viii increased . times if compared with the results in . self-sufficiency has not been reached in plasma derivates in croatia we needed l plasma for gamma globulin and l plasma for concentrate of factor viii for the level of usage of these plasma derivates in . significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than % following transfusion with rhd+ rbc's and as many as % in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital ( beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby . rhd-patients, from to , were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the . rhd-patients we found out identified anti-d, % ( ) detected before any transfusion in our hospital. of the left, nine were excluded because no information was available during some years. had history of pregnancy related to the immunization. patients were exposed to rhd+ blood components: were transfused only with rhd+ platelets (including two women of childbearing age); with rhd + rbc's. the remaining had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period ( ) ( ) ( ) ( ) ( ) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total hospitalized patients who have been analyzed, ( . %) received wb and bp, and there have been transfused units wb and bp. every patient, on an average, has been transfused with . units wb and bp. at the same time ( . %) units have been transfused as a wb, ( . %) have been transfused as red blood cells (rbcs) and ( . %) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during - and to compare them with other studies. one hundred sixty five ( ) unselected patients of whom ( %) were women and ( %) were men with a mean age of years (range - years) were studied retrospectively. seventy six ( %) patients had intracapsular fracture and ( %) intratrochanteric fracture. the mean hb concentration on admission was . g/dl (range . - g/dl) and the mean hb concentration at discharge was . g/dl (range . - . g/dl). a total of units of rbc were transfused (a mean of . units per patient). blood transfusion occurred in patients ( . %), ( - % in other studies) with a mean of . units per patient (range - units), ( . - . in other studies). patients with preoperative hb values < g/dl were transfused more often than those with hb values > g/dl ( % versus %) p: . . women were transfused more often men ( . % versus . %) p: . . patients aged > years were transfused more often than those aged < years ( % versus %) p: . . finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures ( . % versus %) p: . . conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb < g/dl, in patients older than years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of . we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine %, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) . % and trauma/surgery . %, cancer . %. summary/conclusions: the use of ffp corresponds to . % of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from to in our hospital. results: our data are presented in table . conclusion: after , wb usage rates systematically decreased from % to . %. however, in the period of - , the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about % of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since . background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® pre and post a platelet transfusion (n = ) and was compared with healthy controls (n = ). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = ). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < . ). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years ( ) ( ) ( ) , units of wb were collected, among them ( . %) units were collected for pre-storage filtration and in ( . %) of these qc was done. within hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at g for minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume ± . ml ( % corresponds with the guidelines of council of europe), hematocrit . ± . ( %), hemoglobin . ± . g/unit ( %), number of residual leukocytes . ± . ¥ ( %), hemolysis . ± . % ( %), the test of sterility was % negative. during filtration of wb, wbc were removed in approximately . %, the duration of filtration was ± minutes and the loss of hemoglobin was . ± . %. the pre-storage filtration of wb is highly efficient, . % of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from , when the cardiosurgery department was founded in our hospital, to . the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from to were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs - and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about - . % were produced with the use od these filters, and platelets from - % with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts ( ¥ cabg and/or valve; ¥ re-cabg and/or valve; ¥ valve with or without cabg). oncologic surgery patients were also included in three rcts ( ¥ colorectal cancer; ¥ gi-oncology or vascular surgery; ¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in or more of the rcts. multivariate analyses were performed on: in-hospital mortality, -day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from surgery patients were collected ( onco; cardiac; vascular; orthopedic, other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = . ) and at days post-surgery (p = . ). no association between randomization and stay in hospital (p = . ) or stay on icu (p = . ) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = . ), and 'hospital' to be far stronger associated (p < . ). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = . ) and the trial arm became more strongly associated with infections (p = . ). in the analyses of surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = ) and portugal (n = ). in november a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was % (uk) and % (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by % in wales (n = ) and % in england (n = ), but only % in scotland (n = ), % in northern ireland (n = ) and % in eire (n = ). in mainland europe, variation was also observed: % in denmark (n = ), % in portugal (n = ) and % in other countries (n = from countries). fewer laboratories selected c-blood for pmfs: % in england and northern ireland, % in scotland and eire, rising to % in wales. mainland europe showed similar variation: % in denmark, % in portugal (all ccee matched) and % in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged < years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a year old female (with pre-existing antibodies other than anti-k), from % ( ), % ( ) to % in . in this survey, k + blood was selected for a female aged (no antibodies) by %, whilst % selected a k + unit for a female aged (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the and year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data ( ) suggested that > % laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec- ) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c fresh showed ± and after hours storage ± % expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib- ), low (gpib- ) and in-between (gpib- ). the binding and phagocytosis of plt showed an increase of ± % which implicates an indirect and negative relation to gpib- , and direct relation to gpib- expression. anti-human pselectin (cd p) delayed ± % the binding and annexing v ± % the phagocytosis of stored plts, after -hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a -year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured g/l. he received ml . % nacl and ml ringer solutions. after that, he was transfused with units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for hours. two days after the patient was operated. after surgery he was transfused with units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for minutes at °c with fmlp, lps or pma. monoclonal antibodies against hna a and hla-i, or patient serum were incubated with whole blood for min. surface cd b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna a monoclonal antibodies induced neutrophil activation, which were also inhibited by p map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: out of transfusion reactions were febrile, were anaphylactic, were due to circulatory overload, out of transfusion reactions concerned the transfusion of incorrect blood component. out of transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)- beta, il- and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il- has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il- from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from blood donors and frozen at - °c. upon thawing, the level of the il- beta, il- and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il- beta was at the test detection limit in all donor plasma samples. in ( . %) bd, the level of il- was . pg/ml, exceeding the test sensitivity limit of . pg/ml. in ( . %) bd, the level of tnf-alpha was within the range of - pg/ml, with a test sensitivity limit of pg/ml. tnf-alpha levels > pg/ml were measured in plasma samples of ( . %) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il- and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao = mmhg, o saturation = %), hypotension (bp = / mmhg) and fever ( °c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp ( cm h o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for hours the patient improved significantly, was extubated and returned to the surgical unit on the th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a -year old female with a history of multiple abortions whereas the prc unit was from a -year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years - there were . blood components issued in slovenia and atrs were reported ( in blood components issued), in of them the severity grade was ( in . blood components issued). in the year there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from in to atr in blood components issued. . % of all atr were not classified. conclusions: although the number of reported atrs was increased by % and % a year in and respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, . years old, with average weight of kg were used. the animals were dewormed by albendazole ( my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. ( ) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed ( . %) out of ( . %) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa- a in a homozygous hpa- b patient. in a minority of cases the offending antibodies were directed against hpa- b, - a, - b, a, - a, - b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a -year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a -year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a -year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa- a, anti hpa- b and anti hpa- b, respectively. the platelets genotype of the first patient was hpa- b/ b, of the second hpa- a/ a and of the third patient, hpa- a/ a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in . the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: ( ) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. ( ) to use the information acquired to determine the morbidity of transfusion. ( ) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. ( ) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: ( ) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. ( ) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. ( ) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: ( ) the total number of reported cases has steadily increased since the introduction of the programme. ( ) the number of participating healthcare institutions has also increased to % (n = ). please refer to table entitled 'summary of the haemovigilance report - ' . ( ) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. ( ) there has been very good participation from the participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. ( ) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing ( %) were rated for seriousness. of these, ( . %) were rated as grade (moderate to serious morbidity) or worse. ( . %) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in ( . %) and as 'possible' in ( . %). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use ( . per the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested > months following the implicated unit. results: between between - suspected pth cases were reported: ( %) were pth-b, were pth-c ( %) and in patient both hbv and hcv infections were reported. investigation was completed in / ( %) cases, with % of the involved donors ( / ) being retested > months after the implicated donation. hbsag was not detected in any of the retested donors. anti-hbc was detected in donors involved in pth-b cases of which were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 'anti-hbc+ only' donors, and none was found positive. only in / donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in . in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in % ( / ) of the cases where the patient received up to blood components. conclusion: in the past years an average of cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in % of the reported cases. so far, there was no clear evidence of hbv transmission. in cases (out of . million blood units collected nationwide during - ) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. - . , p < . ). the seroprevalence for a-hiv in the bd population was . % (se: . ; ci: . - . ; p < . ) whereas in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-hiv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was . %. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was . , which indicates that the number of bd a-hiv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was . % (se: . ; ci: . - . , p < . ), in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-htlv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was . %. the rp for a-hbc as regards a-htlv was . , which indicates that the number of bd a-htlv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. . despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. . our data show that the real anti-hcv prevalence of our donor population is very low ( . %). . the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. . given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. . donor re-entry in the pool of donors is an issue for further discussion. . the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: . to recognize the epidemiology of hepatitis c and how it differs geographically; . to investigate the risk factors for presence of anti-hcv antibody in blood donors; . to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january , using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was . % out of blood donations. the average prevalence of hcv infection by riba was . %, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was , with cases in males and cases in females. most infections were found among older persons ( % were aged - , and % aged - ). the seropositivity was higher in family/replacement donors ( %) than in volunteers ( %). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion %, hospitalization %, immigrants %, occupational %, sexual transmission %, injection drug use %, household contacts %, other %, tattooing %, unknown %. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested sexual partners and family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using % trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in ) the risk of bacterial contamination, ) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and ) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year . results: in the attached table the estimated risk reduction through % trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by %, the risk of known viral infections by %, and transfusion acquired bacterial infections by %. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly ( : ) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table shows the distribution of the blood donors in different professions. administrative clerks with ( . %) and the workers with ( . %) registered in the group of hbsag(+) blood donors, as well as workers with ( %) and administrative clerks with ( %) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = and p > . there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = . and p < . shows a significant difference, where the workers with ( . %) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table . in the group of hbsag(+) blood donors the number of those who work in production profession- ( . %), is dominant over the number of those that has non-production profession- ( . %). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c = . and p < . there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from confirmed hbsag(+) blood donors and confirmed hbsag(-) blood donors as control group. results: the table shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group ( %) donors that live in a flat dominate compared to ( %) of those that live in a house. having in consideration the presented distribution (table ) for c = . and p < . there is a significant difference, that comes from the bigger number of blood donors ( ) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table ) , we can show that ( . %) of the hbsag(+) donors have less than m living space, in compared to ( %) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between and m , and between and m of living space per person, obviously are not very big. for u = and p > . there is no significant difference in the number of the donors in the two groups, when the available living space in m is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in from cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table shows the distribution of blood donors concerning the use of condoms. table : in the group of hbsag(+) blood donors those who have not used condoms dominate with number of ( . %), in compared to those ( . %) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom- ( %), compared to ( %) who have not. the given distribution of blood donors concerning the use of condoms for c = . and p < . , shows significant difference, which is due to the prevailing of the number of blood donors ( ), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the th- st day and th- th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of - °c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, specimens ( . %) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were . %, . %, and %, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and of them were found positive for one of the three viruses (hiv- , hcv, hbv). with the elisa/axsym assay of the blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those blood units units were given for transfusion following our blood centre protocol and the remaining units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those that were found positive by the procleix ultrio assay was positive for anti-hcv and were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day or and, in case of negative result, prolongation of their storage time to days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to days at to °c with end-over-end agitation. other plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day , and . both aerobic and anaerobic culture bottles were inoculated with a -ml platelet sample. culture bottles were incubated at °c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of plt concentrates were cultured and bacterial contamination was assessed in each unit at day , and after collection. on of storage day two out of ( . %) plt units were found to be positive for bacterial growth. cases of unconfirmed positive results were noted at the beginning of the study. out of the other units which were negative on day and continued to be cultured for the next days, the assessment at day found no other positive. after further storage, at day , defined as the end of the prolonged incubation period, out of the plt concentrates ( . %) grew bacteria although testing of the same units on day and gave no signal. from the platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to days. bacterial contamination testing on day and a storage time of maximum days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from - were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of platelet products were tested during the year period. thirty ( . %) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in ( . %) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with - units of concentrated red cells, depending on their problem. a total units were delivered from the beginning of their problem until december . the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: patients anti-hbc (+) and anti-hbs (+). patients anti-hbc (-) and Ánti-hbs (-) patients anti-hbc (-) and anti-hbs (+) patient anti-hbc (+) and anti-hbs (-) patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is % safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables and ) . for hiv and hcv the specificity was . %. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in % plasma and % ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: - hour after donation, about ml of pc were sampled into the ebds collection pouch and then incubated at °c under continuous agitation (incubator helmer ) for hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso ). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency and run control panels. ( . %) out of samples were positive for anti-hbc. ( . %) out of anti-hbc positive samples were hbsab positive, and ( . %) were hbsab negative. all of samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from . ng/ml bbi to . - . ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than . and . ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in patients referred during the aug. to dec. . all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. ( . %) were hbv dna positive and ( . %) were negative. ( . %) out of patients had evidence of delta infection and ( . %) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in out of hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in out of hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that . % of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker ( . %) than those with hbv dna (pcr) positivity ( . %). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested sera samples from the general population and 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv- , hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b (pb ) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb and hav in portuguese blood donors. we gather, during four months, plasma donations and joined them into pools, with no more than donations each. [ ] [ ] [ ] [ ] [ ] in voluntary donors the anti-hcv prevalence ranged from . % to . %, in family replacement donors from . % to . %, autologous donors from % to . %. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is . % and women . % (p = . ). over the year periods the prevalence in men has a decline tendency ( . % to . %; p = . ) and increasing tendency in women ( . % to . %; p = . ). according to age group the anti-hcv is . % in - age group, . % in - age group, . % in - age group, . % in - age group (p = . ). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [ ] [ ] [ ] [ ] [ ] . a total ftd and ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the -year overall hbsag prevalence among first time blood donors was . %. and ad . %. among autologous blood donors was observed a decreasing hbv prevalence from . % to . % in . according to age the prevalence was higher in - year group . %, while according to sex was higher in man ( . %) than female . % (p < . ). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from - hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. ( : ) . the hbv dna-emia in hbsag negative samples was . ¥ - . ¥ copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high ( . %) therefore the decision to introduce routine hbv nat screening is justified. ( / ) with stored apheresis and whole blood derived platelet concentrates. of these failed results there were confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing / . the bacteria detected were staphylococcus or streptococcus sp. of the fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing . % or / , and were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing / . there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of / , and with a low false positive rate (< . %). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only . % of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot . (genelab diagnostics) results: results from a total of screened donors were analysed. hepatitis b surface antigen rates was . %; anti-hcv seropositivity was . %; anti-hiv seropositivity was . % and tpha seropositivity was . %. one study calculated this risk to be one in for hbv, one in for hiv and one in for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in - was . %. but it had decreased to approximately . % in . anti-hcv positivity has been reported to be . % between and . but it was approximately . % in . rpr positivity in blood donors in turkey was reported to be < . % in and . % in . in , the rpr rates was . %. in our study these rates are . %, . %, . % and . % respectively. anti-hiv seropositivity was found around . introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn , tpn and tpn ) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be %, after testing specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over titer by tppa). the specificity generated from testing hospitalised patients previously screened as tppa negative, was . %. testing a mixture of sera and plasma from random donor specimens, generated donor specificity figures of . %. the precision (cv%) with a positive control was . % ( % confidence interval: . - . %) by the standard -day nccls analysis (ep a ). in a study conducted at asahikawa medical college hospital, in which, positive and negative specimens were tested, concordance with fujirebio tppa was determined to be %. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of - days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns , ns ) and a sandwich test for core antigen detection. results are available within . hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of days earlier than the monolisa hcv plus v test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around . days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products ( ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools ( bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was . ¥ e /pool with mean leucocyte count . ¥ e /pool. ebds installation and training occurred over a day period. platelet pools were tested for bacterial contamination over the subsequent weeks. ebds pouches were sterile connected onto platelet pools hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for hours on a shaking agitator at °c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, technician was able to make oxygen measurements in less than minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. ( ) was . the percentage collected from volunteer blood donors was % (n = ) and the rest % (n = ) was given from patient-related donors. the age of donors ranged from to years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of tested units, of them were found to be positive to at least one marker of hbv infection, that means the . % of the health adult population was infected in the past by the hbv. the . % (n = ) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and . % (n = ) were chronic carriers of the hbv with hbsag(+). the . % (n = ) of the positive donors were patient related donors and . % (n = ) were volunteer donors. in other words, of the not volunteers ( . %) and of the volunteers ( . %) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions ( . %) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, . % compared with the . % of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type , hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv- , hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv- (n = ), hcv (n = ), and hbv (n = ) seroconversion panels were tested neat and diluted ( : and : ) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv- , hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p- effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv- /hcv and hiv- /hcv/hbv (procleix ultrio) assays for the detection of hiv- rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, out of ( % of the positive, . % of total) were reactive for hcv rna only and out of ( % of the positive, . % of total) were reactive for hiv- rna only. none were positive for both hiv- and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv- /hcv assay. of the samples tested by the ultrio assay, ( . %) tested reactive for hiv- /hcv/hbv. in discriminatory assay testing, out of ( . % of the positive, . % of total) was reactive for hiv- rna, out of ( % of the positive, . % of total) were reactive for hcv rna, and out of ( . % of the positive, . % of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than virus. three out of positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in ), and hcv testing (introduced in ). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of patients tested, cases ( %) were hbsag positive, cases ( %) were anti-hcv positive, and cases ( %) were anti-hdv positive. co-infection of hbsag and hcv was found in cases ( %), whereas co-infection of hcv, hdv, and hbv was found in persons ( %). the highest rates of infections and coinfections were found in patients above years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: / ( . %) samples from rbd were anti hiv + nonreactive and rr for p ag both being nonreactive in the neutralization test, they were interpreted as false positives. / ( . %) sample from fbd was rr for p ag/anti hiv + nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion days later: p ag nonreactive, anti hiv + reactive and western blot positive. the only bd p ag positive/anti hiv + nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~ days), this suggests that some - infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in , the general incidence in rbd has dropped from . ‰ to . ‰, suggestive of a : escape rate. the prevalence in ftbd has stabilized at ± ‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that potentially infectious donation in rbd escapes the screening (= to a total of aprox. , annually). a limited investigation using hcv-antigen eia evidenced a ‰ escape rate in ftbds (= to a total of aprox. , annually table and are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than hours. background: the hbsag, anti-hcv, anti-hiv / , p antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: . hcv rna pcr testing in anti-hcv negative blood donations; . correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of samples. pools of ml were tested using the cobas ampliscreen hcv test v. . (roche). results: pools of samples from a-hcv serologically negative donations were tested from october to july . no one pool was initially reactive. invalid tests: ( . %) run failures were observed, due to: invalid internal controls ( . %) and invalid positive controls ( . %). invalid tests were repeated. in none of pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b , such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b by nat since april . methods: over the last years . million donations were screened for b by nat. samples with a virus load over iu/ml were defined as positive, whereas samples with a virus load between the detection limit ( iu/ml) and iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of b positive donors (case group) were determined over a time period of one year. virus load and b antibody status was compared with b negative donors (randomised control group). b antibodies (igg vp , igm vp , ns ) were analysed by two commercial antibody tests. results: overall b nat-positive donors were identified with a virus load over iu/ml out of . million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp igg was detected in . % and % of the case and control group, respectively (p = . ). these data demonstrated statistically significance (p = . ). all donor samples which were b nat positive for more than three months developed neutralizing vp antibodies. in contrast, ns antibodies were observed in % of the case group and in % of the control group (p < . ). ns antibodies were detected more frequently in samples, which were b nat positive for more than six months. conclusion: b nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below iu/ml. these data support our testing algorithm all components of high positive donations (virus load over iu/ml) were discarded. donors with ns antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: . ( . %)cases turned out negative with both technologies; . ( . %) cases were positive in both methods; . cases were positive only using tmpa recombinant [of which ( . %) were confirmed positive by tpha/fta abs. as seen we found samples ( . %) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the ¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between : to : per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least ¥ cfu. the positive control has to be stored at room temperature and is stable for years. after dilution in pbs, the preparation has to be used within hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was . in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per million transfusions, the risk for bacterial infections is estimated to be : to : . especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of cfu/ml to cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of cfu/ml and cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at °c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between cfu/ml for e. coli and cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation ( - h) at °c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within minutes when the sample was taken hours after spiking. background: since year our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from ml plasma by using either the qiagen biorobot and qiamp virus biorobot kit or the manual extraction with cobas ampliscreen hcv pcr kit v . . results: between march and december a total of seronegative donations ( pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs samples in hours where the manual method needs hours for samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than samples/day while preserving the availability of blood products. anti-hcv similarly was high till ( . - . %), but in trend to decrease afterwards ( . %). anti-hiv reflected the low endemicity of the disease in public setting and was % through the mentioned years. rpr test for syphilis was around . %. directed donors were % of all and volunteer donors consisted nearly %. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since . from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was of (% ) were female and (% ) male in age ranged from to years. results: sera tested, ( %) were found to be positive for toxoplasma igg antibodies and ( . %) were igm antibodies positive and of them ( . %) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group to years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are patients suffering from thalassemia major who are aged between and , and who have been receiving erythrocytic transfusion - times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia . ). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least transfusions a year. the tests in all patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years ( ) ( ) ( ) ( ) ( ) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv / was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv / positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: ( ) we observe that there is a decrease in all four infections. ( ) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches % in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least out of the estimate of first-time donors, involving blood transfusion centres from of the italian regions. each centre was required to enrol all first-time donors born before december st, , and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first months of the study the sites saw almost first-time donors, of whom . % belonged to the required age groups. among eligible donors, . % were both hbsag and anti-hbc positive, and . % were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, % were positive for anti-hbe, % had raised alt and % were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, % were negative and % were positive for anti-hbs. among anti-hbs positives, % showed values < miu/ml and % > miu/ml. the avidity index results suggested that approximately % of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately % of the italian first-time donors are older than years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that . % of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly % of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv / and tp ab. to c + ns + ns , ( . %) to c + ns , ( . %) to c + ns + ns , ( %) to ns + ns + ns , ( %) to c + ns + ns , ( %) to ns + ns . the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients ( . %) had anti-hcv ab for or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv & , anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october and january . the multi-marker qc sample 'pelispy s type ' (s ), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > ) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s lots were exported from edcnet and analysed. results: nearly results were submitted: all results were reactive (s/co > ). fifty of these results ( . %) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = ) or due to unacceptable results (n = )]. a further results were excluded because data provided by laboratories were inconsistent or incorrect. a total of results, reported using different prism reagent lots ( for anti-hiv, for anti-hcv, for anti-htlv and for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of < % for all s lots. results from prism anti-hcv and anti-htlv produced cvs between . % and . % for all s lots. data reported for s lot ps (n = , range for anti-htlv to for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with < % bias for the prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a + % bias. prism anti-hcv showed the greatest variability within reagent lot with results from of reagent lots showing a cv between % and %. conclusion: in results in a qc sample distributed to laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in , the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv- rna screening of all blood donors started late . aim of the study: to implement hiv- rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv- rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer ( ml/ml of each). from the final volume of the nucleic acid eluate ( ml) ml was used for the detection of hiv- rna (roche cobas ampliscreen) and ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv- rna ( iu/ml) and hcv rna ( iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv- rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = ) and minipool samples (n = ). results: detection limits of the hiv- and hcv assays ( % hit rate) were calculated to be . iu/ml and . iu/ml respectively. specificity for both assays was % and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of was below the recommendations by the council of europe for blood donor screening (for hiv- rna iu/ml and for hcv iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv- nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of donations, ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • out of donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in to the relevant institutions of the eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the - age limits for blood donation ( - for a first donation) were the rule in countries. in other countries the age limit could be brought forward to and extended to years old. the time interval between donations was identical for men and women in countries, and varied from to weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the / /ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be % correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from to months. previous transfusion history could not be a ci or could be one varying from months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the /ec/ directive on these practices in the eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average whole-blood donations are performed per inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection . % -bruise assessed using post-donation interview . %, sore arm - . %, nerve irritation . %, arterial puncture/pseudoaneurysm/arteriovenous fistula . - . % etc) are most commonly reported, while negative systemic reactions (vasovagal reactions . - . %, syncope . - . %) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol ; : - .) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare ( . %). fatigue ( . %- . %) and diminished physical working capacity ( . %) are reported to occur during days after the donation. a recent study of a consecutive sample of swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - .) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common ( % of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; % of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - ). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa- establishing a national adverse event reporting system for blood donors -a prospective study of . there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: . to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; . to define and categorise adverse events; . to record data systematically and prospectively using the existing computerised donor database. methods: from summer , a small project team of senior clinical and operational staff took months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over staff working on mobile collection teams, static sites and blood centres. daer was fully implemented by january . adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first months . million donors attended sessions throughout england and north wales. results were very consistent month on month. donors ( : ) had vasovagal symptoms but only % of these suffered syncope. % of all vasovagal reactions occurred in women. % occurred in donors aged - and a further % in donors aged - . (donors aged - represent only % of the total donor base.) donors ( : ) reported a delayed reaction, % of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in donors ( : ) and, more rarely, arterial puncture was diagnosed in donors ( : ). bruising was reported after the session by : donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about % of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only % of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around % of the global blood population has access to only % of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population ( participants). the survey was performed in november . results: five percent reported being active donors (had donated during the last months), % were passive donors (had not donated during the last months), % were non-donors with a positive attitude towards becoming donors, and % non-donors with no intentions ever to donate blood. in the youngest age group (age - ), % reported being active donors and % were passive donors. however, % of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and % reported they did not care about blood donation. in comparison only % of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and % reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion ( %) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: . a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. . specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. . consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to first-time and repeat blood donors, in order to be used as an indicant of donor's satisfaction. . first-time donors increased . % in the months of application. among them, . % were donors 'for relatives or friends' turned into volunteer donors and . % were first-time volunteer donors. the questionnaire analysis further revealed: • % were motivated by the use of magnetic card. • % appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • % were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • % considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. . the turnover of repeat donors also increased . % after replacing their plain old paper cards with new ones. further analysis revealed that: • % appreciated the quick cross-checking of donor's identity. • % were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in , greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa- emerging technologies in transfusion. dna based assays until the late s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short ( - nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing donors were genotyped for hpa- through - and ; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and , all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested ( epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons , , , , and . in all known weak d types, all six rhd specific exons are amplified (except for weak d type which lacks rhd exons and ), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between and , samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types ( t > g), ( g > c), ( c > g) and ( c > a). weak d type was identified by the combination of serology and absence of exons and by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all samples were subjected to the rhd mpx pcr and sample showed absence of rhd exons and , indicative of a weak d type when combined with serology. the remaining samples were analyzed by the weak d pcr-ssps, resulting in weak d type samples, weak d type samples, weak d type samples and weak d type sample. two samples remained undetermined and were sequenced for all rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau partial d phenotype ( g>a, g>a and c>t). the other sample had only one, not previously known mutation ( a>t), which is located intracellularly at the coohtail. extensive serology using the epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type . conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types , and should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, out of patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a . % incidence of anti-kel is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel acquired by transfusion/previous pregnancies: st case: in july , a -year-old woman (gravida , para ), rhdnegative, kel -negative was referred at weeks gestation. the father's phenotype was rhd-positive, kel -positive. a maternal antibody screen revealed d and kel alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron and exon of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel homozygosity; nd case: in august , a -year-old woman (gravida , para ) was referred at weeks gestation. she had a history of transfusion with rbcs units in -one of the donors was kel positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel -negative. the maternal antibody screen revealed anti-kel . doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron , exons , , and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel /kel by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel /kel result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of . g/dl was found to be favourable to one of g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger < . mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < . mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of months (hb transfusion trigger of hb < . mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then months. hrqol is measured after inclusion, after randomisation, weeks, , , , and months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july till june patients were included ( ra, rars, rcmd, raeb, cmmol) in general hospitals and university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and patients for the liberal transfusion policy. the mean follow up period in the liberal group was . months (inclusive run in period) and . months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group patients finished the study, received growth factors and patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about % reduction in amount of transfused red cells was found ( . units per pt per month in the liberal group vs . in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa- table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < . ). t-pa- thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches % at age . this rate is even better in well-treated patients, almost % of whom survive at age . regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the -year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from % to . % per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of adult patients with b-thalassaemia major transfused with a total of . rbc units from january to december . all units were fresh, stored less than days. . units were lr-rbc, . units were ld-rbc and . were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was . % and . %/per unit respectively, in those receiving ld-rbc was . % and . %/per unit respectively, while in those receiving washed lr-rbcs was . % and . %/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients . times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa- viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b ). pasteurisation (liquid heat treatment at °c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than nm pore size was introduced in the early s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately ml of plasma, whereas the improved set accommodates up to ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo ( ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below - °c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from % to %. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts- ), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained % to %. plasminogen and alpha -antiplasmin were retained % and %, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive / /ec require that fresh plasma prior to freezing contains < residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in fresh plasmas divided into groups: group : plasmas from leucoreduced whole blood, group : plasmas from packed cells after removal of buffy coat and specific filtration, group : : apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is . ¥ residual rbcs/l; linearity study with concentrations of . , . , . , . , . , . , and . ¥ rbcs/l showed excellent correlation between observed and expected values (r > . ); reproducibility study showed c.v of respectively . % and %. for values > ¥ rbcs/l, it appeared necessary to introduce a correction factor of . for the anti gpa pharmingen. quantification of rbcs in plasma: group (plasma from leucoreduced whole blood): . ± . rbcs/l; group (plasma from packed cells after removal of buffy coat and filtration): < . °¥ rbcs/l in all the cases; group (apheresis plasma): < . °¥ rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. . improving basic transfusion knowledge amongst health workers; . improving pre-operative preparation for surgery . strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao ) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o (hyperoxic ventilation) can be employed to rapidly raise cao by increasing the amount of physically dissolved o in plasma (hyperoxia). however, molecular o causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received homologous units in addition. the patients undergoing tkr and the patients undergoing removal of implants predeposited and units respectively, but finally and of them have been used. according to our patients data, the . % of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate autologous units. all of them underwent thr except one woman who also had a tkr months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of . homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran % is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f + . in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes / . subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo units three times per week (group b). rhuepo was given for a minimum of weeks or until icu discharge or death. the maximum duration of therapy was weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is > % chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa bb. ten percent of these women make anti-hpa a antibodies in their first hpa a incompatible pregnancy. in - new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of pregnancies show that only babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count < ¥ e /l. in previous studies, it is reported the ich appears in - % of the pregnancies where antibodies are present and that % of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets ( ¥ e mean) from donors with same blood group were pooled and divided equally into two bags, po- and control (pl , baxter), which have and ml/m *day*atm of oxygen permeability, respectively. on days , , , , , and of storage, swirling, mean platelet volume, po , pco , ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to days in po- ( / ) than in control ( / ) bags. dropped ph less than . on day was observed / in po- whereas / in the control. aggressive drop of glucose ( mmol/l) with prominent lactate accumulation ( mg/l) was also observed on day in of control bags. the po level in the control dropped more significantly by % ( . mmhg) on day than in po- ( . mmhg) compared with the initial level ( . mmhg) (p < . ). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po- bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day , and became a detrimental defect in of control bags on day . p-selectin expression was higher in control bags than in po- on days and with no statistical difference. in two control bags p-selectin expression reached > % and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for - days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa- background: maintenance of a neutral ph in the range of . - . is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of . - . cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph . ) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of bcs obtained from standard day -old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from - %. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields ( . ± . vs. . ± . °¥ e ) were similar (pags vs t-sol). statistically significant (p < . with paired ttest) improved platelet quality at days , , and of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at and days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = . for esc, and r = . for hsr at days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from to days provided the quality has been validated and bacterial screening is performed. irradiation up to gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to days is not known. method: two wbc reduced pcs, each made from buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at - °c. swirl, ph and cd p expression were determined on day , and . twelve experiments were performed and compared with a paired t-test, p < . was considered significant. results: see table (day values; mean ± sd; n = ). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day , the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day was < . in / pcs, versus / in group 'b' (not significant). the cd p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to days after blood collection. - b, - a, - b, pra %, donors. in patient -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient and were refractory to two and one hpa compatible platelet units respectively. in patient and compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: . the frequency of occurrence of anti-hpa antibodies in transfused patients was: - b, - b, - b, - a, - a; in three patients they were monospecific, in four polyspecific. . in patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. .the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa- the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b ). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen- or s- . the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about / of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of reactions in years with reactions more common in platelet pools ( : transfusions) than with single donor platelets ( : transfusions). these reactions caused fatalities in of cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the / of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march , the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of : true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: / repeats of x dilution and / repeats of x dilution were hcv rna negative, whereas / repeat of x dilution and / repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype a. the low hcv viremia (below iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a . % take-up rate ( . % where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd -enriched cord blood cells maintained at °c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at °c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd cells were cultured continuously at °c or °c for days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to °c, the cultures maintained at °c produced significantly more total cells ( . fold) and total mks ( fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets ( . fold). accordingly, the cells cultured at °c contained an increased frequency of cfc-mk ( fold) at day . cultures done at °c and °c were also more efficient than at °c but less than at °c. platelets produced in °c cultures could be normally activated by thrombin. as expected, the cells cultured at °c contained an increased amount of the heat shock protein hsp . control experiments showed that the culture of several cell lines was inhibited or unaffected by the °c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of > °c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at °c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than °c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only - % of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a . -year period. requests were received for deliveries from mothers and of these, collections were successful including pairs of twins. failed collection was most often due to a damaged cord at delivery. collections were made for siblings possibly requiring transplant (median age ) with for leukaemia, for erythroid disorders, for immune deficiency, for enzyme deficiency and others. the remaining collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was ml (range - , % exceeded mls) and mean tnc count was . ¥ (range . - . , % exceeded . ¥ ^ ). the mean cd +ve count was . ¥ (range . - . ). all collections were cryopreserved within hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was % (range - %) and the mean cd +ve viability post freezing was % (range - %). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v. . in an abi (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than %. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day . both test and control samples (n = ) were tested on day for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd +, cd +cd +, cd +cd +, cd + and cd +cd + human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd , was observed in . % (sd = . %) of control t cells upon activation with pma, while only a . % (sd = . %) of the test t cells increased cd expression. proliferation assays showed that h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il- , il- , il- b and il- cytokines after -h incubation in culture media increased significantly to pg/ml (sd = ), > pg/ml, pg/ml (sd = ) and > pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of . pg/ml (sd = . ) for il- , . pg/ml (sd = . ) for il- , pg/ml (sd = ) for il- b and pg/ml (sd = ) for il- . addition of lps further stimulated the release of tnf-a, il- , il- , il- b and il- in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k cells (k -rna). methods: dcs were derived from human pbmncs, which were incubated for days in the presence of gm-csf and il- , and then were transfected with k -rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k -rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared hours later, while the cells were expressing p bcr-abl protein and expressing increased cd , cd , cd , hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k cells. conclusion: human dendritic cells transfected with total rna of k cells in vitro could induce effective p bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl - donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in - % of relapsed patients. the success rate is % or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (< %) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately % of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately % to % of patients, is generally mild, and transient, but in < % of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to . j/ml uv light in the presence of um riboflavin, followed by storage under blood bank conditions with various concentrations of -deoxyglucose from to mm for days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without -dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days , , and . results: lactate production and glucose consumption increased from . mmol/ cells/h (sd = . ) and . mmol/ cells/h (sd = . ) for control samples to . (sd = . ) and . (sd = . ) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from . (sd = . ) for controls to . (sd = . ) for treated platelets at day , hsr from % (sd = . ) to % (sd = . ), esc from . % (sd = . ) to . % (sd = . ), swirl from . (sd = . ) to . (sd = . ), and increased p-selectin expression from . % (sd = . ) to . % (sd = . ). addition of -dog up to mm significantly reduced lactate production rate to . mmol/ cells/h (sd = . ) and glucose consumption rate to . mmol/ cells/h (sd = . ), and maintained ph above . (sd = . ) for days of storage. the effect of -dog exhibited a dose-dependent response. however, the addition of -dog had no effects on hsr ( . + . % at day ), esc ( . + . % at day ), swirl ( . + . at day ) and p-selectin expression ( . + . % at day ) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above % of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of -dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv- ( / ), international standards for hcv ( / ) and hbv ( / ) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv- subtypes other than hiv- b, for hiv- and for hepatitis b and c genotypes as well as specificity was evaluated with > specimens. results: results from this study indicate that high analytical sensitivities ( iu/ml hiv- m, cp/ml hiv- o and . cp/ml hiv- , iu/ml hcv and iu/ml hbv) and a specificity of > . % are accomplishable for the mpx test. the % detection rate for hiv- m subtype isolates (a through h) was between to iu/ml, for hcv genotype isolates ( a through ) between to iu/ml and for hbv genotype isolates (a through g and precore mutant) between to iu/ml. investigating seroconversion panels, hiv- rna was detected an average of and days earlier than hiv- antigen with abbott hivag- monoclonal and coulter p antigen tests, respectively, hcv rna an average of or days earlier than hcv antibody with the abbott hcv eia . or ortho eia . tests, hbv dna an average of days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october and september , apheresis ip units have been transfused in sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated ( . %). the population of patients receiving at least one transfusion (n = ) included . % of males, . % of females, the median age was (range - ). the most frequent broad diagnostic categories were hematology-oncology ( . %) and cardiovascular surgery ( . %). the patients received their transfusions either in regular hospital wards ( . %), intensive care units ( . %) or as outpatients ( . %). the number of transfusions by patient ranged from to (mean . ± . , median ). half of the patients ( . %) had previous transfusion experience and . % had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in . % (n = ) of the transfusions ( % ci . - . ), and . % of patients. only ( . %) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, . % ( % ci . - . ) of the transfusions were confirmed as having caused reactions in . % of patients, none of them serious. the most often reported symptoms were chills ( . %) and fever ( . %). itching, skin rash or urticaria was observed in . % of transfusions. of the serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio . , p = . ). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n mÜller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using single donor plasma units. for the evaluation of the plasma factors ml were drawn at different stages (before treatment, after plasma filtration with plas , after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha -antitrypsin complement: ch activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen ( . %), factor viii ( . %), and factor x ( . %). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days , and . the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was . ¥ ( . - . ¥ ; sd- . ). platelet pool volume was . ml (sd- . ). plasma % was within . % and . %. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had . ¥ (± . ). the average platelet loss was . ¥ . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for days. patients were randomized to receive one -day ip transfusion and one -day cp transfusion in random order. after each study transfusion, the hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, -hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included patients, randomized to the ip-cp sequence and to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence ( % vs %; p = . ). mean platelet dose (¥ e ) was . for ip and . for cp (p = . ). there was a significant period by treatment interaction (p = . ) at the . significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) -hour cci (¥ e ) was . ± . for ip vs . ± . for cp. the mean paired difference for both sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). for the first period only, mean -hour cci (¥ e ) was . ± . for ip vs . ± . for cp) the mean paired difference for the first period sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). the non-inferiority margin for the study was . ¥ e for mean treatment difference in cci (cp-ip). median time to next transfusion was h for ip vs h for cp following the first transfusion (p = . log-rank test; data censored at days after transfusion) and h ip vs h cp after the second transfusion (p = . ). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding -hour cci was not met. however, transfusion with -day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower -hour cci compared to -day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood ( ml/kg), across a major antigen (hgd) mismatch. high anti-s- ab titers (≥ : ) were induced after repeated immunization (days , , , , ) with klh-(s- ) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc ( ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken , , , , and days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = , g ), or o-srbc (n = , g ). no ab against o-srbc developed after biweekly transfusions over weeks in g rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = ; g ). transfused rabbits (g & g ) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s- abs were then induced by klh-hapten immunization in rabbits from g (n = ) and g (n = ), and in a group (n = , g ) of naïve rabbits. non-immunized rabbits (g , n = ), and (g , n = ) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g rabbits, but not any of the g rabbits. g rabbits exposed to o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g and g were transfused with m-srbc. viability of m-srbc in all g rabbits (hyper-immune or not) and the hyperimmune g rabbits was equivalent to crbc circulation in g rabbits. in pre-immunized rabbits with high titer anti-s- ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s- ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s- rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed + reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; + reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year are kept in computerized system that has patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr , rzr , r¢r¢, r¢r≤, r≤r≤, ge- , , and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge , anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally . examinees were typified. with o blood group were . ( . %); with blood group a were . ( . %); b blood group . ( . %) and ab blood group were . ( . %) examinees. totally . ( . %) were d positive and . ( . %) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system . ( . %) and d blood group in rh system with . ( . ) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence . % of anti-d in females versus . % in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than . %. however, the rare phenotype jk(a-b-) is more common in polynesians ( . %). individuals with jk(a-b-) phenotypes typically form anti-jk with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was . g/dl. she was given units of crossmatchcompatible rbc. on day her haemoglobin was . g/dl, with a total bilirubin of . mg/dl and lactate dehydrogenase of u/l. on th day an unexpected fall in hb ( . g/dl) occurred with an increase of bilirubin to . mg/dl and of lactate dehydrogenase to u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka: / and jkb: / ). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was g/dl. two months later her haemoglobin was . g/dl and anti-jk was present in the same titer. a year later her blood cell count was normal and the anti-jk was detected in a lessened titer ( / ). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at °c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer ( / ) were revealed. the rare anti-jk alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case . a years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer / ) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated . % red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated . % red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared . % enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc month to months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d( ), c( ), e( ), k( ), cw( )] and were tested with enzyme treated red cells in use and rbc to months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells to months post-expiration, except anti-k antibodies (negative with k positive cells - months post expiration). conclusion: most rbc antigens studied were detectable months after rbcs expiration date. tests with . % cells were valid in gel test (nacl/enzyme) for at least months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated . % red cells. background: wr(a) is a low-incidence blood group antigen ( : ) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: -year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in blood centres using different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard ng/ml anti rh (provided by cnrgs), a positive control anti kel and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions ( tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a + intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving blood donors from the university hospital of coimbra during the year . the incidence of weak d and rh (d) phenotype was determined in individuals who were rh (d) negative. the ab and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented . % of the total sample. we also describe ab , rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in s and s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from to times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about / of tested samples the number of igg molecules per rbc was small (< ) and the laboratory signs of haemolysis were present in . % of them as well as in . % samples with moderately coated red cells ( - igg/rbc). the large number of igg molecules per rbc (> ) was significantly associated with high frequency ( . %) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c d. in % of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in % of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting + with o rbcs at all phases and + with a rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c b,-c d (table ) . prewarming of test system did not change the reactivity ( + at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from + to + when tested with o red cells, as well as a significant reduction in antibody titer from : to : tested at immediate spin (table ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied . women, - years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in cases; moreover a rbc autoimmunization was found in women. anti-d ( cases), e ( ), c ( ), k ( ), c ( ), s ( ), d + jka ( ), d + s + e ( ), d + c + k ( ), m ( ), c + e ( ), d + c + g ( ) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in . % of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department ( . %) and ( %) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types , and were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum- , th- , ms- (bioscot/serologicals) and d e / - (immucor). results: out of samples molecular typing results were as follows: in samples dw were not determined, in samples, weak d type ; weak d type ; weak d type , weak d type ; weak d type and weak d type were determined. by all monoclonal reagents % weak d type , % weak d type , % weak d type and % weak d type negative results were given. by all monoclonal reagents weak d type and weak d type positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type , type and type . such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in > % of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in . according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting + - + s at c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs ( . % ficin) and no reactivity using . m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity > grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment ( . m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in samples from patients; ( . %) patients had > episode. these findings comprised . % of unexpected results found at pt and bg. incidences were . % at routine and . % at urgent bg ( and bgs, respectively), and . % both at routine and urgent pt ( and pts, respectively). . % patients were female, . % over , but . % < years, coming mostly from surgery ( . %), internal medicine ( . %), hematology ( . %), ginecology ( . %), cardiac diseases ( . %) and cardiac surgery ( . % patients). frequent diagnosis were solid tumors ( . %), cardiac diseases ( . %), hematologic malignancies ( . %), uraemia ( . %), orthopedic surgery ( . %) and hepatic diseases ( . % patients). . % patients were previously transfused, with only . % patients proved as not transfused or pregnant. subsequently positive antibody test during the study had . % tested patients. at pt positive crossmatch was found in . %, antibody screening in . % and both tests in . % cases. majority of reactions were ' +' ( % at pt and . % at bg); reactions ' +' or ' +' were found in only . % cases at pt, compared to % at bg. one crossmatch only was positive in . % positive crossmatches, with / patients having > crossmatched unit. ahg identification was positive in . % tested patients; in % of them papainized panel was also positive. lymphocytotoxic antibodies were found in . % tested patients; . % ( %- %) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in . %, hla lymphocytotoxic antibodies in . %, 'igg antibodies of unknown specificity' in . % (hla noncytotoxic antibodies in / elisa tested samples!), contaminated sample in . %, anti-bga in . %, non-specific cold antibodies in . %, subsequently recognized specific antibodies in . % ( lua, m, kpa, yka), non-specific autoantibodies in . %, carry-over of dat-positive cells and antibody to reagent in . % cases each, while in . % cases antibody screening and in . % cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < . ) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt ( . % vs . %), positive lct ( . % vs . %), lymphocytotoxic hla antibodies (lytxab) ( . % vs . %) and reactivity 'positive screening, negative panels' ( . % vs . %); in males non-specific cold antibodies ( . % vs . %) and antibodies to reagents ( . % vs ) were noted. age. in patients > vs < reactivity was often found at pt ( . % vs . %), caused by lytxab ( . % vs . %), anti-bga ( . % vs . %) or 'positive crossmatch, negative panels' ( . % vs ), but rarely by laboratory mistake ( . % vs . %) or later recognized antibody ( of patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt ( . % vs . % at bg), as positive antibody screening ( . % vs . % if positive crossmatch), with positive panels ( . % vs . % if negative). subsequent tests were positive in only . % patients with lytxab, % with anti-bga, of with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in % and . % tested samples found at urgent and routine pt (diamed), vs at bg. all cases due to lytxab, of anti-bga and of 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) . % cold antibodies and . % laboratory mistakes were found. positive antibody test: identification was negative in . % screening-only cases; % of them were caused by laboratory mistake. lytxab were found in . % crossmatch-only cases; . % reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in % cases with lytxab (in with papainized panel) and often due to 'igg antibodies of unknown specificity' ( . %), anti-bga ( . %), contaminated sample ( . %), but also to later recognized specific antibody ( . % cases). strength of reaction: laboratory mistake was noted in . % of 'w', . % of ' +', . % of ' +' and . % of ' + and +' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt ( % and . % of patients, respectively), due to positive crossmatch ( % and %; and . % patients with liver and cardiac diseases) and caused by lytxab ( . % and . %) or 'crossmatch/screening positive, panels negative' ( . % patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg ( . % and . % of patients), due to laboratory mistake ( % and %) or cold antibodies ( . % and %), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of patients ( male and female) were evaluated. the mean age of the patients was years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal ( / ) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in patients with the incidence of . %. antibodies were registered more frequently in females ( / , . %) than in males ( / , . %). patients ( . %) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table ). in patients ( . %) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. patients ( . %) were dat positive. autoantibodies were found in patients ( . %), all of which had specificity to rhesus system. cold agglutinins were positive in patients ( . %). no specificity could be assigned in patients ( . %), while in patients ( . %) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was . %. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about % of women form red cell allo-antibodies in pregnancy and . % of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that % of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and % of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case : a -year-old asian woman had three previous uneventful pregnancies. in her th pregnancy she presented with miscarriage at weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique ( . iu/ml by quantification using r r cells). in a th pregnancy, no antibodies were detected until weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case : a -year-old asian woman had a positive antibody screen post caesarean section in jan . no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at and weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was . iu/ml using rr cells and the anti-d level was < . iu/ml using r r cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of / . in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and ( ) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > % being s+. conclusion: there is a unique distribution of red cell antigen groups in the races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table . conjointly, in order to obtain the frequency of mi.v phenotype, we screened samples among with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype ( . %), there were also mi.i/ii phenotype ( . %), mi.v phenotype ( . %), mi.vi phenotype ( . %), mi.x phenotype ( . %), and most interestingly two miltenberger related not yet classified variants ( %). related variant a ( . %) was phenotypes as mia+, anek+ and hil+. related variant b ( . %) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore . % (table ). the discovery of unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period patient samples were screened, of the positive results were evaluated. blood samples with positive screen tests by the ortho auto vue tm method (av) performed with % cell suspension, and patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into categories according to antibody specificity; . rh system antibodies (n = ), . clinically significant non rh system antibodies (n = ), . clinically non significant antibodies (n = ), . auto antibodies (n = ), . not identified (ni) antibodies (n = ), . negative screening results by the diamed technique (n = ). the categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of samples were compared, . % of them gave stronger reactions by av, . % gave equal reaction strength and . % of them gave weaker reactions by av. positive screen tests by av only were detected in samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in samples, were rh system related, kell system related and not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to . % suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh and rh rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh or -rh antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague , † blood bank, faculty hospital m, prague , czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab grouping was performed with registered immucor reagents (immuclone: anti-a birma , anti-b lb ) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre . titration of reagents /+ aliquot of serum added/: anti-a: titre (both patient's serum and control); anti-b + patient's serum: titre , anti-b + control: titre . conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed % agreement between all three methods. samples were tested per min with the autovue innova, compared to min required for the same number of tests performed with manual testing and min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcÍa borrÁs, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples ( - years old, n = and > years old, n = ) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for h at °c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . , nrbc: . + . ; srbc: . + . . the values of am obtained with young individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . . nrbc: . + . ; srbc: . + . . conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < . ) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < . ) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < . ) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < . ). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification ¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by d-fluorescence microscopy after lipid extraction with triton x- in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa- tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at nm the flim-fret from alexa tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from to %, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in transfused sickle cell patients. methods: all the patients included in the study had received at least unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was - units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c c, c d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the patients. the dat was positive in ( %) of the patients. in cases ( %), the dat was igg type, in case ( %) igg + c d and in case ( %) igg + c c + c d. the acid elution was performed in cases. the eluate was positive in cases ( %). in ( %), of the cases, autoantibodies were pointed out whose specificity were specificity public, anti-e (rh ), anti-c (rh ), anti-ce (rh ). in cases ( %) we found alloantibodies whose specificity were anti-e(rh ), anti-k(k ), anti-c (rh ) and anti-jka (jk ). in the cases ( %) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in ( . %) of the patients. of these, ( %) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of patients was %. seventeen patients ( %) of the patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang , (suppl. ), ). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having distinct incubation wells, application zones and detection areas. microliters of % suspensions of reagent red cells for reverse grouping (reverse-cyte a , a , b, , medion diagnostics, switzerland) are mixed in each incubation well with microliters of plasma. the resulting suspensions are incubated for min, followed by the transfer of microliters each to the application zones, where migration starts immediately. results can be read after min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho screening rbc since the year . aim: (i) to summarize the performance of the autovuetm system for rbc abs, during years ( ) ( ) ( ) ( ) ( ) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in . % of , blood donations that were screened, during - , using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in . % ( . % of the donations). an increased percentage of confirmed tests is noted, since , probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, samples with low positive results (agglutination degree of . ) were retested twice on the autovuetm. / ( %) gave negative results in both repeats. only / was confirmed positive and anti-m was identified by diamed gel test. the remaining samples had at least one positive repeat. / ( . %) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and blood donors of azeri nationality were examined. patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee ( . %) •• ccdee ( . %) phenotypes were twice as much higher than the occurrence of those in the donors ( % and . % respectively). phenotype ccdee occurs more frequently in the donors ( %), whereas it is significantly rare in the patients ( . %). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors ( . % and . %) than in thalassemic patients ( . % and . %). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c c and c d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c c or anti-c d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of heterozygous sickle patients and homozygous patients were analysed. of the homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. ( ) and are present in more than % of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a years old swedish woman, a rh negative, at th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre ( : ) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from . /ul to . /ul) was observed. this pattern showed no variation until the end of the pregnancy. at th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to . /ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p- rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of alloantibodies on blood samples over . these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia ( . %), e ( %), le a ( . %), le b ( . %), p ( . %), m ( . %), d ( . %), c ( . %), jka ( . %) and c ( . %). within the chinese community, the most frequent alloantibodies were similar: mia ( . %), e ( . %), le a ( %), le b ( . %) and p ( . %). in the malays, the most frequent alloantibodies were le a ( %), le b ( . %), mia ( . %), e( . %) and p ( . %); while in the indians, these were mia ( . %), le b ( . %), le a ( . %), d ( . %), and e ( . %), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians ( %) compared to chinese ( . %). anti jka tended to occur mainly in the malay race and anti-c was rare in all (< %) reflecting the high prevalence of c in the singapore population (r r phenotype). the ratio of alloimmunised male to female (m : f) is : . most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at : . . alloimmunisation increased with age for mia, e, k, p , jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies ( or more) within the alloimmunised subjects is . %. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a years period, from / / - / / , we studied newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in ( %) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in ( . %) (newborns of group a with mothers of group o), anti-b antibody in ( %) (newborns of group b with mothers of group o), anti-d antibody in ( . %) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in newborns and in the rest , no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d ( ), anti-e ( ), anti-k ( ), anti-fya ( ), anti-s ( ), anti-jkb ( ), anti-n ( ), anti-kpb ( ). in two patients antibodies were identified, while in / ( . %) no antibody was identified (unspecific). it is remarkable that only in out of patients with both dat and iat positive, an irregular antibody was identified, while the rest patients had unspecific antibodies. in patients with only iat positive, had an irregular antibody and had unspecific antibodies. in out of patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is . % and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. . % of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than % in china) were reported from % to %. moreover all del individuals in this population were found mainly carrying a same allele, rhd a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in rh-positive donors with cde/cde and cde/cde genotypes, respectively, and del phenotype individuals carrying rhd a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and isoforms were detected in rh-positive and del, respectively. among them, isoforms have identical sequences, which are transcripts with exon , exons and , exons and , and exons to spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon or exons - were spliced out, but both possess a bp segment of sequence from intron of rhd. through additional reversedtrancriptase pcrs, which amplified exon to ¢-region and exon to ¢-region, the results showed that exon did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd a allele since the exon was always spliced out in all isoforms. all transcripts in del maintain a normal open reading frame and encode proteins with different numbers of amino acid residues and different c-terminals (genbank ay , ay , ay , ay , ay , ay ). among them, the sequence of del (isoform with exon spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in . it encodes amino acid residues and has amino acids more than normal rhd. it is different from rhd after codon . in normal rhd protein, the amino acids after ( residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del that was found in del. (iii) there is only one polymorphism in the region of bp segment between rhd intron and of the del transcripts, which indicated that other polymorphisms may exist in intron of rhd a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh ( pooled we reported earlier that metabolic arrest followed by incubation at °c reduces the platelet storage lesion (badlou et al. transfusion ) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium ( min, °c) followed by storage ( h, °c) and recovery with glucose ( h, °c). controls were (i) platelets in glucose-rich medium stored for h at °c and recovery with glucose (c ) and (ii) platelets stored for h at °c (c ) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. binding of msp, c , c was ± , ± , ± % of total platelets. phagocytosis of msp, c and c was ± , ± , ± % of total macrophages (means ± sem, n = ). before recovery of msp, binding/phagocytosis was % higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in patients with leukemia and lymphoma, males and females with mean age ± years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of compatibility tests were performed, of which were compatible. twenty five compatible platelet concentrates out of were clinically evaluated. twenty from compatible crossmatches ( %) were resulted in successful transfusion while only from ( %) in unsuccessful. the incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~ . ). additionally patients out of ( . %) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june and is still in course: in january , blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the blood samples analysed, were reactive and the specificity of the antibodies were: anti hpa a: , anti hpa b: , anti hpa a + hla: , anti hpa b. , anti hla: , auto hpa- b: . specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa- , , , , , ) using pcr-ssp or pcr-rflp. the infants with hpa immunization suffered from severe (plt count - /ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa- and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa- a is the most frequently implicated antigen. other antigens such as hpa- a, or hpa- a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa- bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa- bw (maxa+) mismatch as the sole antigenic incompatibility in out of families. in the last one, incompatibility was found for hpa- without anti hpa- b maternal alloantibody. as the father was found to be hpa- bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa- bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa- bw (maxa+) fmait is not uncommon and was found to be around % of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa- a antibodies after non-myeloablative allogeneic hsct. a year-old male patient was diagnosed with multiple myeloma in / . treatment consisted of cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received cycles of bortezomib, and after complete remission a stem cell allograft ( . ¥ /kgbw cd + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine ( ¥ mg/m ) and alkeran ( mg/m ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d but was accompanied with a continuous decrease of plt counts. between d and d the patient received seven plt transfusions, containing a median of . ¥ plt/unit (range , - , ¥ plt/unit) from random donors. the corrected plt count increments at to h after these transfusions were < /ml. therefore, and because of even a further decline of platelet counts to /ml on d we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak ®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa- a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa- , - , - and - of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d after hsct reacted strongly with the donor's plt due to anti-hpa- a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa- bb, - aa, - ab, and - aa; the donor was hpa- ab, - aa, - aa, and - aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d , d and d after hsct contained antibodies against hla class i antigens but hpa- a antibodies were not anymore detectable. no hla antibodies were detectable on d after hsct. the severe thrombocytopenia was caused by hostderived hpa- a antibodies. fortunately, plt counts started to increase on d spontaneously and the patient could be discharged at d (plt . /ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa- b antibodies: case report p moncharmont, m vignal, y mÉrieux and d rigal efs rhone alpes site de lyon, lyon cedex , france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa- b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a -year old mother, gestation /partum , gave birth to a male neonate by caesarean section at weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted . g, was . cm tall and had a head circumference of . cm. the apgar score was , , , at , , and min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during h. the plt count which was . , . and . giga/l at day (d) , d and d respectively dropped dramatically at . giga/l at d . simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa- a/a and anti-hpa- b ab were detected in her serum. the baby was heterozygous, hpa- a/b. plt were transfused to the baby and the plt count rose to . , . and . giga/l at d , and respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa- b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor antibodies. hit type ii occurs in up to % of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf antibody test (diamed id-card). results: from the studied patients, ( . %) developed thrombocytopenia (determined by a decrease in the platelet count below %, after the introduction of heparin therapy); ( . %) did not show decrease in the platelet count. six ( . %) out of thrombocytopenic patients were positive for anti-heparin/pf antibody. three ( . %) out of non thrombocytopenic patients were positive for anti-heparin/pf antibody. the results demonstrate that ( . %) patients were positive for anti-heparin/pf antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects . - in live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to % of reported cases). the commonest antibodies are anti-hpa- a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week of gestation to a healthy mother after first uneventful pregnancy; birth weight g, apgar score . immediately after birth, severe thrombocytopenia ( ¥ /l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa- a/a) were given. twenty-four hours later platelet count rose to ¥ /l and no new petechiae were observed. on third day of life the blood platelet count was ¥ /l and the newborn received ivig g/kg and corticosteroides. twenty-four hours later the platelet count rose to ¥ /l and further clinical course was uneventful. natp due to hpa- a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa- a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of . % to % in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: samples were referred after being characterized as weak d. in cases we obtained a positive result, with a preponderance of weak d type ( . %) over type ( . %), ( . %) and ( . %). two samples were not categorized. the high incidence of weak d type in our population is in marked contrast to studies performed in other european populations where weak d type was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a -bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to ¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the ¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a -bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = ). results: ( . %) rhd-positive and ( . %) rhd-negative samples were phenotyped. of the rhd-positive donors, ( . %) were rhd homozygous and ( . %) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee ( . % vs . %) and dccee ( . % vs . %) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in . % of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v. . in an abi (applied biosystems). results: see table . the substitutions of rhce-specific nucleotides in exons , and with their rhd-specific counterparts lead to different new rhc-antigens with weakened expression. since one amino acid change in allele lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a -year old mother (w.o.g. ) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron , exons and were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron , exon and exon while sample of fetus ii lacked rhd-specific intron . therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons - , & ; polymorphisms for dweak type - , d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi . rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons , , , and were possible and failed only in exon - . determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron pcr. results: weak d type was not identified in our research population. weak d type was identified in cn, weak d type was identified in cn and weak d type was found in cn, bsa, be, bc and saa. conclusions: although weak d types to represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type in all populations. however, regarding rhd phylogeny, the weak d type mutations ( c > g and t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from a, b, ab subgroups, and provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon and , and allele separation were performed on these samples. results: abo*a allele was observed in an aint subgroup. two new a alleles that showed g > a base change and c > t of intron and a polymorphism of c > t in a(pro) intron were discovered. o and o alleles were also observed. in b subgroups, a silent substitution c > t (leu leu) was observed as a new b allele. another new b allele which showed g > a was also found in b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > . ) verifying that the monoclonal anti-ab associa- study aim: to prevent the sensitization to rh (d), to a d-patient who was transfused with d+ blood. material and method: on september , ( / / ), we admitted to our hospital through air-carriage, a female of years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: %) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally units ( ml) of d+ condensed red cells, out of that were initially asked. before the h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh (d). the indicted dose was - iu/ ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = ) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table ) . for the long-term follow-up samples, additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with copies of negative dna in a single amplification tube for all primer pairs. we were also able to calculated the clinical sensitivity that using donor-recipient pairs. . % of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with . % of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to -chloro- - dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag- /common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with million viral particles of murine cmv (mcmv). at different post-transfusion time-points ( h, weeks, weeks, weeks, weeks), different female recipients plus non-transfused female knock-out controls were challenged with million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp ( mg sc once or q weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day and day after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ % during blood donations and blood donation was not performed if ht < %. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was . € in the aranesp group and . € in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from operations in patients with anti-erythroid antibodies. materials: three patients ( male and females), aged between - years old, had to undergo selective operations, total hip replacement surgery and aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of consecutive patients (group ) who donated two red cell units (rbc) of autologous blood and weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ ) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ . ; postop-hb = hb ( - h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as g/l) < (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next patients who were also assigned for pabd (group ). pabd was refused if the probability of rbc requirement (prr) was < %. between - % one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded % two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group ( vs and vs g/l). % of autologous blood of group were discarded, / patients needed additional allogeneic rbc. hb-loss caused by rp was ± g. mean prr in group was . %. / patients donated one rbc, which was later discarded, and no patients donated two rbc. / of group needed allogeneic rbc. mean prr of these patients was % (range . - . ). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other ( . %) patients who required autologous and homologous blood, had average predonation hb level of (sd ± . ) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = . ), predonation htc values (p = . ), weight (p = . ) and gender (p = . ): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = . ). ( . %) patients with ptka were transfused with autologous blood only, and had average predonation hb level of (sd ± . ) g/l. other ( . %) patients transfused with autologous and homologous blood had average predonation hb level of (sd ± . ) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = . ): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = . ) predonation htc values (p = . ), gender (p = . ) and age (p = . ) of patients and the need for postoperative transfusion. conclusions: our results show that over % of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight - g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, cm proximal and cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, patients, including males and females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about - percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at - day intervals. the blood volume taken from patients in every collection differed from - ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, cc to cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately % of the total blood volume withdrawn] had platelet counts of - /mm . the procedures of agp labelling were performed in laminar flow chamber. to seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed h after application of labelled agp (early scan) and at , , , h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at , h and at lapse time = h for days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every - weeks. by the th week of gestation the patient had donated four units of blood, her hematocrit was %, anti-ku titre was / and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were and at and min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day . the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above °c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+ °c, - °c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature + °c, + °c and + °c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+ °c) or freezer (- °c). the temperature was observed in min intervals for three hours, first measurement was min after putting into freezer or termostat. results: (see table ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since , we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between - %, the mean volume about ml and the mean platelet-content is . ¥ per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from to days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: twins made out of pools from buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at °c, and sampled at day , day and day . ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day with cb was . , with nfb . ; glucose with cb . mmol/l, with nfb . mmol/l; lactate with cb . mmol/l, and with nfb . mmol/l, hsr with cb %, with nfb %; p-selectin with cb % and with nfb %. the new platelet storage bag showed better results of in vitro quality markers, especially after day of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at °c or rt simplifies the preparation process. filtration at + °c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, % of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by . mm. a decrease of the process time is expected. methods: units of whole blood were collected with the new t 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within min in all cases. after donation whole blood units were stored at room temperature for h. subsequently, whole blood filtration was performed by gravity at a standard height of cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at g ¥ min by an heraeus cryofuge i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx -abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table . results are given as mean and standard deviation. whole blood filtration was completed within min in all cases. red cell units were transfused after a mean of days to patients affected from transfusion dependent ( %), post-surgery ( %), and post chemotherapy anemias ( %). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day , , and , and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day , and , and cd expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years ( , .) aberrations from these principles have taken place (self-sufficiency). nbti collected x = ( ) blood units into blood bags. in serbia x = ( ) blood units. retrospective analysis: ldpc-bc was administered x = % with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted ( %). increase of the use of leukocyte poor red blood cells was also registered ( % introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at °c with platelets stored at °c, and to determine the in vitro-effects of pre-incubation at °c for h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at - degree on a flat bed agitator; (ii) at - degree on a flat bed agitator and pre-incubated for h prior to analysis; (iii) at °c; and (iv) at °c and pre-incubated for h prior to analysis. this paired in vitro study (n = ) over days include volume, platelet counts, mpv, volume, ph, po , pco , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf ). results: platelet count (day and ; p < . ), mpv (day ; p < . ), ph (day and ; p < . ), pco (day and ; p < . ), bicarbonate (day ; p < . ), glucose (day , , , and ; p < . ), atp (day and ; p < . ) was significantly higher in platelets stored at °c and platelets stored at °c with preincubation. ldh (day ; p < . ), bicarbonate (day and ; p < . ), lactate (day , , , and ; p < . ), ph (day and ; p < . ), esc (day , , , and ; p < . ), hsr (day , and ; p < . ) was significantly lower in platelets stored at °c and platelets stored at °c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf was significantly higher in platelets stored at °c than in platelets stored at °c (day , , , and ; p < . ). hsr (day and ; p < . ) and esc (day , , and ; p < . ) was significantly higher in preincubated platelets stored at °c compared with platelets stored at °c. conclusion: platelets stored at °c maintain metabolic and cellular characteristics to a great extent during days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at °c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: whole blood donations were collected in a whole blood filtration set with cpda- and stored at room temperature for h before filtration at room temperature. the leucodepleted whole blood was stored for days. following parameters were analysed on day , , : free haemoglobin in%, k. in addition leucocyte count was performed on day and a blood culture on day (see table) . blood cultures on day remained negative and all counts of residual leucocytes were below ¥ (exponential) /unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day . however the results were within the required specifications from the european and swiss guidelines up to day . we conclude that autologous leucodepleted whole blood can be stored in cpda- -for days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a -month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: ( ) blood of female donors was not used for ffp production, and plasma was only used for fractionation ( ) plasma of female donors was not used for kt-bc pools ( ) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: %*; %** ffp produced by plasmapheresis: %*; . %** female donor units on cell separator: . %*; . %** ffp from total plasma units: %*; %** plasma units used for bc-pc pools: %*; %** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~ %), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of days. extended storage to days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd p and cd b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = ) using apheresis technique. the pc from each donor was divided in units, one served as control and the other was gamma irradiated with gy. the pcs were stored on a flatbed agitator at °c for days. samples were taken on day (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day , and were also analysed for hrs, cd p (p-selectin) and cd b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day , and . the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd b expressing cells were well maintained for days of storage. glucose decreased and lactate increased significantly during the storage period, from . mmol/l to . mmol/l for lactate and from . mmol/l to . mmol/l for glucose. the percent cd p expressing cells increased significantly during storage from % on day to % on day . po was well maintained but ph increased and pco decreased significantly between day and whereafter ph decreased and pco continued to decrease. the for parameters g'max and time to g'max increased significantly between day and and the time to g'max continued to increase significantly between day and . the results indicate a well preserved platelet quality after storage for days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b (transforming growth factor b ) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from buffy coats. following overnight storage at - °c, buffy coats were pooled with ml t-sol (baxter). forty-two pcs were produced either manually (n = ) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = ) using the orbisac validation bc set (gambro) equipped with the lrp leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl series analyser (radiometer). samples for testing of cytokines were centrifuged for min at g, °c; supernatants were harvested and frozen at - °c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days , and . results: platelet concentrate volume (mean): m-pcs: ml, a-pcs: ml. platelet yield was found to be . ¥ for m-pcs and . ¥ for a-pcs (p < . ). in all pcs ph levels were between . - . . glucose consumption and lactate production from days - and days - did not differ significantly. rantes levels (pg pr plts) were significantly higher in a-pcs than in m-pcs (p = . , repeated measures analysis of variance), but no significant difference was found in tgf-b levels (pg pr plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/ - . pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd + cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in °c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd + cell count were evaluated by flow cytometry with a no-wash, single-platform technique and aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h , stemcell technologies) and colonies were counted after days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables , and . post-thawing tnc and mnc, as well as cd + cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd + cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd + cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: cord blood units ( from males and from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb . blood units were also randomly typed for hla-dqa and dqb at high resolution. results: mean birth weight was g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was . g. babies were < th centile ( g) and were > th centile ( g). comparing the hla allele distribution in these extreme bands we found that hla drb * was significantly associated with high relative birth weight: . % in th centile vs . % in th centile, p = . . on the contrary, hla-drb * and dqb * were associated with low relative birth weight: . % and . % respectively in th centile vs . % and . % in th centile p = . and p = . . all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb * and higher relative birth weight (mean . vs . ; p = . ) as well as the association of hla-drb * with lower relative birth weight (mean . vs . , p = . ). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb * and hla-drb * genes encode for different amino acid sequences in the pocket of the molecule (aa , , ). this implies distinct functional restriction patterns. the sequence motif of hla-dr is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retarda- which provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a %- % dextran solution in . % saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation °c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was % to % when stained with gimsa or wright staining method. the viability of isolated pmnls was also % too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during to . at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged g to g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: exchange transfusions were carried out. four out of et due to abo incompatibility mother-newborn. five out of due to rhesus incompatibility mother-newborn and eight out of due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at °c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: samples were taken from newborns at first h of life for abo blood group typing during - . simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in out of newborns were found detected antibodies anti-a, anti-b. in out of newborns no antibodies were found. in out of were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from + . to + . conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab incompatibilities, we have discovered alloimmunized women with the following specificities: anti-d, anti-c, anti-c, anti-e, anti-jka, anti-d + anti-jka, anti-d + anti-s, anti-d + anti-c, anti-d + anti-c + anti-k, anti-s, anti-k, anti-m, anti-c + anti-e and anti-d + anti-c + anti-g. the most severe hdn were the d + c + g, the c + e and out of c newborns, with mean hemoglobin between and g/l, bilirubin = . g/l, reticulocyte count = %. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, years of age, in th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the th gestation week covered with mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < % or hemoglobin (hb) < g/dl and reticulocytes < %; b) hct < % or hb < g/dl in these conditions: o required < %, recurrent apnoea and bradicardia, cardiac rate > bpm and respiratory rate > bpm for more h; c) hct < % or hb < g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of % after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume ( ml per kg if premature or ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of - weeks) show a range of weight from to . g, while at term newborns from . to . g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always - ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of neonates admitted during one year, ( male, female) received blood components. fifty four percent received one, % two and % received three types of blood components. the frequency of transfusions were times. the most common used blood products were fresh frozen plasma ( %), red blood cell ( %), whole blood ( %) and platelets ( %). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were %, %, % and % respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january and december we studied d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of d-negative studied samples, ( . %) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that patients ( . %) had in their serum other anti-d associated alloantibodies: anti-k ( . %), anti-c ( . %), anti-e ( . %) and anti-c + e ( . %). during the immunohematologic follow up, it was determined that in / patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (- °c and - °c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at ¥ g for min within h of collection. plasma was removed. four units of plasma were pooled into a ml bag, mixed, divided into parts (aprox. . ml) and immediately frozen. two of these units were stored at - °c and units of ffp at - °c. after one month the plasma was thawed at °c during - h. the fibrinogen concentrates ( - ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at - °c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored - °c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center ( donations per year and utilization of approximately of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in the national center for the production of blood products was established, under the direction of elias politis and years later in begun the production of dried plasma from greek donors. by the year the center started the production of fibrinogen and by the year the production of antihaemophilc factor. in all the activities of the center settled down due to administrative aspects. at the beginning of s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early s, when techniques for the isolation and concentration of clotting factors were improved. in , matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl , is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation ( ¥ g for min) of whole blood. four units of plasma were pooled into a ml bag, mixed, divided into parts and immediately frozen. one of them was stored at - °c and after one month the plasma was thawed at °c during - h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well- (light cycler, roche diagnostic systems, nj) was used for the identification of the c y, h d and the s c point mutations of the hemochromatosis gene, and were based on protocols developed, for c y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c y allele was obtained ( . %) in individuals with genotype , however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than . patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c y, h d and s c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from % to - %, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of samples ( mothers and newborns), from / / and / / , were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the cases studied it was found cases without incompatibility ( %). from the incompatibilities, . % were abo, . % were rhd, . % were other rh incompatibilities, and . % were due to auto-antibodies. % of the mothers were rhd+ and % rhd-. conclusion: of the pregnant women studied, only were rhd-. from this group ( %) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the pregnant women rhd-, % had incompatibility abo, which decreases to near % the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a haplotype expression has been found about - % in caucasian and in greek population particularly, . %. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a expression. in of these donors the expression of cd , cd , cd , cd , cd , cd , hla dr, cd +cd +, cd +cd +, cd -cd +, cd +cd -cd +, cd -cd -cd +, cd -cd -cd + was examined. meanwhile, patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a expression, while in of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a expression in healthy donors was . % and in cancer patients % (p > . ). in table the mean, standard error, t-test and p of the two groups are included. (see table ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd expression, which was higher in cancer patients. summary and conclusion: the expression of hla a in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in ), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in , dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march . after keeping it for days at storage, blood was forwarded on a two-day journey to a small town, kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are - nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deter- objective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd ) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = ) and umbilical cordon (n = ) were used. several dilutions oh hailuronic acid sodium salt solution % (sigma l- h ) were confronted with % erythrocyte suspension in phosphate saline buffer (pbs) ph . . the rbcs were previously treated with an enzymatic solution of % bromeline in pbs ph . (sigma l h ). agglutination readings' were been by slow sharking after of h incubation at °c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à si. di- . - (i = , , . . .) where si represent the score and di- is dilution inverse. the adult' rbcs showed a = ± , while en the newborn the parameter was a = ± . our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd ) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during min at °c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p -dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig , is induced by p through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele ( tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (< %) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele ( repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a +/ + reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a + reaction in a test with monoclonal anti-d and a +/ + reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against anti-d reagents in the id-partial d typing system. one of these (number ) gave negative reactions and the remaining five gave positive reactions (ranging from +/ + to +), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a +/ + reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk applied conventional and no conventional (hemolytic medium time) techniques in sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in ae from b patients and in ae from ab patients; a epithopes in ae from ab patient and in ae from a patients; p and p epithopes in ae and only p epithopes in ae. these patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in sera and anti a immune antibodies in sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c a and c a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during - period all anemic patients on hemodialysis were divided in groups. the first group pts with febrile non-hemolitic transfusion reaction. the second group- pts immunized toward leukocyte and hla antigen. the third group young candidates for kidney transplantation for prevention of hla immunization. the fourth group pts with sle (for immune-complexes and autoantibodies). total patients ( males and females) received units of rbc with residual number of leukocytes. commercial filters of baxterÔ (lekostop lds) and terumoÔ (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with - microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until days of collection. result: aabb permits maximum < ¥ wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of ¥ the le reduction of - . %. the number of rbc after filtration is minimum % - g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g a, mthfr c t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients ( . % heterozygous, . % homozygous) compared to controls ( . % heterozygous), p < . . the or for heterozygous carriers was . ( % ci . - . ), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients ( . %) and controls ( . %), or . ( % ci . - . ), p = . . although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c t mthfr than the control group ( . % vs . %), or was not significant ( . , % ci . - . ), p = . . analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c t mthfr gene (tt and ct) (or . , % ci . - . ), p < . . conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to ¥ /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease ( % atipical plasma cells). to treat heavy anaemia (hgb g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol mg per day). coagulation parameters were tested: pt . inr, aptt s, fibrinogen . g/l, trb ¥ /l, d-dimer mg/l, atiii %. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with dose of cryoprecipitate, dose of fresh frozen plasma, iu atiii, doses of red blood cells and vitamin k mg. two days after the substitution therapy we saw pt . inr, aptt s, fibrinogen < . g/l, trb ¥ /l, atiii %. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till th day of therapy, fibrinogen was below . g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with mg/kg/dose of rfviia (*) for days, after which there was clinical and endoscopic recovery, and an inh decrease to . ub/ml (fviii dosage %) . he began to take meprednisone ( mg/kg/day) for days, after which the inh titre was . ub/ml. (table a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of . ub/ml and was treated with mg/kg/dose of rfviia (*) for days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to . ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was . ub/ml. (table a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a -year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio ( : ). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, to november, , we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than °b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo < ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of patients (ra, rars and raeb < % blasts) receiving epo ± gcsf between and and having serum epo < ui/l, the response rate at week (iwg criteria) was %. six variables were associated with response to epo ± g-csf for mds: age > years (p = . ), number of prior red blood cell transfusions < packs/months (p = . ), serum epo level < ui/l (p = . ), percentage of blasts < % (p = . ), percentage of erythroblasts > % (p = . ) and low ipss score (p = . ). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p- pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on february , during the course of the first general meeting of the pbsa, which comprised of founding patient organizations. the objectives of the pbsa are: . to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. . to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl - gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl - gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to - u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than patients with haemophilia have now been treated in phase gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, < u/dl rise, in / patients enrolled were observed, although less factor ix concentrate was needed in / subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (> % of normal) for > year in mice following a single treatment. in / patients treated repeated factor viii rises ( . - . u/dl) were seen, but no improvements lasted beyond months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. / patients enrolled sustained levels of factor viii > u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~ u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. con- reference: petz ld, garratty g. immune hemolytic anemias. nd ed. philadelphia: churchill livingstone, , pp - . w-pa- autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [ ] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [ ] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [ ]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (< h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [ ] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about % of cases. gift detects igg antibodies and is positive in about % of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa- practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab ( + = adsorption; + = adsorptions; + = adsorptions; + = or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time ( - h) does not allow for adsorptions, one can dilute the patient's serum (e.g. in , or in ) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that - % of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [ ] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [ ] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [ ] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [ ] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb (cd ) and gp - . the respective antigens, frequencies and alleles are illustrated in table . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [ ] . references are available from the author upon request. w-pa- a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a -yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c -independent binding of c b complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia ( and neutrophils/ml). the diagnosis of annanti hna- a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day and , respectively. omphalitis was successfully treated with -day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for days without success. this was followed by an attempt at neutrophil count increase with -week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at -week intervals. in spite of prolonged neutropenia ( and months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: ( ) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; ( ) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna- a immunization, and ( ) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors ( . %), aged between and years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during , % of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the % of them were successfully processed to ffp. during , % of the donations were fulfilling the specifications and . % of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc / ). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about % during - , suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc / hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa- transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september , , with almost deaths and more than injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the -year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than units of blood used in the first to h. in in new york, the five hospitals closest to the disaster site admitted only disaster victims. the new york blood center, which supplies percent of blood for the city's hospitals, added units to routine inventory at hospitals. the center received telephone calls and collected more than units of blood in the first h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september , america's blood centers collected more units of blood, and the american national red cross collected more units than in the same period the previous year. more than units were collected for the disaster victims, but only units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach %s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june , , a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august , , as shown in the following sections: . the development of a voluntary blood donor system. . removal of the upper age limit for blood donors. . funding for blood donations. from january , , each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa- can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from to %. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around . % of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv , hhv ), parvovirus b , hepatitis g ( . %) and tt (transfusion transmitted) virus ( . %). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around : to : depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values < . g/dl in the first and third trimesters, and < . g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa- impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris , bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach / above years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than % bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts > %) evolving in a short timespan with severe cytopenias, and to aml in approximately % of cases. at diagnosis, % of mds patients are anemic, with an hemoglobin level less than g/l, and % of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of - units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) ( - u/kg tiw, or a - u as single weekly dose) are typically used in low-risk mds. the response rate to epo is - %, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than g/l). absent or low rbc transfusion needs and a serum epo level less than u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable ( - months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to - %, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in , compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin or ug/week) with response rates varying from % to % in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over years and backed by postmarketing experience in countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa- iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss > l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (< weeks) of the anemia is desirable in cases with transferrin saturation < % and ferritin levels < mg/l. with this regime it is possible to collect up to autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed red cell apheresis in patients with diagnosis of hereditary hemochromatosis ( ¥ c y homozygotes, ¥ c y + h d heterozygote) using haemonetics mcs p cell separator (protocol tae) in which red cells are removed from patients in - cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december , and february , have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter and days after drbc procedure. donors suitable to drbc apheresis had to have: age between and years, weight > kg, hb > . g/dl and serum ferritin between and ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of donors who donated drbc during the study period, only males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb . ± . g/dl, ferritin ± ng/ml, serum iron ± microg/dl. on day mean hb was . ± . g/dl (p < . ). on day mean hb was . ± . g/dl (p < . ), ferritin ± ng/ml (p < . ), serum iron ± (p ns). only out of donors ( %) had a ferritin value > ng/ml. in the studied donors the collection of units of rbcs induced an expected reduction of about grams of hb, however only % of this reduction was recovered after days (p < . ). similarly, also iron stores have not been restored after months from donation, as shown by a % reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of units of rbcs (approximately - mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively % of subjects at first blood donation and, in these subjects, hfe mutations prevalence was . for c y and . for h d (velati et al., ) . aims: the role of the c y mutation is well known in the patho-genesis of iron overload, whereas the role of the h d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h d mutation in comparison to a population of blood donors wt/wt for the h d mutation. methods: a total of repeat blood donors were examined in italian transfusion centers ( in northern italy and in southern) for c y and h d mutations. out of those, blood donors heterozygous for the h d mutation and wt/wt for the same hfe mutation, both groups wt/wt for the c y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table summarizes the allelic frequencies in the blood donors. table reports the haematological evaluation in the subjects heterozigous for h d mutation and the wt/wt for the same mutation. conclusions: these data suggest that subjects with h d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h d heteroxygotes or even homozygotes when available, in order to get further insights on the h d role in iron metabolism. background: cd is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and females aged to (median ). twenty of them had never been transfused (group a) and were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd b, anti-cd , anti-cd , and anti-cd . leukocytes were gated according to cd . we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < . ) and controls (p = . ). this downregulation concerned the proportion of cd + cells, that was lower in the transfused patients than the non-transfused (p < . ) and controls (p = . ), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < . ) and group c (p = . ). negative correlation was observed between the cd expression and cd b (p = . ) and cd (p = . ). cd b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = . and . respectively) while the percentage of cd b cells did not differ significantly between the various groups. increased expression of cd was also found in the group a compared to group b (p < . ) and c (p = . ). the proportion of cd + cells did not differ between the various groups. the levels of immuno-reactive svcam- as determined by elisa were found . + . in group a, . + in group b and . + . in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in centers and the gfm has the intention to extend the study to a larger population in at least centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after blood donations or plasma donations. before , each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g % for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g % for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g % for intravenous use (ivig) has been used in the study. the preparation is liquid, % stabilised with glucose of a ph value of . ± . . it is used in those cases where sepsis developed after surgery. both an ivig group (n = ) and a control group (n = ) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = ), and the control group (n = ); pancreatitis (n = ) control group (n = ); intestinal obstruction (n = ) control group (n = ); abdominal organ perforation (n = ) control group (n = ); abdominal perforate injuries (n = ) control group (n = ); serious abdominal interventions (n = ) control group (n = ). the period of hospitalisation of the patients in the ivig group was ± days while the period of hospitalisation in the control group was ± days. the mortality rate in the ivig group was % counter . % in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g % for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: pc were produced by this device and some parameters were studied. for the preparation of pc, bc were pooled using the orbisac set, with an integrated filter (pall lrp ). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol / . the pc was stored in a gambro elp bag. results: the average platelet count per unit was . ¥ e . the platelet recovery from pooled bc was . % (range . %- . %). all products of the tested pc containing < ¥ e wbc (by flow cytometry). the values of ph on day and of storage were . and . . the swirling phenomenon was good until day °. the average loss of haemoglobin per bc was . g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood ( ml) was collected in top-and-bottom bags, and randomly selected to be processed by either ( ) current routine or ( ) atreus c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc d (atreus). lr-rccs were stored at °c and sampled until day . various in vitro measurements were performed (n = per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was % lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained ± ¥ platelets in ± ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. . ± . . ± . < < . aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day . on day , either , , , or pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days , and for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein b (gp b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of pools were studied, each of , , and pc's. the mean platelet yield was . ¥ e with a range of . - . ¥ e . the five pc's had a mean yield of . ¥ e and all maintained a ph > . on day . all products had less than ¥ e residual wbc. platelet quality data is presented in the table. data are the mean ± sd, n = . conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may . the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were blood units (ek) issued for the mentioned period to pediatric clinic for pediatric patients (~ , % units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to - bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was - ml er (depends on age and body weigh). ek was issued as washed concentrates, ek were filtered and ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than times than obstetric ones ( vs ) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk is an immunohaematological analyser for detection of red blood cells antigens of ab , rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: blood samples of donors were tested between july and january . all samples were analysed for ab blood group. samples were tested for rhd antigen and ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab , rh (d, c, c, e, e) and k were investigated truly by first testing at . % samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about . % samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify bp in sregion of hbv genome in the first pcr and bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to . iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of % (hc-ii; %) and a specificity of % (hc-ii; %) (p < . ). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p- blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in . since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from . january to . december . results: in whole blood participated in the consumption with . %, packed red blood cells with (rbc) only . %, washed rbc were used in . % of the cases. in whole blood participated in the consumption with . %, packed rbc with . %, washed rbc with . % and rbc in additive solution with . %. as far as plasma preparations are concerned, there has been, since , a great consumption of plasma -witch was separated from whole blood in period up to five day in . % cases, and small consumption of fresh frozen plasma (ffp) only . %. since , there has completely been cancelled the production of five day old plasma, only ffp is being used. from to for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from units to units per year. in , after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from units in to units in . conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we total febrile non haemolitic male transfusion reaction f e m a l e alloimunisation on le/hla male antigens f e m a l e kandidates of renal male transplantation female lupus nephritis male f e m a l e total female male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, cgy of gamma radiation reduce lymphocyte response to mitogens by %.the aim of the study: . to estimate potassium level increment in stored irradiation blood units. . to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. . to evaluate the expiratory date of blood units post irradiation. the study included units of blood collected in cpd-adsol (as- ). in twenty units the blood collection bag was with inline leucodepletion, while the other units were non leucodepleted. all the units were irradiated using caesium as a source of irradiation, with a dose of - cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from st day post irradiation and continues to day post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day , p value of more than . . . gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. . there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within h post irradiation. . there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. . an out date of days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within - h post irradiation. . the percentage of irradiated blood units requested by our physicians ( . %) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from % ( ) to . % ( ) and increasing trend of using packed red cells component therapy from . % ( ) to . % ( ) . there is also increasing trend of using fresh frozen plasma (ffp) from . % ( ) to . % ( ) . comparing and year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during year period, a total of units of blood and units of f.f.p were used. more specifically, the results can be shown in the following table . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a -year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt s, pt %, fibrinogen . g/l, fv % (other factors were in normal range), platelet count ¥ /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about . bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of mg/kg (~ ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding ( %), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted ( %) along with the presence of factor v inhibitor at a level of . bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of : respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr t/t cpd/sagm (macopharma). whole blood ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. ± °c for to h. after component separation (beckman coulter j mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. ± °c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit . wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr and abc leucoflex lcr ( . and . ) is highly efficient. the use of abc leucoflex lcr showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = . < . ). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = . < . ). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable : , rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst system and leucoflex lst system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed donations with the lst system and donations with the abc lst system. all the donors were random male volunteers and they were meant to donate mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows: . no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst system compared to the abc lst system. . the duration of filtration has been found without statistically significant differences between the two systems. . the loss of volume in the filter of lst is higher than in the abc lst (p = . < . , which is statistically significant). . there was very good leucodepletion with the lst systems (median reduction of the wb cells . log) but there was a superiority of the abc lst over the lst concerning the leucodepletion per litter and per unit (p: . and p: . respectively). . the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst . evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged - days, filtered and consecutively washed with ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were: . the duration of priming and filtering the rccs. . the loss of volume in the filter. . the efficiency of the filtration. . the acceptance of the personnel of a new (to them) filtration system.our results have as follows: . we counted the duration of priming and filtration. median time of priming was s (range - ) and of filtration was min (range - ). . the median volume lost in the filter (as calculated) was . ml (ranging from . to . ). this narrow range is apparently due to the non-flexible cell of the filter. . the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was . ¥ (range . - . ¥ ) and per unit was . ¥ (range . - . ¥ ). the median reduction of the wbc count was . log (range . - . ). . the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly blood banks and blood stations in whole country. the overall blood collected at those centers is about . . units. nearly one in third is being collected at government hospitals ( . %), nearly the same is collected at university hospital blood banks ( . %). the third major group is the red crescent society blood centers-rcsbc ( . %), followed by the social security hospitals ( . %). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) > . . . - . . . - . . . - . . < . . conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among topics in five broad categories. the top two topics were selected for the forum from the completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord- -x t h gu authors: madariaga, m. l. l.; guthmiller, j.; schrantz, s.; jansen, m.; christenson, c.; kumar, m.; prochaska, m.; wool, g.; durkin, a.; oh, w. h.; trockman, l.; vigneswaran, j.; keskey, r.; shaw, d. g.; dugan, h.; zheng, n.; cobb, m.; utset, h.; wang, j.; stovicek, o.; bethel, c.; matushek, s.; giurcanu, m.; beavis, k.; disabato, d.; meltzer, d.; ferguson, m.; kress, j. p.; shanmugarajah, k.; matthews, j.; fung, j.; wilson, p.; alverdy, j. c.; donington, j. title: clinical predictors of donor antibody titer and correlation with recipient antibody response in a covid- convalescent plasma clinical trial date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: x t h gu background: convalescent plasma therapy for covid- relies on the transfer of anti-viral antibody from donors to recipients via plasma transfusion. the relationship between clinical characteristics and antibody response to covid- is not well defined. we investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. methods: multivariable analysis of clinical and serological parameters in confirmed covid- convalescent plasma donors days or more following symptom resolution was performed. mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in convalescent plasma recipients with severe covid- . results: mean symptom duration of plasma donors was . and . % ( / ) had been hospitalized. antibody titers ranged from to : , (anti-receptor binding domain (rbd)) and to : , (anti-spike). multivariable analysis demonstrated that higher anti-rbd and anti-spike titer were associated with increased age, hospitalization for covid- , fever, and absence of myalgia (all p< . ). fatigue was significantly associated with anti-rbd (p= . ) but not anti-spike antibody titer (p= . ). in pairwise comparison among abo blood types, ab donors had higher anti-rbd titer than o negative donors (p= . ) and higher anti-spike titer than o negative (p= . ) or o positive (p= . ) donors. eight of the ten recipients were discharged, one remains on ecmo and one died on ecmo. no toxicity was associated with plasma transfusion. after excluding two ecmo patients and adjusting for donor antibody titer, recipient anti-rbd antibody titer increased on average % per day during the first three days post-transfusion (p= . ) and anti-spike antibody titer by . % (p= . ). conclusion: advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to covid- . despite variability in donor titer, % of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. a more complete understanding of the dose-response effect of plasma transfusion among covid- patients is needed to determine the clinical efficacy of this therapy. convalescent plasma therapy has historically been used as a treatment during epidemics ( ) . in this therapy, neutralizing anti-viral antibodies, as well as non-neutralizing antibodies and other immunomodulators, are transferred via plasma transfusion from those who have recovered from disease to those currently infected ( ) ( ) ( ) . for patients with severe covid- , convalescent plasma therapy has safely led to improvement in clinical and radiographic parameters ( ) ( ) ( ) ( ) ( ) ( ) . once adequate numbers of people convalesced and supply chain logistics were established, providing plasma therapy to a large number of patients has proven feasible ( ) . efficacy of convalescent plasma therapy relies on a robust antibody response in convalescent plasma donors. measurements of antibody response among patients with covid- demonstrate that the majority develop igm and igg within weeks of symptom onset, with specificity towards receptor binding domain (rbd) and spike protein viral epitopes correlating with virus neutralization ( ) ( ) ( ) . strikingly, a small proportion of recovered covid- patients show no detectable antibodies to these epitopes ( , ) . the relationship between host characteristics, disease course and variability in antibody response to covid- is poorly understood. the aim of this study was to establish a translational convalescent plasma program to investigate the relationship between clinical and serological parameters in convalescent plasma donors and define the antibody response of convalescent plasma recipients. this was a prospective open label clinical study to assess the feasibility, safety and immunological impact of delivering anti-sars-cov- convalescent plasma to hospitalized patients aged years or older with severe or life-threatening covid- disease within days from the onset of their illness. this study was conducted at university of chicago medicine (ucm) from april , to may , . the final date of follow-up was may , . we used existing hospital infrastructure and personnel to build the convalescent plasma program at a time when state-wide shelter-in-place orders were active, elective procedures were not being performed, and non-covid- -related research activities were halted. the donor enrollment team consisted of two surgeons, two surgical residents, and three physician assistants. a dedicated study coordinator was present at the ucm blood donation center to facilitate whole blood donation and collect research samples. recipients were selected during daily videoconference with infectious disease. one surgeon visited the hospital covid- unit daily to obtain consent and research samples. plasma donors were age or older, able to donate blood per standard ucm blood donation center guidelines, had a documented covid- polymerase chain reaction (pcr) positive test, and complete resolution of symptoms at least days prior to donation. recruitment occurred via social media, news outlets, word-of-mouth and announcements in university and community bulletins. the ucm infectious disease team provided an institutional list of patients with a positive pcr test for covid- , and their physicians were emailed to request permission to contact the patient for donor participation. interested plasma donors were directed to fill out a short screening survey online. potential donors meeting study criteria were screened for eligibility, reported symptoms and comorbidities, consented, and were scheduled for donation at the ucm blood donation center in a single telephone encounter. after meeting the ucm blood donation center eligibility, whole blood was collected and processed according to standard ucm blood donation center procedures. standard whole blood donation was used for plasma collection because it fit into preexisting ucm blood bank infrastructure and workflow therefore facilitating rapid deployment of a collection process, and allowed red blood cell and unused plasma units to be used in the regular blood bank inventory. during blood donation, a single research sample was collected at the same time as blood samples for standard immunohematology testing and infectious disease screening. leukocyte filters used in separation of constituent blood parts were also collected for research. eligibility for convalescent plasma recipients included: age or older, laboratoryconfirmed covid- , within days from the start of illness and severe or life-threatening covid- as defined by the united states food and drug administration (fda) ( ). severe covid- was defined as dyspnea, respiratory frequency ≥ /min, blood oxygen saturation ≤ %, partial pressure of arterial oxygen to fraction of inspired oxygen ratio < , and/or lung infiltrates > % within to hours. life-threatening covid- was defined as respiratory failure, septic shock, and/or multiple organ dysfunction or failure. patients who were pregnant, received pooled immunoglobulin in the past days or had a history of transfusion reaction were excluded from this study. recipients had routine pre-transfusion testing, in keeping with institution policies. on the day of enrollment, an emergency investigational new drug (eind) application was filed and approved for each recipient by the fda ( ). subsequently, one abo-compatible unit of convalescent plasma (~ ml) was transfused over hours. repeat administration of convalescent plasma occurred in one recipient (r ). blood samples and nasopharyngeal swabs were obtained at day , , , , post transfusion. the primary outcome was feasibility as defined by the collection of convalescent plasma and its administration into hospitalized patients. secondary outcomes included type and duration of respiratory support, cardiac arrest, transfer to intensive care unit (icu), length of stay, mortality, complications of plasma administration, process outcomes, and antibody titer of plasma donors and recipients. levels of anti-rbd and anti-spike antibodies were measured by enzyme-linked immunosorbent assay (elisa) in blood samples at time of donation and plasma recipients, as previously described ( ) . nasopharyngeal specimens were obtained by flocked swabs in plasma recipients and analyzed by rt-pcr to detect sars-cov- rna. study data were collected and managed using redcap electronic data capture tools hosted at ucm ( , ) . donor patient characteristics were compared using the chi-squared test for categorical variables and the two-sample t test for continuous variables. univariate regression analysis for antibody titer (anti-rbd and anti-spike) was conducted against age, sex, body mass index (bmi), previous pregnancy, previous blood donation, blood type, symptoms (fever, cough, sore throat, dyspnea, abdominal pain, aguesia, anosmia, fatigue, myalgia, headache), co-morbidities (respiratory, cardiovascular, renal, diabetes, autoimmune disease, cancer, liver disease), smoking history, travel in the past months to the united states, asia or europe, symptom duration, interval from symptoms resolution to plasma donation, and hospitalization. pairwise comparison using t tests without adjusting for multiple comparisons was used to compare antibody titers among different abo blood groups. we conducted multivariable analyses to identify prediction models for anti-rbd and anti-spike antibody titers among convalescent plasma donors. best subset variable selection method was chosen to identify the subset of predictors that maximizes the adjusted r-squared among all possible models. to compare daily change in recipient antibody response, we fit mixed effects regression models with piecewise linear trend with a change point at days after intervention for log-transformed antibody titers. we considered recipients on extra-corporeal membrane oxygenation (ecmo) (r and r ) separately from recipients not on ecmo (r , , , , , , , ), because ecmo recipients had different baseline characteristics. data analysis was performed using software r, version . . . mixed effects regression models were fit using the lmer function of the lme package ( ) . data analysis was conducted within rstudio environment, and r markdown files with fully reproducible data analysis can be obtained from the authors upon request. this study was approved by the institutional review board (irb - ). all participants (plasma donors and plasma recipients) gave written informed consent prior to inclusion in the study. analysis was performed by mlm and mg. this clinical trial was registered at clinicaltrials.gov with identifier nct . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint potential plasma donors were recruited to our study over days (table ). the average age was . years (range to ), the majority were female ( . %), and % had never donated blood before. potential donors with confirmed positive covid- pcr (n= , %) were more likely to be male, have ageusia and anosmia, and lack cough, sore throat and dyspnea compared to the symptomatic patients who had clinical signs of covid- but were never tested (table ) . among plasma donors (n= ) who donated as of publication, average symptom duration was . ± . days, ( . %) had respiratory comorbidities such as asthma, chronic obstructive pulmonary disease or obstructive sleep apnea, and ( . %) had been previously hospitalized for covid- ( table ). the average interval between symptom start and plasma donation was . ± . days. donor antibody titers measured on day of plasma donation ranged from to : , (anti-rbd) and from to : , . (anti-spike) ( table ). in univariable regression analysis, higher average anti-rbd and anti-spike antibody titers were associated with plasma donors who were older, male, had higher bmi, had fever and had been hospitalized (p< . , supplemental table ). in a pairwise comparison among abo groups without adjusting for multiple comparisons, ab donors had higher anti-rbd titer than o negative donors (p= . ) and higher anti-spike titer than o negative (p= . ) or o positive (p= . ) donors. to determine predictors of anti-rbd and anti-spike antibody titer, we performed best subset multivariable analysis including age, sex, blood type, history of previous blood donation, fever, cough, fatigue, myalgia, symptom duration, hospitalization and travel in the united states within the past months. significant predictors of anti-rbd antibody titer were age (p= . ), fever (p< . ), previous hospitalization (p< . ), lack of myalgia (p= . ), and fatigue (p= . ) (r-squared= . , adjusted r-squared= . , table ). significant predictors of anti-spike antibody titer were age (p= . ), fever (p= . ), previous hospitalization (p= . ), and absence of myalgia (p< . ) (r-squared= . , adjusted r-squared= . , table ). o positive blood type was associated with lower anti-rbd (p= . ) but did not meet significance threshold for antispike (p= . ). ten hospitalized patients with severe or life-threatening covid- received plasma on day ( figure , table ). plasma recipients were on average . years old (range to ) and % female. the average time from start of symptoms to plasma transfusion was days (range to ) and the average time from hospital admission to plasma transfusion was days (range to ). at the time of plasma transfusion, two patients were on ecmo, one patient was mechanically ventilated, two patients were on high-flow nasal cannula (hfnc), four patients were on nasal cannula and one patient was on room air. five patients had received other therapies for covid- before transfusion, including remdesivir, tocilizumab, anakinra and hydroxychloroquine. two plasma recipients were on chronic immunosuppression after transplantation. figure shows selected clinical and laboratory parameters of convalescent plasma recipients. only one recipient (r ) had fever prior to transfusion and this resolved by day post-transfusion. r and r remained on ecmo throughout the study period. in the remaining recipients, oxygen requirements improved to room air or nasal cannula. the sequential organ failure assessment (sofa) score ( ) was calculated for recipients on mechanical ventilation or ecmo and showed a general trend towards improvement; notably both ecmo patients were weaned off vasopressor and intra-aortic balloon pump support by days post-transfusion. levels of inflammatory marker c-reactive protein (crp) were variable. crp decreased in six recipients (r , r , r , r , r , r ). sars-cov np swab pcr remained positive in patients and turned negative in patients; patient (r ) had been positive for sars-cov days prior to plasma transfusion but was negative for sars-cov on day of transfusion ( figure ). at last follow-up, patient on ecmo remained in the hospital (r ), patient on ecmo was transitioned to comfort care and died on day after plasma transfusion (r ), patients were discharged to rehabilitation facilities and patients were discharged to their place of residence ( figure ). on day of transfusion, anti-rbd antibody titers were undetectable in recipients (r , r , r ) and anti-spike antibody titers were undetectable in recipients (r , r , r ) ( table and figure ). both patients on ecmo had very high antibody titer at day which decreased in the days after transfusion (figure ). the remaining plasma recipients showed increase in antibody titer within the first three days after transfusion (r , , , , , , ) with the exception of r who did not show any antibody titer until day (anti-spike) and day (anti-rbd) after transfusion ( figure ). we performed a mixed effects model for log-transformed reciprocal antibody titer adjusting for donor antibody titer level looking at the first days post-transfusion among the non-ecmo patients. after plasma transfusion, recipient anti-rbd antibody titer increased on average by % per day (p= . ) and recipient anti-spike antibody titer increased on average by . % per day (p= . ) (figure ). among the two ecmo recipients, recipient antibody response was not significantly changed until three days after plasma transfusion (decreasing by . % per day for anti-rbd titer and . % per day for anti-spike titer, p< . ) (figure ) . we monitored the clinical status of the recipients before, during and immediately after transfusion. no recipients experienced toxicity associated with plasma transfusion. there was no clinical deterioration or worsening of disease status immediately related to plasma transfusion. convalescent plasma transfusion was safe in high-risk individuals in our study: immunosuppressed patients after stem cell and lung transplants and a patient with end-stage renal disease on dialysis. we developed a translational convalescent plasma treatment program within the existing hospital infrastructure during the covid- pandemic that provided a new therapeutic option for patients while assessing the antibody profile of both convalescent and hospitalized patient populations. our multivariable analysis demonstrated that clinical characteristics can predict serological response of antibodies associated with virus neutralization ( ) . higher anti-rbd and anti-spike antibody were more likely found in convalescents who were older, hospitalized, had fever, and lacked myalgia. fatigue also significantly predicted higher anti-rbd but not antispike antibody titer. variability in convalescent populations and immune response to viral infection may explain why recovery is not always marked by seroconversion ( , ) . indeed, in our study four plasma donors (as well as four plasma recipients) had undetectable antibody titers. disparate plasma donor populations and geography may explain why symptom duration and elapsed time from symptom onset was associated with antibody response in new york city ( ) but not among our patients in chicago. disparate plasma donor populations and geography may also explain antibody variability. these data highlights that the impact of variability in antibody type and titer on virus-neutralizing activity and long-term immunity is unknown. interestingly, we found that antibody titers significantly differed across abo blood type groups, ( ) . further studies on the relationship between abo polymorphism and antibody titer may uncover genetic determinants of the host response to covid- . recipients received plasma with a range of antibody titer from : to : , (anti-rbd) and : to : , (anti-spike). despite this, % of recipients demonstrated a significant increase in anti-spike and anti-rbd antibody titer in the days post transfusion that was independent of donor antibody titer and were discharged after clinical improvement. interestingly, recipient antibody titer continued to increase up to days in four recipients (r , , , ); in contrast, the two most severely ill patients on ecmo who had the highest antibody titers (up to : , anti-spike antibody in r ) showed a decrease in antibody titer after receiving plasma on day - of illness. importantly we demonstrate the safety of transfusing convalescent plasma in immunosuppressed patients after lung transplantation and stem cell transplantation. none of the plasma recipients in this study deteriorated after convalescent plasma transfusion, consistent with the safety profile of other trials ( ) ( ) ( ) ( ) ( ) ) . repeat plasma dose in recipient r was also welltolerated. pre-clinical models of sars-cov and clinical experience of other viral illness had raised concern about the potential for non-neutralizing antibody to cause antibody dependent enhancement of disease, which was not seen here despite variable titers of donor antibodies ( ) ( ) ( ) . the variability in post-transfusion recipient antibody titer and clinical response seen here and in other studies ( , , , ) indicates that the therapeutic activity of convalescent plasma depends on the timing of treatment and composition of convalescent plasma. indeed, plasma contains more than , proteins, including albumin, immunoglobulins, complement, and coagulation factors as well as organic compounds such as cytokines ( ) . convalescent plasma drawn shortly after natural infection ( , ( ) ( ) ( ) ( ) may be enriched for populations of protective antibodies not present in plasma derived from long-recovered or rarely-hospitalized donors studied here. furthermore, immunomodulatory and non-virus neutralizing antibody effects such as stimulation of the host humoral immune response and facilitating viral uptake into cells via fc-receptors to increase viral antigen presentation to other effector cells may contribute to disease recovery. taken together, while randomized controlled efficacy trials for convalescent plasma therapy in covid- are currently underway, establishing effective anti-covid- plasma-based therapy will require both an understanding of the precise dose and type of virusneutralizing antibody and in-depth characterization of plasma donor-recipient pairs. the availability of a pre-existing hospital-based blood collection facility within our medical center significantly eased the procurement of convalescent plasma and will allow us to assess immunological characteristics of donor-recipient pairs in future studies. such hospitalbased blood collection facilities have been declining in number across the united states for several decades ( ) . cultivating region-specific convalescent plasma inventory may potentially facilitate the identification and isolation of antibodies with specific activity against local virus strains and be a useful model for future outbreaks. in addition, convalescent plasma derived from whole blood collection is a rapidly scalable technique that requires basic phlebotomy and blood separation rather than a dedicated apheresis personnel and equipment. furthermore, a significant proportion ( . %) of our plasma donors had never donated blood before, indicating that a convalescent plasma donation program can serve as important community outreach during a time when patients avoid hospitals that are perceived as unsafe ( ) . in summary, development of a convalescent plasma program is feasible, rapidly deployable and economical when existing resources of equipment, space and personnel are used. establishing the clinical predictors of high antibody titer and understanding the serological posttransfusion response may guide patient selection and shed light on antibody response to covid- . further work characterizing convalescent plasma donor and recipient pairs is needed to elucidate mechanisms of convalescent plasma therapy and demonstrate optimal viral epitope therapeutic targets. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . , interval of symptoms to plasma donation, blood type were not significantly associated with anti-rbd or anti-spike antibody titer. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint treatment of influenza pneumonia by the use of convalescent human serum: preliminary report the convalescent sera option for containing covid- convalescent plasma as a potential therapy for covid- convalescent plasma in covid- : possible mechanisms of action effectiveness of convalescent plasma therapy in severe covid- patients treatment of critically ill patients with covid- with convalescent plasma treatment with convalescent plasma for critically ill patients with sars-cov- infection use of convalescent plasma therapy in two covid- patients with acute respiratory distress syndrome in korea patients with convalescent plasma in convalescent plasma treatment of severe covid- : a matched control study early safety indicators of covid- convalescent plasma in , patients humoral immune response and prolonged pcr positivity in a cohort of sars-cov patients in the new york city region temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study antibody responses to sars-cov- in patients of novel coronavirus disease seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support the redcap consortium: building an international community of software platform partners fitting linear mixed-effects models using lme the sofa (sepsisrelated organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid- susceptibility inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection current studies of convalescent plasma therapy for covid- may underestimate risk of antibody-dependent enhancement treatment with convalescent plasma for influenza a (h n ) infection use of convalescent plasma therapy in sars patients in hong kong continued decline in blood collection and transfusion in the united states- delayed access or provision of care in italy resulting from fear of covid- we thank all the plasma donors for their willingness to help in a time of need and the blood bank staff for their excellent care. we thank samantha guerrero, alyssa anneken, bruce boehrnsen and rohit allada for helping us establish the infrastructure for this study. we thank the university of chicago and department of surgery, university of chicago for providing support for this study. this study was funded by the department of surgery, university of chicago and the national institute of allergy and infectious diseases (niaid) collaborative influenza aka, above the knee amputation; chf, congestive heart failure; dm, diabetes mellitus; dvt, deep venous thrombosis; esrd, endstage renal disease; htn, hypertension; nafld, non-alcoholic fatty liver disease; pe, pulmonary embolism; pvd, peripheral vascular disease.all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this prep this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- -l hsik authors: chan, conrad e. z.; lim, angeline p. c.; macary, paul a.; hanson, brendon j. title: the role of phage display in therapeutic antibody discovery date: - - journal: int immunol doi: . /intimm/dxu sha: doc_id: cord_uid: l hsik phage display involves the expression of selected proteins on the surface of filamentous phage through fusion with phage coat protein, with the genetic sequence packaged within, linking phenotype to genotype selection. when combined with antibody libraries, phage display allows for rapid in vitro selection of antigen-specific antibodies and recovery of their corresponding coding sequence. large non-immune and synthetic human libraries have been constructed as well as smaller immune libraries based on capturing a single individual’s immune repertoire. this completely in vitro process allows for isolation of antibodies against poorly immunogenic targets as well as those that cannot be obtained by animal immunization, thus further expanding the utility of the approach. phage antibody display represents the first developed methodology for high throughput screening for human therapeutic antibody candidates. recently, other methods have been developed for generation of fully human therapeutic antibodies, such as single b-cell screening, next-generation genome sequencing and transgenic mice with human germline b-cell genes. while each of these have their particular advantages, phage display has remained a key methodology for human antibody discovery due its in vitro process. here, we review the continuing role of this technique alongside other developing technologies for therapeutic antibody discovery. antibodies are one of primary effector molecules of the human immune system and bind their target antigen through their complementarity-determining regions (cdrs) displayed on the fab (antigen-binding fragment) portion of the antibody (fig. ) . the cdrs encode unique structural diversity and provide antibodies with the ability to recognize a wide variety of targets. the protective efficacy of an antibody can be achieved through binding alone if it results in neutralization of a function of the target or may require additional immune functions provided by the fc (crystallizable fragment) portion of the antibody molecule with receptors found on immune cells or complement proteins in serum. antibody fc region functionality can trigger or augment a number of different host defence mechanisms including phagocytosis, release of cytotoxic or immunomodulatory biomolecules and the complement cascade and formation of the membrane attack complex. these properties potentially make antibodies extremely potent molecules for therapy and they have been developed into an important class of drugs for the treatment of numerous cancers, autoimmune and infectious diseases ( ) . display of properly folded proteins on phage through fusion with phage coat proteins was initially conceived as a means to identify the sequence of the protein target which binds to an antibody ( ) . ironically, one of the most common uses of phage display today, almost years after its initially development, is to discover antibodies against selected protein targets. originally developed by greg winter et al. at the mrc laboratory of molecular biology and richard lerner, carlos barbas et al. at scripps research institute, the basic concept that a vast antibody repertoire could be displayed on phage with their genetic sequences packaged within has allowed for the robust screening of a variety of target antigens and rapid recovery of specific antibody sequences for recombinant expression ( ) ( ) ( ) . today, a wide variety of antibody phage display libraries exist, with the antibodies displayed either as fabs, scfvs (single-chain variable fragments) or sdabs (single-domain antibodies) in fusion with typically the gene iii coat protein ( , ) (fig. ). the small size and solubility of the phage particle (up to particles/ml) has allowed repertoire sizes of up to - to be efficiently displayed and manipulated, which improves the likelihood of finding suitable antibodies ( ) . these antibody sequences can be obtained from natural sources (animal or human) or synthetically constructed [usually a mix of pre-defined framework regions (frs) with randomized sequences in the cdrs] and isolation of specific sequences is carried out by repeated enrichment against the target of interest. the defining attribute of all phage display libraries is the physical linking of antibody phenotype (specificity and affinity) with genotype (sequence) via the phage particle-this allows for in vitro selection on immobilized antigen or whole cells (fig. ) . as this method by-passes the immune system, the usual factors that influence antibody development and limit diversity based on those that can be isolated in animals are not applicable. as such, antibodies can be isolated against targets that have low immunogenicity in animals (nonproteinaceous antigens), to regions of a protein not usually targeted due to the presence of immunodominant epitopes elsewhere, or to hydrophobic targets that are challenging for in vivo manipulation ( ) ( ) ( ) . as such, phage display offers high potential for discovery of novel therapeutic antibodies that cannot be generated naturally through in vivo immunization or infection. furthermore, the ability to display synthetic repertoires devoid of any negative selection has made this technique a key method for the generation of potent fully human antibody therapeutics against self-antigens ( ). nixon et al. provide an in-depth review of the clinical development of such drugs ( ). each antibody comprises two heavy and two light chains each of which have four and two immunoglobulin domains, respectively. the first domain is variable and determines specificity (vl and vh) while the second domain of the light chain (cl) and the second to fourth domains of the heavy (ch - ) are constant across all antibodies of the same isotype. the light chain and first two domains of the heavy chain form the fab, which is the portion expressed on the phage. the last two domains of the heavy chain form the fc and are responsible for immune function through engagement of receptors on immune cells. heavy and light chains are linked through a single disulfide bond (orange) between the cl and ch domains and the two heavy chains have multiple disulfide bonds at the hinge region between the ch and ch . an scfv consists of just variable light and variable heavy domains joined by a flexible polypeptide linker while an sdab as the name implies is only a single immunoglobulin (usually vh) domain which is sufficient for binding. (b) variable domain genetic structure and construction of a natural phage display library. each variable domain consists of three hypervariable cdrs interspersed between the more conserved frs. the immunoglobulin domain folds such that the cdrs are brought together to form the antigen-binding surface at the tip of the fab. degenerate primers (arrows) are used to amplify the entire variable heavy and light chains (or alternatively variable and first constant domain) from a source of b cells and cloned in-frame with the phage coat protein (usually gene iii) into e. coli to produce an fab, scfv or sdab library. the rest of the phage genome is supplied through replication defective helper phage to produce antibody-displaying phage. general strategy for phage panning. polyclonal phage expressing recombinant antibodies on their surface is applied to target antigen presented as either immobilized on a magnetic bead, polystyrene surface or on the surface of a whole cell. phage carrying antigen-binding fab bind and non-specific fab are removed through stringent washing. antigen-bound phage is eluted off either typically through ph change or protease digestion and re-infected into e. coli, from which a new library enriched for antigen-binding clones can be made. after several cycles, the library would be sufficiently enriched so that the individual clones can be isolated from e. coli stock, expressed as monoclonal phage, tested, sequenced and the specific antibodies expressed recombinantly. another key attribute of phage display is that selection can be far more rapid as compared to the natural immune response, and in our laboratory, we have been able to identify monoclonal antibodies (mabs) in as short a period as week ( ) . this is particularly useful for generation of antibodies against novel or genetically modified pathogens in an emergency outbreak scenario. in recent years, however, alternative antibody discovery systems have been developed, such as high-throughput testing of individual b cells and next-generation sequencing of b-cell repertoires. we review here the use of phage display against various types of targets, such as self-antigens, non-protein targets and infectious disease agents, in relation to on-going developments in both phage display and other antibody discovery technologies. in the human immune system, naive b cells targeting selfantigens are deleted by negative selection and immunization in a different host is therefore required to generate specific antibodies against human targets. however, the resultant antibodies, even after humanization by replacement of constant regions or of frs with human equivalents, are usually more immunogenic than a human-derived antibody ( ) . while this can result in increased toxicity, the primary concern is the potential for reduced clinical efficacy of the therapy which can occur with development of a humoral immune response against the antibody. for self-antigens, such as tumour cell markers, phage display is an ideal technique for the generation of fully human antibodies due to the absence of tolerance mechanisms. semi-synthetic or synthetic libraries, whose sequences have not been negatively selected for any antigen, are usually the first choice. an additional advantage of these libraries is that design features can be incorporated to allow facile affinity maturation, such as cdrs flanked by restriction sites for easy replacement or appropriate consensus primers for targeting of specific cdrs for directed mutagenesis ( ) . several fully human synthetic library-derived mabs have been approved by the us food and drugs administration or completed clinical trials. the first of these was adalimumab, an anti-tnf-α antibody developed by cambridge antibody technologies for the treatment of rheumatic arthritis and crohn's disease ( , ) . subsequently, other antibodies were developed for the treatment of cancer and autoimmune disease such as necitumumab (non-small cell lung cancer), ramucirumab (gastrointestinal cancers) and belimumab (systemic lupus erythematosus) ( , , ) . many others are currently at advanced stages of clinical development and may reach market approval within the next few years ( , ) . as mabs gain popularity as a form of alternative drug therapy, the use of phage displayed antibody libraries has also gained a reputation as the premier technology for antibody discovery. semi-synthetic libraries, which were the first to be made, typically consist of frs sourced from naturally occurring antibodies and cdr regions synthesized from degenerate oligonucleotides ( , ) . in many instances, to simplify construction, only the cdr region was fully synthetic. subsequently, libraries with fully synthetic variable sequences were introduced, with the fr based on consensus sequences from natural antibodies but optimized for high expression on phage in escherichia coli to reduce the effect of variable expression of the human framework sequences on selection. the first fully synthetic human combinatorial antibody libraries (hucal) generated to produce therapeutic antibodies was reported by morphosys in with their first version of the hucal ( ) . several versions of the hucal has since been constructed that incorporate additional features such as improved phage elution by disulphide bond cleavage, selection for in-frame clones, additional cdr loop length and diversity and optimization for expression in mammalian cell culture through removal of unwanted potential post-translational modification sites ( , ) . these modifications have resulted in an increase in the number of antibodies isolated per target antigen. there are several antibodies generated from the morphosys libraries currently in various stages of clinical development; a recent example being the potent anti-cancer activity of omp- r , with activity in multiple preclinical human tumour models ( ) . this antibody targeting the wnt pathway was isolated from the hucal gold library and is currently in phase clinical testing. while synthetic libraries have allowed the development of human antibodies with reduced immunogenicity relative to humanized animal antibodies against self-antigens, the cdrs of these libraries can also be immunogenic as often the sequences are not naturally present in the human antibody repertoire ( ) . with this in mind, another approach to build synthetic libraries has been to generate novel variable region sequences by overlapping pcr of individual amplified germline cdrs with respective fr ( ) . while allowing generation of novel sequences that target self-antigens, it also prevents the generation of synthetic sequences that could contain high levels of t-cell epitopes and be strongly immunogenic. indeed, antibodies obtained from this library had a proportion of t-cell epitopes no higher than that of naturally occurring antibodies. an antibody against icam- was isolated from this library and shown to have potent anti-myeloma activity in vivo ( ) . over the years, semi-synthetic and fully synthetic libraries have shown utility beyond targeting of self-antigens, as these libraries are also capable of generating antibodies of high affinity against a wide range of target antigens ( , , ) . one such high-affinity antibody which was recently approved for clinical use is raxibacumab for the treatment of inhalational anthrax ( ) . extremely high diversity synthetic libraries have also been generated with the goal of providing higher affinity antibodies than currently approved therapeutic antibodies, thus circumventing the need for further improvement by affinity maturation. hoet et al. at dyax corp reported the isolation of very high-affinity antibodies through synthetic diversity introduced into the key antigen-contact sites of cdr and cdr within the heavy chain in combination with naturally occurring diversity in the heavy chain cdr and light chains from donors with autoimmune diseases ( ) . their human antibody phage display libraries have produced multiple drug candidates currently in different phases of clinical development. non-protein targets: carbohydrates, lipids and posttranslationally modified proteins while antibodies are typically raised against proteins, the first therapeutic antibodies in clinical use in the early th century, prior to the discovery of antibiotics, were actually polyclonal preparations that recognized the carbohydrate coat of pathogenic bacteria such as neisseria meningitidis and streptococcus pneumoniae. these were raised in animals against inactivated whole bacteria for treatment of severe bacterial infections and each preparation was effective only on bacterial strains with homologous carbohydrate structures ( , ) . this lead to a realization that antibody recognition of the coat structure was an important correlate for efficacy, and the functional classification of bacteria into serotypes for proper matching of antibody and bacterial strain. the requirement for multiple polyclonal antibodies for treatment of even one bacterial species due to large numbers of serotypes was one of the reasons for the switch to more broadly acting antibiotics once they became available ( ) . nonetheless, their effective use is an indication of the therapeutic potential of non-protein targets. to generate high-affinity antibodies against such targets, a strong immune response is required which is usually stimulated through conjugation with a protein carrier, delivery with an adjuvant such as lipid a or as an antigen complex with proteins such as whole inactivated bacteria ( ) ( ) ( ) ( ) . even so, the antibodies obtained can often be low affinity igm due to the inability to engage the mechanisms required for affinity maturation and class switching, hence limiting the utility of animal immunization for antibody generation ( , ) . on the other hand, phage display has been used to produce anti-carbohydrate antibodies of reasonable affinity in the low nanomolar range ( ) . in particular, a phage library constructed with the heavy chain cdr enriched for basic residues to improve binding to negatively charged carbohydrates produced anti-carbohydrate antibodies that had relatively high affinity (k d ≈ nm) and excellent specificity ( ) . although no anti-carbohydrate antibodies have been approved for routine clinical use, such antibodies have been shown to be able to target both carbohydrate-rich tumour antigen and bacterial lipopolysaccharides, the latter having been used on occasion to treat severe cases of sepsis ( , ) . anti-lipid/lipoprotein antibodies are fairly uncommon in nature and in humans, they are usually associated with metabolic, autoimmune and neurodegenerative diseases; perhaps unsurprisingly given the key role lipids play in these processes. cerebrospinal fluid and serum antibodies against myelin basic protein, sulfatide and oxidized lipids have been found in patients with multiple sclerosis, and serum antibodies against neuronal gangliosides are directly implicated in the pathology of the peripheral neuropathy guillain-barré syndrome ( ) ( ) ( ) . anti-oxidized low-density lipoprotein antibodies on the other hand may play a role in the development of atherosclerosis ( ) . isolation of such antibodies via phage display and the use of recombinant versions in animal models of disease may help determine their pathological role and potentially reverse its effects ( ) . for example, in anti-phospholipid syndrome, antibodies against phospholipid-binding proteins such as β -glycoprotein i are considered to be a major driver of pathology through inappropriate complement fixation. the use of phage display to obtain human antibodies of the same specificity has enabled the development of blocking antibodies that are defective in fixing complement which were shown to inhibit the effect of endogenous antibodies in animal models ( ) . in another example, with certain animal models of atherosclerosis, a high-titre antibody response to the malondialdehyde low-density lipoprotein immunogen also appears to correlate with reduced disease severity, suggesting that such antibodies could be used therapeutically ( ) . phage display can also be used to develop anti-idiotypic antibodies; these have been shown to have therapeutic potential by binding and blocking the effects of naturally occurring autoantibodies ( ) . using phage display, we and others have also been able to generate anti-lipid antibodies even against pure lipids completely insoluble in aqueous solution, through enrichment against crystalline microdroplets formed following evaporation of organic solvents ( , , ) . while having potential for use as diagnostic reagents to identify targeted lipids in crude mixtures, it remains unclear whether such antibodies can recognize lipids in their natural biological environment and the therapeutic potential of anti-lipid antibodies generated through such modalities remains an open question. another type of target uniquely suited to phage display is proteins with post-translational modifications such as glycosylation or phosphorylation. in such cases, the approach is to engineer a specific recognition domain into a particular cdr region which significantly increases the likelihood that the recovered antibody will recognize the modification in addition to the protein epitope ( ) . for recognition of phosphorylated peptides, a phosphate-binding motif from a naturally occurring antibody was used to generate a mutant antibody library from which antibodies binding specifically to either phosphoserine, phosphotyrosine or phosphothreonine were identified. the cdr amino acids responsible for phosphorylated amino acid recognition were identified and then used to generate a second set of libraries which preserved these structures but had the rest of the cdr randomized; these libraries were then used to screen phosphorylated peptide targets. a similar strategy was used to identify glycan-binding antibodies based on a motif isolated from the anti-hiv antibody g , which binds the hiv- glycoprotein gp primarily through its glycosylation ( ) . a key advantage of phage display is the in vitro nature of the antibody identification process, as this allows for selection methodologies not possible with in vivo antibody generation. by carefully controlling the selection and screening conditions on immobilized antigen, for example, by the presentation of specific antigen conformations or the inclusion of competitors for direct selection towards specific targets or epitopes, the generation of antibodies can be biased towards desired regions of the target ( ) ( ) ( ) . moreover, binding affinity and on and off rates of recovered antibodies can be optimized independently according to the selection strategy employed, enabling tailoring to the desired biomedical application ( , ) . this can be further enhanced during recombinant expression as full antibodies where various immunoglobulin isotypes and constructs with alternative functionality can be easily generated. another screening modality available to phage display is the targeting of antigen presented on the surface of whole cells or tissue. this is especially useful for membrane-bound antigen such as cell surface receptors and transmembrane proteins whose structure is not easily recapitulated by free antigen and is particularly useful for tumour-specific surface antigens. however, this approach is complicated by the diversity of antigens on the cell surface which may result in selection of phage against common surface epitopes ( ). one means to overcome this is to leverage on unique biological properties of the target antigen. for example, signalling receptors such as her , epidermal growth factor receptor and p neurotrophin receptor, cycle from the cell surface to the interior and recovery of phage from the cell interior has allowed specific phage enrichment ( ) ( ) ( ) . however, most cell types will carry multiple membrane proteins capable of internalizing bound phage. an alternative and complementary means for selection is to pan against alternating cell types expressing the same target antigen to prevent selection of common surface antigens; this can be achieved through the use of mammalian or yeast expression vectors for expression of recombinant antigen on a suitable host cell ( , ) . in addition to these in vitro techniques, phage library approaches with in vivo binding modalities have also been explored. first described for organ targeting using peptide phage libraries where the library was injected intravenously into mice and the phage were subsequently rescued from the brain and kidneys ( ) , the technique has also been successfully carried out in tumour-bearing mice ( ) . in recent years, similar methodologies have also been employed with antibody phage libraries not only to identify potentially therapeutic antibodies but also for determining the accessibility of a given target ( ) , further expanding the boundaries of phage display technology. today, almost all widely accessible commercial libraries are based on non-immune repertoires of high complexity to enable use in the selection of antibodies against a virtually unlimited number of target antigens. the latest state-of-art libraries reach complexities of up to - clones and thus may even exceed the size of individual human cell repertoires ( , ) . while high-affinity antibodies can be isolated from these libraries, occasionally the affinity of the obtained antibodies is low as the genes are obtained from naive b cells that have not yet undergone affinity maturation. in such cases, where the affinity required for therapeutic efficacy is higher than that of the antibody obtained, in vitro affinity maturation may be performed. this is often accomplished by introducing random or specific mutations in the cdrs or the exchange of whole heavy or light chain domains by chain shuffling ( ) ( ) ( ) ( ) ( ) . recently, li et al. described an alternative phage display approach for affinity maturation of a natural human antibody msl- targeting human cytomegalovirus (cmv), where each of the cdrs was allowed to vary in only one amino acid residue at a time ( ) . higher affinity antibodies were isolated which had improved cmv neutralization potency when expressed as fab fragments in bacterial cells. in another novel application of in vitro phage library screening, single antibodies capable of dual epitope recognition have also been developed. carried out by bostrom et al., a subset of solvent exposed residues of the light chain cdrs of the anti-her antibody herceptin were randomized to generate a highly diverse combinatorial phage library ( particles) ( ) . by subjecting the library to selection for vegf binding while maintaining her binding, dual antigen-binding clones were identified which inhibited both vegf and erb-b -mediated cell proliferation in vitro and tumour progression in mouse models ( ) . such 'two-in-one' antibodies may represent a paradigm shift and provide exciting new opportunities for therapeutics. in contrast to lipids, carbohydrates and self-antigens, highaffinity antibodies against pathogen-derived protein antigens are easily generated through immunization or during the course of a natural infection ( , ) . present at high levels during the course of the immune response due to secretion from short-lived plasma cells and plasma blasts, lower levels are maintained in the serum by secretion from long-lived plasma cells and usually have therapeutic or prophylactic efficacy. this was put to good use in the preantibiotic era through serum therapy, where patients were given pathogen-specific polyclonal serum raised in animals ( ) . more recently, human-derived antibodies have been shown to be efficacious against a number of highly pathogenic diseases such as severe acute respiratory syndrome (sars) coronavirus, ebola and h n avian influenza ( ) ( ) ( ) ( ) . recovery of such naturally occurring antibodies from convalescent patients is clearly of prime importance for developing drugs against pathogens to which there are no other effective treatments. in addition, they provide a wealth of information on the humoral immune response, its duration and maintenance over time, and how it can be effective, or in some cases, ineffective, against infection ( , ) . to capture these antibodies, immune libraries can be constructed relatively quickly, allowing identification of the high-affinity antibodies that can be used to generate diagnostic reagents and also tested for their therapeutic potential. in addition, novel in vitro screening strategies can be employed to isolate rare broadly neutralizing antibodies, as has been highlighted in the hunt for antibodies against pandemic influenza. kashyap et al. reported the generation of combinatorial antibody libraries from the bone marrow of five survivors of a h n avian influenza outbreak in turkey yielding > unique antibodies against h n viral antigens, several of which were shown to have broadly neutralizing properties across various h subtypes ( ) . similarly, throsby et al., using combinatorial display libraries constructed from human memory b cells elicited by seasonal influenza vaccination, isolated antibodies exhibiting broad heterosubtypic neutralizing activity against antigenically diverse h , h , h , h , h and h influenza subtypes ( ) . the most potent antibody was also protective in mice when given before and after lethal h n or h n challenge and the epitope was localized to the highly conserved therapeutic antibodies from phage display membrane-proximal stem of the ha and ha subunits of hemagglutinin ( , ) . using a combination of panning strategies against the hemagglutinin from several antigenically distinct h n isolates to bias-selection of antibodies towards more conserved domains of the protein, we were able to isolate similar antibodies to that described by throsby et al. from a non-immune fab phage display library ( ) . the selection of such antibodies from even non-immune phage displayed libraries highlights the potential of these readily available libraries for the development of therapeutically relevant antibodies against emerging pathogens of high concern and may enable more rapid development of therapies against novel emergent pathogens in the future. the main competition for phage libraries in the area of cancer and autoimmune disease therapeutics where self-antigens are the primary target are transgenic mice whose immunoglobulin germline genes have been replaced with human equivalents. this technology, developed in the s, has been driven mainly by two biotech companies-mederax and abgenix-which licence the humab and xenomouse strains, respectively ( ) ( ) ( ) . during the course of v(d)j recombination, these reassemble into fully human antibodies although they are carried on murine b cells and the mice can generate functional immune responses (including somatic hypermutation) during immunization, from which antibody-secreting hybridomas can be made. some of these antibodies are already approved for clinical use and more are in the clinical pipeline ( , ) . one disadvantage of these first-generation strains appears to be the use of human constant regions, which has been suggested to affect downstream signalling in b cells and result in defective immune responses against particular antigens; newer strains of transgenic mice have been developed that have only the variable regions replaced and utilize murine constant regions ( ) . currently, more antibodies in clinical use or trials are derived from transgenic mice as compared to phage display, but this may not be an accurate comparison of their utility as the technology for transgenic mice was developed several years ahead of the first truly diverse synthetic antibody libraries ( ) . additionally, as antibody generation is governed by the rules of the immune response, their utility for generating high-affinity antibodies against epitopes common to humans and mice, non-protein targets, or antibodies with novel binding characteristics such as those achieved by in vitro selection under non-physiological conditions, is limited. for isolation of antibodies from human subjects, while phage display was the first technique suitable for the screening of large numbers of b cells, in recent years, additional techniques of similar robustness and efficacy have been developed. these fall into two main categories: analysis of single b cells through in vitro immortalization, activation into antibody-secreting cells, or recovery of their individual antibody transcripts; or alternatively, high-throughput analysis of polyclonal antibodies. the latter approach can be carried out at either the proteome or transcriptome level through mass spectrometry of digested peptides or next generation sequencing of harvested b-cell mrna, respectively. ( , ( ) ( ) ( ) ( ) . both these techniques have one main advantage over phage display; they allow elucidation of the heavy and light chain pairings and frequencies of the b cells of interest. as such, they may be more useful for characterizing the dynamics of humoral immune responses ( ) . on the other hand, they require significant investment in specialist equipment, such as a high-speed fluorescenceactivated cell sorter (facs) or next generation sequencer, as well as robotic handling equipment if large numbers of cells are to be screened ( ) . phage display does not require any such equipment and can be performed with basic microbiological and molecular biology apparatus with the exception of an electroporator if high efficiency cloning is required ( ) . another advantage is that a phage library once created is usually sufficient for numerous screenings and can be stored almost indefinitely, while the various omics procedures involve destruction of the sample and single b-cell cultures, unlike murine hybridomas, do not allow for indefinite antibody production ( ) . even antibodies produced by b cells immortalized with epstein-barr virus can gradually lose affinity due to re-activation of somatic hypermutation (p. a. macary, unpublished data). hence, while these techniques require a finely honed screening strategy only available with well-characterized pathogens, phage display may be more suited towards investigation of poorly understood or novel pathogens where multiple screening targets and conditions need to be investigated. in addition, sideby-side comparison of phage display and next generation sequencing produced completely different sets of antibodies that were attributed to the poor expression of particular antibodies on phage. nevertheless, the antibodies produced by either technique had comparable specificity and affinity ( ) . while for some applications, such as isolation of antibodies from patients, phage display may be complemented or substituted with other techniques, an area where phage display remains critical is for directed evolution of antibodies for increased stability under non-physiological conditions of ph and temperature-such selection is simply not possible in vivo. neither are other in vitro alternatives such as cell surface display (mammalian, yeast or bacterial) or ribosome display feasible due to their lower resistance to high temperatures or extreme ph. phage can tolerate temperatures of up to °c and a ph range of - ( ) . table provides a summary comparison of the advantages and disadvantages of these various in vitro techniques. phage display is also likely to remain useful for discovery of antibodies against non-protein targets, evolution of dual-binding antibodies and for affinity maturation, due to the limitations of the natural immune system. however, antibodies against non-protein targets such as lipids and carbohydrates remain a fairly unexplored area, partially due to the lack of validated therapeutic targets aside from bacterial membrane surface carbohydrates. one area that could be of particular interest is lipids involved in autoimmune and neurodegenerative disorders, for which there is some evidence that antibodies could have both protective and pathological roles and which a therapeutic antibody designed to suppress immune function, e.g. of the igg isotype, could have benefits. for regular protein antigens, there may still be one particular niche for phage display based on its strengths: the rapid development of therapeutic antibodies against emerging diseases or a genetically modified pathogen in an emergency situation. antibodies are a natural and well-studied component of the immune system and use of a naturally derived antibody should have minimal side effects and thus be ideal for emergency use. in such a situation, rapid antibody isolation is critical. phage display allows for the screening of an extremely large collection of antibodies of up to sequences, far more than that possible by next generation sequencing or single b-cell analysis, thus improving the chance of isolating rare antibodies. even if an immune library is required, rapid construction and screening is possible using cloning protocols such as megawhop which do not require prior digestion of pcr immunoglobulin sequences and new panning strategies that enable effective enrichment over only one cycle of selection ( , ) . in addition, vectors have been developed that can express antibodies both on phage as well as in mammalian cell culture as full length igg through the use of intron-mediated differential splicing without any need for sub-cloning, which enables rapid highthroughput expression analysis of the full length antibody ( ) . as such, phage display is likely to remain a key technique for antibody discovery for the foreseeable future. isolation of naturally paired vh-vl immunoglobulin genes that have gone through affinity maturation. lacks post-translational modification of antibody fragments. requires optimization due to relative instability of rnaribosomal complex. smaller library size compared to phage/ribosome display. labour intensive and technically challenging. smallest library size. affinity maturation can be performed in vitro. can be used to select for highaffinity binders. can be used to select for high-affinity binders during facs sorting. not required. therapeutic antibodies from phage display the use of antibodies in the treatment of infectious diseases filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface phage antibodies: filamentous phage displaying antibody variable domains generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda assembly of combinatorial antibody libraries on phage surfaces: the gene iii site selecting and screening recombinant antibody libraries selection of non-aggregating vh binders from synthetic vh phage-display libraries high affinity, developability and functional size: the holy grail of combinatorial antibody library generation novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis generation and characterization of a novel recombinant antibody against -ketocholestane isolated by phage-display neutralizing human monoclonal antibody against h n influenza ha selected from a fab-phage display library development trends for human monoclonal antibody therapeutics drugs derived from phage display: from candidate identification to clinical practice comparison of the efficiency of antibody selection from semi-synthetic scfv and non-immune fab phage display libraries against protein targets for rapid development of diagnostic immunoassays immunogenicity of engineered antibodies fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen technology evaluation: adalimumab, abbott laboratories antibody-based therapeutics to watch in antibody phage display libraries: contributions to oncology beyond natural antibodies: the power of in vitro display technologies in vitro display technologies reveal novel biopharmaceutics selection and application of human single chain fv antibody fragments from a semisynthetic phage antibody display library with designed cdr regions isolation of high affinity human antibodies directly from large synthetic repertoires hucal platinum, a synthetic fab library optimized for sequence diversity and superior performance in mammalian expression systems the human combinatorial antibody library hucal gold combines diversification of all six cdrs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies wnt pathway inhibition via the targeting of frizzled receptors results in decreased growth and tumorigenicity of human tumors the immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the cdr regions recombining germline-derived cdr sequences for creating diverse singleframework antibody libraries a human icam- antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity serum therapy revisited: animal models of infection and development of passive antibody therapy synthesis and characterization of hapten-protein conjugates for antibody production against small molecules conjugate vaccines-a breakthrough in vaccine development characterization and functional activity of murine monoclonal antibodies specific for α , -glucan chain of helicobacter pylori lipopolysaccharide the binding sites of monoclonal antibodies to the non-reducing end of francisella tularensis o-antigen accommodate mainly the terminal saccharide isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. . expression, purification, and characterization of recombinant single chain antibodies engineering antibody heavy chain cdr to create a phage display fab library rich in antibodies that bind charged carbohydrates isolation and characterization of antibodies against three consecutive tn-antigen clusters from a phage library displaying human single-chain variable fragments treatment of gram-negative bacteremia and septic shock with ha- a human monoclonal antibody against endotoxin. a randomized, doubleblind, placebo-controlled trial. the ha- a sepsis study group lipid microarrays identify key mediators of autoimmune brain inflammation peripheral neuropathies and anti-glycolipid antibodies combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and ebv antigen human-derived anti-oxidized ldl autoantibody blocks uptake of oxidized ldl by macrophages and localizes to atherosclerotic lesions in vivo anti-beta( )gp-i and antiprothrombin antibodies generated by phage display a non-complement-fixing antibody to β glycoprotein i as a novel therapy for antiphospholipid syndrome hyperimmunization of apo-e-deficient mice with homologous malondialdehyde low-density lipoprotein suppresses early atherogenesis efficacy of intravenous immunoglobulin (ivig) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris characterization of -dehydro-thromboxane b recombinant antibody obtained by phage display technology nature-inspired design of motif-specific antibody scaffolds a strategy for phage display selection of functional domain-exchanged immunoglobulin scaffolds with high affinity for glycan targets directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries directed selection of mip- alpha neutralizing ccr antibodies from a phage display human antibody library directing phage selections towards specific epitopes tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor for enhanced neutralizing activity improved affinity selection using phage display technology and off-rate based selection selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library a novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p toward selection of internalizing antibodies from phage libraries selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens organ targeting in vivo using phage display peptide libraries vascular ligand-receptor mapping by direct combinatorial selection in cancer patients proteasome activator complex pa identified as an accessible target in prostate cancer by in vivo selection of human antibodies construction of large naïve fab libraries modelling the human immune response: performance of a human antibody repertoire against a broad panel of therapeutically relevant antigens in vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity crystal structure of an in vitro affinity-and specificity-matured anti-testosterone fab in complex with testosterone. improved affinity results from small structural changes within the variable domains in vitro antibody affinity maturation targeting germline hotspots antibody affinity maturation by chain shuffling variants of the antibody herceptin that interact with her and vegf at the antigen binding site appearance of peripheral blood plasma cells and memory b cells in a primary and secondary immune response in humans duration of humoral immunity to common viral and vaccine antigens a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants ebola virus can be effectively neutralized by antibody produced in natural human infection an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus the human immune response to dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity neutralizing antibodies derived from the b cells of influenza pandemic survivors combinatorial antibody libraries from survivors of the turkish h n avian influenza outbreak reveal virus neutralization strategies heterosubtypic neutralizing monoclonal antibodies cross-protective against h n and h n recovered from human igm+ memory b cells antibody recognition of a highly conserved influenza virus epitope functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice antigenspecific human antibodies from mice comprising four distinct genetic modifications functional expression and germline transmission of a human chromosome fragment in chimaeric mice human antibodies from transgenic animals mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice monoclonal antibodies isolated without screening by analyzing the variablegene repertoire of plasma cells rapid cloning of high-affinity human monoclonal antibodies against influenza virus ex vivo characterization and isolation of rare memory b cells with antigen tetramers rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array profiling human antibody responses by integrated single-cell analysis an in vitro model of differentiation of memory b cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization antibody isolation from immunized animals: comparison of phage display and antibody discovery via v gene repertoire mining stability engineering of antibody single-chain fv fragments harnessing phage and ribosome display for antibody optimisation engineering antibodies by yeast display single b cell antibody technologies a general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition megawhop cloning: a method of creating random mutagenesis libraries via megaprimer pcr of whole plasmids a dual host vector for fab phage display and expression of native igg in mammalian cells the authors declared no conflicts of interest. key: cord- - esdjy authors: delhalle, sylvie; schmit, jean-claude; chevigné, andy title: phages and hiv- : from display to interplay date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: esdjy the complex hide-and-seek game between hiv- and the host immune system has impaired the development of an efficient vaccine. in addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. for more than years, phage display technology has been intensively used in the field of hiv- to explore the epitope landscape recognized by monoclonal and polyclonal hiv- -specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. in parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify hiv- inhibitors. besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the hiv- protease. phage particles also represent valuable alternative carriers displaying various hiv- antigens to the immune system and eliciting antiviral responses. this review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures. in , the human immunodeficiency virus (hiv- ) was identified as the causative agent of the acquired immunodeficiency syndrome (aids) [ , ] . in years of pandemic, hiv- has infected more than million individuals and killed million. thirty-three million individuals are currently living with hiv- making this disease a major worldwide public health problem (unaids ). natural sterilizing immune response against hiv- has never been described and despite decades of intensive research, a vaccine against hiv- is still lacking, mainly due to the high ability of the virus to escape from the immune response. in the absence of a vaccine, combinations of small antiviral molecules are intensively used to control hiv- infection. the majority of these drugs are reverse transcriptase and protease inhibitors [ ] . more recently, new molecules targeting the fusion step, ccr or integrase were licensed for clinical use [ ] [ ] [ ] . despite the increased life expectancy observed with the advent of these therapies, severe side effects, lack of adherence and emergence of drug-resistant virus strains still limit the long-term control of the infection [ ] . hiv- is an enveloped virus whose genetic material consists of two identical rna strands coding for the structural genes gag, pol and env as well as the accessory genes tat, rev, nef, vif, vpr and vpu. the gag gene codes for structural proteins p and p , while pol codes for viral enzymes (reverse transcriptase, integrase and protease) and env for the gp envelope protein precursor that is subsequently cleaved into gp and gp . gp and gp proteins assemble at the surface of hiv- into trimeric spikes composed of three monomers of membrane-embedded gp complexed to free gp . these two proteins are involved in virus entry and represent the principal targets for the humoral response. upon cd receptor binding, glycoprotein gp undergoes conformational changes exposing the v loop, a region that further interacts with the chemokine receptors ccr or cxcr thereby promoting viral entry [ ] (figure ). coreceptor binding leads to the insertion of the gp fusion peptide into the cell membrane, the creation of a hairpin loop intermediate and finally the fusion of both viral and cell membranes. the viral capsid then enters the cell and the genetic material is released in the cytoplasm. most viral strains use only one coreceptor to enter host cells and are classified accordingly as ccr -(r strains) or cxcr -tropic (x strains), although viruses with broadened coreceptor usage (dual-tropic) have also been described. r viruses infect macrophages and ccr -expressing t lymphocytes, and are mainly associated with transmission. in contrast, x viruses infect cxcr -expressing t-cells and t-cell lines, and often appear at the later stages of infection. the envelope glycoprotein gp is composed of variable and more constant regions. several studies demonstrated that the elicitation or binding of effective neutralizing antibodies are impaired by the gp glycan shield or steric hindrance of its constant regions [ ] . moreover, variable immunodominant domains were shown to be recognized by non-neutralizing antibodies. nonetheless, it is estimated that % to % of hiv- -positive subjects develop neutralizing antibodies (ntabs) appearing at least year after infection. only % of infected patients develop a broad neutralizing response against heterologous virus strains [ ] . among hiv- -infected patients, such antibodies arise only rarely and tardily, thus inefficiently controlling viral replication. however, the recent identification of broadly neutralizing antibodies (bntabs) and mapping of their epitopes fueled interest in the humoral immune response against hiv- (reviewed by overbaugh [ ] ). to better understand the reasons underlying the persistance of viral infection despite the strong and sustained immune response on the one hand, and to identify new protective immunogens, numerous studies were conducted to map the epitope landscape of both hiv- -neutralizing and non-neutralizing antibodies isolated from infected patients. in parallel, the development of new molecules or antibody fragments capable of blocking either viral proteins or host receptors has been widely investigated. to serve this purpose, the phage display technology has been extensively exploited in the field of hiv- as it represents one of the most powerful technologies for epitope mapping as well as for the identification of ligand binding to many types of targets. bacteriophages (phages) are bacteria-infecting viruses whose dna or rna genome is packed in a capsid composed exclusively of surface proteins. the principle of phage display relies on cloning of exogenous dna in fusion with the phage genetic material allowing the display of foreign peptides in an immunologically and biologically competent form at the surface of phage capsid proteins [ ] . the significance of phage display was first demonstrated for filamentous phages such as m , fd or related phagemids and later extended to lytic bacteriophages λ, t and t (reviewed by beghetto [ ] ). the phage biopanning process consists of iterative cycles of binding, washing and elution steps leading to the progressive selection of phages displaying peptides/proteins binding to the target of interest [ ] . the target is usually immobilized on a solid support which can be plastic, beads or even cells. a significant advantage of this technology is that phages may be used to display a collection of sequences (phage library), reaching up to billions of distinct sequences. phage libraries can be constructed to express combinatorial peptides or proteins/immunoglobulin fragments/variants that may be further screened for many different purposes including drug discovery, epitope mapping, diagnosis as well as identification of therapeutic antibodies. the phage display technology is versatile as it can be applied to different domains of research; it allows the easy handling and high-throughput screening of billions of sequences. it is affordable and enables the identification of linear as well as conformational epitopes when applied to an antibody target. numerous studies have described the use of the phage display technology in the field of hiv- were reported. they can be classified in four main applications ( figure ): i. epitope mapping, which relies on the screening of random peptide libraries on immobilized monoclonal or polyclonal antibodies to determine the linear and/or conformational epitopes recognized by these antibodies (linear epitope: sequence of continuous amino acids recognized by the paratope of a given antibody; conformational/discontinuous epitope: group of amino acids scattered along a protein sequence which come together in the folded protein and are recognized by the paratope of a given antibody). such screening usually results in the identification of sequences mimicking the natural epitope (mimotopes) and provides precise information about the location of residues forming the natural epitope. these mimotopes may in turn be used as valuable immunogens to elicit antibodies targeting the original epitope, an approach referred to as -reverse vaccinology‖. ii. inhibitor discovery, based on screening of phage libraries displaying random peptides or antibody fragments against viral or host proteins critical for viral replication. iii. -phage substrate‖ approach in which potential substrate sequences are displayed at the phage surface to enzymes such as proteases. this approach not only allows for proteolysis specificity profiling, but provides information and data to develop specific inhibitors. iv. carrier phage, in which the phage functions as a -carrier‖, -vehicle‖ or -virus-like particle‖ to display exogenous peptides such as mimotopes or even full size antigens to the immune system, to elicit specific humoral and/or cytotoxic t-cells responses. this review is intended as an overview of the different studies conducted using phages in the field of hiv- , laying special emphasis on the nature of the phage libraries used, the target display mode, the biopanning procedure as well as the results obtained. these studies are classified according to the applications described above and the main results are presented in tables. monoclonal antibodies (mabs) or polyclonal antibodies (pabs) epitopes can be identified through screening either combinatorial or antigen-fragment libraries displayed at the surface of phages. antibodies may be derived from infected/immunized animals or from hiv seropositive patients with peculiar immunological profiles, such as the long term non progressors (ltnp) [ ] , leading to the identification and characterization of hiv mimotopes. the seminal paper characterizing the epitope recognized by a mab directed against hiv- using the phage display technology was published in . keller et al. screened a -mer random peptide library (rpl) against the bntab - d which targets the v loop of gp (krkrihigpgrafy) [ ] (figure ) and identified clones presenting a gpxr consensus sequence [ ] (table ). these mimotopes were further used in rabbit immunization experiments and elicited neutralizing responses. boots et al. later investigated the linear epitope recognized by the mab - d by combining gp competition and panning of v -region biased/constrained libraries. such a set-up favors the selection of mimotopes in which residues surrounding the gpgr crown motif are similar to those present in the gp used for competition, suggesting that the use of strain-specific competitors with a mab of broad specificity can select for strain-specific mimotopes [ ] . [ , ] . in their study, a -mer rpl was constructed and panned against mab . according to two different protocols, either streptavidin capture of phages mixed with biotin-labeled mab . (sa-bio) or panning against mab . immobilized on microwells (micropan) [ ] . phages selected with the sa-bio protocol shared a consensus sequence (y/l)(v/l/i)gpgrxf homologous to the v loop. the micropan protocol allowed for the identification of sequences sharing the same motif, of which two were also identified in the sa-bio panning. biopanning results were further validated in peptide array hybridization assays. hybridization of mab . to -mer peptides containing all possible point substitutions within the v loop sequence demonstrated that both phage display and peptide array experiments identified the same critical amino acids, thereby confirming the quality of the -mer rpl and the validity of the screenings performed. epitope mapping was also performed on a monoclonal antibody (mab b) isolated from an asymptomatic hiv- -infected patient and recognizing the xxix pgrafytt motif within the v loop sequence (krihigpgrafytt) [ ] . binding of mab b to viral isolates presenting mutations in this sequence revealed that not all residues within this recognition motif were crucial for reactivity [ ] . biopanning with a -mer rpl resulted in the selection of sequences compatible with the minimal binding site (-i----g--fy-t) inferred from gp sequence alignment from clades a to f which bound mab b. taken together, data from binding assays as well as phage biopanning experiments demonstrated that the mab b epitope spans both sides of the v loop. substitutions within the residues located at the crown of the loop are however tolerated, provided that the formation of a β-turn induced by the gpgr crown motif is allowed. however, one exception was reported by boots et al. who reported that the phe to trp substitution may be tolerated in the absence of a β-turn [ ] . in parallel, grihalde et al. constructed and panned a -mer rpl against mab , which recognizes a constrained linear epitope on the v loop [ ] . several clones were obtained and presented the common motif (r/k/h)xgr mimicking the crown of the v loop sequence, thereby confirming the epitope sequence of mab . to assess the reactivity of peptides deprived of the phage scaffold, the mimotope with the highest affinity for the mab was expressed in fusion with the e. coli alkaline phosphatase. binding of the phage and fusion protein to the mab was assessed by elisa, western blot and spr assays and highlighted that binding was independent from the scaffold, although interactions were weaker when the peptide was displayed in the fusion protein format than in the phage scaffold. in another study, laisney et al. investigated the minimal epitopes recognized by two mabs interacting with the v loop, -a and . . , whose specificity is strictly restricted to the x -tropic lai isolate [ ] . the screening of a -mer rpl on the mab -a allows the selection of numerous sequences with a consensus motif. binding assays with synthetic peptides further showed that both mabs reacted with residues - of the lai gp . in this narrow region, the minimal epitope deduced for the mab -a was hyxrgp, whereas the mab . . recognized the xq(r/k)gp motif (hy: non-aromatic aa, underlined: aa tolerating substitutions). interestingly, the essential qr residues located at positions - correspond to a qr insertion located upstream of the v loop gpgr crown motif that is characteristic of the lai isolate and may thus explain the restricted specificity of the two mabs. the same authors screened a -mer rpl on the mab , specific to the v loop of the mn isolate, and identified two groups of sequences [ ] . a representative sequence from the first group ( . , hlgpgr), corresponded to the crown of the v loop, a linear epitope, while two sequences of the second group ( . , kaihri and . , kslhrh), showed no homology to linear hiv- epitopes. both peptides . and . nevertheless inhibited the interaction of mab with gp , and were even able to compete with each other for binding to the antibody, indicating that peptide . was also a mimotope of peptide . . when conjugated to klh and injected separately into rabbits, both peptides - and - were able to elicit gp -reacting antibodies that partially competed with the homologous peptide, confirming that . and . peptides are both antigenic and immunogenic mimics of the gp mn v loop. the isolation of the bntab f , which interacts with an epitope (eldkwa) located on the gp mper was reported in [ ] . conley et al. further characterized this epitope by biopanning a -mer rpl on immobilized f ab. different sequences were obtained and classified in four groups, whose consensus motifs, dkw, ldxw, ed(k/r)w and eldkw, revealed information on the residues involved in f ab recognition [ ] . immunization attempts with eldkwa peptides failed to elicit f -like ntabs, suggesting that the epitope necessitates additional residues in order to be immunogenic. therefore, menendez et al. screened a panel of libraries of linear and constrained peptides against the mab f and deduced that residues flanking the dkw core at the c-terminal side region were important for high-affinity binding to the mab [ ] . they subsequently constructed and screened two phage sublibraries displaying random residues either upstream or downstream of the dkw core (x -aadkw and aadkw-x ) and isolated three peptides displaying high affinity for f from the aadkw-x library. ala substitution and deletion studies revealed that each clone bound f according to a different mechanism. this data led the authors to postulate that the f paratope was composed of two binding domains either recognizing the dkw core with strong specificity or multispecifically binding to the residues located at its c-terminus. based on this study, additional investigations were recently conducted on the bntab f epitope to assess the importance of structural constraints for mab f recognition [ ] . a linear -mer rpl and a constrained -mer rpl were screened against this antibody and all the sequences selected from the -mer rpl contained the d(k/r)w core motif, with flanking residues l, a and s present at different frequencies. analysis of the sequence representation compared to their estimated probability of occurrence indicated a trend towards enrichment for sequences such as dkwa or ldkwa throughout panning of the -mer library, while all sequences selected from the constrained library contained dkwa or ldkwa. these results demonstrated that the strong epitope specificity postulated by menendez [ ] is only displayed when the epitope sequence is presented in a certain structural context provided in the constrained -mer peptides. immunization studies performed with both linear and constrained forms of the peptide in mice and rabbits resulted in the inhibition of cell fusion only with sera of rabbits immunized with the linear peptide. rpl screening may also contribute to the elucidation of the antigen structure. to that purpose, stern et al. used a -mer rpl to analyze two different mouse mabs (gv a and gv d ) recognizing non-overlapping sequences between residues and of gp [ , ] . biopanning performed on gv a allowed for identification of mimotopes sharing a (l/i)w motif identical to residues - of the gp c domain and highlighted a hxxixxlw motif compatible with two turns of an α-helix. computer modeling confirmed that such a structure placed the residues recognized by gv a contiguously on one face of the helix while other secondary structures did not. similarly, biopanning on mab gv d yielded sequences with a trend towards an nx wxxd motif. the epitope maps to the fnmwknd sequence satisfying the helical motif fxxwxxd. in this study, the use of phage display not only predicted the α-helix structure of the c domain of gp , but also pinpointed the contact residues defining the surface of the helix. phage display was also applied to epitope mapping of antibody-dependent cellular cytotoxicity (adcc)-inducing mabs since hiv- infected cells may be targets for fc receptor-bearing effector cells interacting with hiv- -specific abs. screening of -mer, -mer-c and -mer rpls against the adcc-inducing mab id resulted in the identification of phages with txxfxxwxxd ( -mer rpl) and fxdwxf ( -mer and -mer-c rpls) motifs homologous to the c domain of gp [ ] . competition assays showed that binding of mab id to gp or gp was abrogated in the presence of -mer mimotopes. in contrast, heptapeptide mimics only slightly impaired this binding, supporting the hypothesis that the mab-id epitope probably encompasses residues additional to the fxdwxf motif. as this epitope is highly conserved among circulating hiv- subtypes, it might be useful to induce mab id -like antibodies. the bntab igg b was the first neutralizing mab selected from a phage-displayed fab (antibody fragment composed of one constant and one variable domain of the heavy (ch and vh) and the light (cl and vl) chains linked together) library derived from an hiv- -infected donor (see section . . . . .) [ ] . this antibody recognizes a conformational epitope overlapping the cd -binding site of gp [ ] . attempts to precisely map the residues interacting with the igg b mab with -mer and -mer rpls provided no consensus sequence [ ] . as previous screening of cysteine-enriched peptide libraries resulted in the identification of two sequences bearing an sdl motif flanked by one or two cysteine residues (rekrwifsdlthtci and tclwsdlraqci) [ ] , zwick et al. constructed two sublibraries (x sdlx ci and xcxxsdlx ci) sharing the sdl motif and reflecting the cysteine content of the two clones [ ] . a b . peptide (hersymfsdlenrci) containing a unique cysteine bound b in fab as well as igg formats with a much higher affinity than the other clones. moreover, the phage-borne b . peptide was used to screen the fab library from which b was identified. this -reverse panning‖ experiment showed that b . was able to select only the fab sequence corresponding to b , confirming the specificity of the b . mimotope towards the b ab. b . peptide was immunogenic in mice and rabbits but did not elicit significant anti-gp cross-reactive abs titers. dorgham et al. attempted to map the b epitope with a rpl of two random -mers joined through an allry spacer (x allryx ) [ ] . selection resulted in the identification of clones sharing a m/varsd consensus motif (ar standing for any aromatic residue) as previously observed [ ] . a second-and a third-generation of semi-rpl containing fixed consensus motifs identified in the previous panning surrounded by randomized residues were constructed (x (m/v)wsdx and xlxvwxdexx). phagotopes (phage particle displaying a particular peptide sequence selected on a given target) obtained from the first, second and third generation libraries showed increasing binding affinity for b , respectively. phagotopes were able to compete with gp for b binding and triggered the production of abs capable of recognizing at least five distinct, unrelated hiv- strains. in contrast, the corresponding peptides were not able to compete for b binding and did not elicit anti-gp mabs. such discrepancies between phagotopes and peptides might be explained by constraints imposed by the phage scaffold. detailed characterization of the bntab b was conducted with the mapitope algorithm developed by enshell-seijffers et al. to facilitate the identification of discontinuous epitopes. this approach is based on the assumption that the collection of mimotopes recognized by a given antibody must in some manners reflect the antibody's paratope [ ] . a constrained -mer rpl was screened against b and selected sequences were compared to those obtained from previous panning experiments performed against b [ , , , ] . although no similarity was observed with the mimotopes selected by boots et al. [ ] , a consensus wsdl motif was observed in the newly identified mimotopes and the sequences isolated by bonnycastle et al. [ , ] . mapitope analysis conducted on these sequences as well as on the peptide sets isolated by boots and bonnycastle resulted for each of the three panels in the prediction of two clusters located at the periphery of the cd binding site. at the same time, both a linear -mer rpl and a constrained -mer rpl were used in panning experiments against another gp cd binding site mab ( a) [ ] . screening of the -mer rpl resulted in selection of a single sequence (wkpvvidfe), while screening of the -mer-c rpl on a allowed for identification of a gpxepxgxwxc consensus motif. peptides were synthesized as peptide-piii fusion proteins and their affinity for a was assessed in phage/mab and gp /mab binding inhibition assays. the two most affine peptides (aecgpaeprgawvc and aecgpyeprgdwtcc) were used to immunize rabbits and elicited antibodies binding to recombinant monomeric gp . nevertheless, generated abs seemed to target a different epitope since they were unable to compete with the a cd -binding site specific mab. the extreme c-terminus of gp forms a pocket which may interact with gp and was suggested to undergo conformational changes weakening the interaction between gp and gp upon cd binding. in the absence of available crystallographic information, ferrer et al. utilized the mouse mab - . to analyze an epitope overlapping with this pocket region [ ] . epitope mapping of mab - . achieved by cross-blocking experiments on gp suggested that the ab recognized residues - while the screening of an heptapeptide rpl against mab - . preincubated with gp allowed for the recovery of phages presenting an axxkxrh motif homologous to residues - . affinity studies confirmed that ala was the n-terminal residue of the mab - . epitope and showed that affinity increased when c-terminal residues were added. the mapitope algorithm designed by enshell-seijffers et al. was initially developed to elucidate the cd -induced epitope recognized by the mab b [ ] . screening of a -mer-c rpl yielded sequences with no homology to gp . comparison of the mimotopes to the gp structure in complex with mab b and scd predicted candidate epitopes that were in agreement with the actual b contact residues. for further validation of the algorithm, rpl libraries were screened against the p -specific mab b and analysis of the selected sequences predicted four clusters, the largest of which corresponded to the genuine epitope. the algorithm was finally applied to the mab cg , an ab with an unknown epitope competing with the mab b for the binding to the cd /gp complex. mimotopes sequences were analyzed and produced seven clusters, one of them being in accordance with previous mutation analysis impeding mab cg binding [ ] . noteworthingly, when reconstituted in a phage scaffold, the epitope was capable of binding mab cg . after having successfully identified linear or nearly linear epitopes [ , , ] , boots et al. extended the use of the phage display technology to the identification of epitopes recognized by mabs binding to discontinuous sequences [ ] . one of these abs (mab a ) binds to a cd -induced discontinuous epitope involving residues within the c , c and c regions of isolates from clades b, c, d, e and f [ , ] . panning of a -mer rpl yielded several phages which only shared a trp residue. in the same study, panning of a -mer rpl against mab - , which reacts with the id gklic region of gp , resulted in the identification of sequences sharing a common trp within motifs wgcx(k/r)xlxc and fgxwfxmp. the selected consensus sequences were however not further characterized. the bntab g presents the typical feature of recognizing a cluster of high-mannose oligosaccharides of gp [ ] [ ] [ ] . in an attempt to identify peptidic immunogens capable of eliciting g -like abs, menendez et al. screened a set of previously described rpls [ ] against g and identified one phagotope specifically binding to g ( g . ) [ ] . the crystal structure of mab g complexed to the synthetic g . peptide was compared to structures of g -oligomannose epitopes and revealed that interactions with the abs were different for the two ligands. these results showed that the peptide selected from rpl panning experiments is not a structural mimic of the g oligomannose epitope. the phagotope g . was used in rabbit immunization experiments and elicited high titers of peptide-specific antibodies, but no cross-reactivity with gp was obtained, further supporting that peptide g . is not an immunogenic mimic of the mab g epitope. the first attempt at identifying epitopes recognized by hiv-specific pabs was performed in on plasma igg from two ltnp patients (table ). using linear and constrained -mer rpls, scala et al. identified mimotopes of the linear immunodominant (id) gklic region of gp or the v and c domains of gp [ ] . these mimotopes were immunogenic when injected to mice and elicited an ntab response against hiv- . moreover, the same mimotopes reduced viraemia to undetectable levels in immunized monkeys as shown in a subsequent study [ ] . the same year, a similar study conducted on one ltnp with an rpl library of cysteine-constrained -mers selected for peptides defining the gp id epitope csgklic. the levels of reactivity of these phagotopes were further assessed against a panel of hiv positive plasma to evaluate the plasticity and polyclonality of the immune response mounted by infected individuals [ ] . later, palacios-rodriguez et al. evaluated the impact of factors such as highly active antiretroviral treatment (haart) or ab titers on a selection of peptides mimicking the id epitope csgklic [ ] . in their study, a mix of linear -mer as well as linear and constrained -mer rpl was screened against the individual plasma samples of four hiv- infected patients initiating haart and presenting different titers of anti-gklic antibodies. a consensus motif cxxkxxc was obtained from the -mer linear rpl, and the percentage of occurrence of the motif in the selected sequences was proportional to the anti-gklic ab titers of each sample, indicating that these abs are involved in selection of the consensus motif. mice immunization experiments with the two mimotopes resembling most to the gp id parental epitope as well as with pools of phages eluted from the panning experiments showed that all phages elicited reactivity, and that immunization with the phage eluates induced the strongest recognition. these findings indicate that the immunogenic properties of mimotopes are different and additive, opening the possibility of immunizing animals with different mimotope combinations (see section ). in , humbert et al. investigated the immune response of eight ltnp patients presenting bntabs. by using linear and constrained rpls they identified epitopes recognized by plasma iggs captured on tosylactivated beads [ ] . each panning round consisted of a positive selection performed on ltnp iggs followed by a negative selection on the iggs of healthy donors. homologies of some selected sequences to immunodominant regions such as the gp v loop or the gp gklic region were observed, as reported in previous studies [ , , ] . further homologies to linear motifs located near the v loop (nnnt), downstream of the id gklic region (avpw motif) and overlapping with the f bntab epitope (ppwx w motif) were also identified. additionally, the authors applied the dex software to compare the phage insert sequences to hiv- protein structure files from the rcsb protein data bank (www.pdb.org) [ , ] . phage pools corresponding to the linear v loop, gklic domain and wxxxw motif, as well as pools representing potential conformational epitopes, were selected for mice immunization assays, and elicited plasma-associated neutralizing activity against primary hiv- strains. the highest neutralizing ability was obtained with mice immunized with the v mimotopes, although immunization with potential conformational epitopes also provided a modest neutralizing response. a similar approach was used by the same authors on a rhesus macaque infected with an shiv chimera encoding the env of a clade c hiv- strain (shiv ip) and presenting a broad neutralizing response against homologous shiv-c as well as heterologous hiv- strains of different subtypes [ ] . biopanning yielded clones similar to gp (v and v loops or c-terminal domain) or to regions of gp (id gklic region, other id regions and mper domain) [ ] . remaining clones showed no significant homology to linear hiv- regions and were analyzed with the dex software, which allowed the identification of a discontinuous mimotope located near the v loop crown. the antibodies binding to this phagotope were affinity-purified and subsequent assays demonstrated that recognition was conformation-dependent. an immunofocused immunization of mice primed with a dna vector coding for the gp shiv ip and boosted with pools of phage particles corresponding to the v loop, the gp c-terminus, the gp id region, the gklic region and the mper domain was set up. almost all mice developed anti-env abs and % of them presented a neutralizing activity. in , dieltjens et al. applied the phage display technology to identify the epitopes potentially involved in the bntabs response of an hiv- crf ag-infected individual (itm ) and to monitor the evolution of humoral response and viral escape through the course of infection [ ] . biopanning of a -mer rpl against plasma samples from itm resulted in the identification of different peptide sequences. half of these sequences were homologous to linear epitopes on gp , i.e., the e epitope region in the mper domain (nwfnltqtlmpr) or the lentivirus lytic peptide (llp ) (slxxlrl) while the other peptides shared homologies with the c domain (kxwwxa) and the crown of the v loop (kx igphxxy) of gp . further analysis of the levels of reactivity of the phage groups against itm six-year follow-up samples revealed different temporal patterns of recognition, confirming the dynamic nature of the immune response. interestingly, the mper region was the only epitope retaining immunogenic properties during this period. in a more recent study, the same group investigated the antigenic landscape of an hiv- subtype a-infected individual with bntabs by screening an rpl library against a pool of sequential samples drawn from to [ ] . the biopanning procedure yielded sequences predicted to represent autologous v sequence (kx hx y), v loop (kxxhxgpx f) and gp id domain (cxgxlxctxnxp). again, follow-up sample recognition of the four phage groups showed different patterns. antibody reactivity towards gp id region fluctuated slightly in all plasma samples. reactivity against the v loop-like phages decreased over time. in contrast, the v loop mimotopes were not recognized before , but once emerged, reactivity persisted until . env sequence analysis of the follow-up samples showed that a tyr to his mutation in the v loop sequence coincided with the emerging antibody response against this sequence. additionally, the authors highlighted that the neutralizing activity observed in the samples was partially due to antibodies recognizing the v mimotopes. besides the multiple reports on the use of rpl to characterize the humoral response against hiv- env proteins, gupta et al. evaluated the reliability of using targeted antigen gene fragment libraries for the identification of epitopes recognized by antibodies elicited in rabbits immunized with p . to this end, they constructed a phage library composed of dnase-digested fragments of gag dna [ ] . phagotopes obtained after the first panning round displayed mainly - -mer peptides, % of which mapped to of the n-terminus of p ( - of gag) and % corresponded to the c-terminal region of p ( - of gag). only one phagotope mapped to the central region of gag ( - ). at the end of the second round, selected phages displaying longer inserts of to aa corresponding to the n-and c-terminal regions of gag were identified, revealing the presence of two distinct antigenic regions in gag. this study demonstrated that gene-fragment phage display could be used to identify epitopes targeted by polyclonal abs. although they occur at a very low frequency in humans, antibodies targeting host proteins involved in hiv- infection have been reported in immunized animals. given their potential value for viral entry inhibition and the general understanding of this mechanism, rpls were screened on these mabs to gain better knowledge of their epitopes (table ) . na: data not available. the murine mabs a and c were raised against cells transfected with the seven transmembrane-spanning domains chemokine receptor ccr , one of the main coreceptors for hiv- . they recognize a common epitope located near the ccr n-terminus [ , ] . both mabs were used to screen a constrained -mer rpl [ ] . phagotopes selected on a displayed the sequence chasiydfgsc while cphwlrdlrvc was the most prevalent sequence isolated on c . these sequences showed homologies to residues located at the n-terminus but also within the first or third extracellular loop (ecl) of ccr . both reacted against the targeted mab either in phage, cyclic peptide or linear peptide formats. moreover, they were able to bind to gp and the peptide selected on a inhibited binding of the mab to a cell line expressing ccr . to further characterize the conformational epitope recognized by a , additional screening rounds of -mer, -mer and -mer-c rpls were performed [ ] . sequences with an hw motif homologous to the cphwlrdlrvc motif selected on the mab c were identified, and ala-scanning confirmed the importance of the hw motif and siyd motifs previously identified for a binding [ ] . another murine antibody (mab d ) recognizing a conformational epitope on the second ecl of ccr [ ] was explored by screening a linear -mer rpl [ ] . three phagotopes (m , m and m ) were isolated and one of them (m ) was able to inhibit cell infection by the hiv- sf isolate. the corresponding peptide (fcaldgdfgwlapac) fused to the piii phage coat protein neutralized infection mediated by the jr-fl but not the iiib strain. the fusion protein specifically bound d and was recognized in a dose-dependent manner by three ccr chemokine ligands, i.e., ccl (rantes), ccl (mip α) and ccl (mip ß), confirming its ccr mimicry. six years later, another screening campaign was conducted with a linear -mer rpl on d and the ewqkeglvtlwl sequence of a high-affinity binding peptide was obtained [ ] , revealing that this peptide presented homologies to the n-terminal ( -qkegl- ) and c-terminal regions of the ccr ecl . ala substitutions of the tl residues confirmed their crucial role in d binding. the selected peptide was used in rabbit immunization studies and elicited abs with d -like biological functions, i.e., which inhibited hiv- -mediated cell fusion and pbmc infection. the cd cell surface molecule, a part of the lfa- molecule, is involved in the syncytia formation of hiv- -infected lymphocytes [ ] . as mab mhm , a cd binder, inhibits hiv- -mediated cell fusion, poloni et al. applied the phage display technology to map the mhm epitope and thereby identify the cd domains which account for syncytia formation [ ] . linear and constrained -mer rpl were panned on the mhm mab, to allow for the selection of linear and constrained sequences. a ppfxyrk consensus motif was inferred by sequence comparison, assigning the epitope recognized by mhm to residues - of cd . two phagotopes inhibited in vitro hiv- -induced syncytia formation and one of them retained this ability in the peptide format, confirming its role in syncytia formation and highlighting that mimics of this epitope could prevent cell-mediated viral propagation. as summarized in the first section of this review, phage-displayed rpls are powerful tools to determine or to characterize mabs as well as pabs epitopes. besides epitope mapping the phage display technology was also widely applied to the identification of hiv- inhibitors. screening of phage-displayed rpls, antibody-fragment or ligand libraries on viral or host targets contributed to the discovery of molecules interacting with the key players of hiv- infection. antibody libraries were particularly investigated, and repertoires of fab, scfv (antibody fragment corresponding to variable regions of the heavy (vh) and light (vl) chains of antibody connected by a short peptide linker), v hh /nanobodies (single domain antibody fragment (sdab) corresponding to the variable heavy-chain domain of a camelid heavy-chain only antibody (hcabs)) or cdr fragments from naï ve or hiv- infected subjects as well as from immunized animals were displayed at the surface of phages ( figure ). most of the hiv- inhibitors selected with the help of phage display were identified by targeting viral proteins (table ). ( ) -helix, izn ( ) -helix ( ) pie -trimer ( ) tat ( ) cyclin t [ ] [ ] ( ) ncp ( ) psi rna the cd binding site represents one of the main achille's heels of the virus since it is involved in the earliest step of hiv- entry and is conserved in almost all hiv- strains [ , ] . numerous phage display biopannings were performed on gp and are classified here according to the type of antibody libraries used. burton et al. were the first to report the construction of a phage-displayed fab library from the bone marrow of an asymptomatic hiv- -infected patient with high titers of gp -specific abs [ ] . this library was screened against recombinant gp from the iiib isolate and clones displaying high affinities (< nm) for gp were selected [ ] . one fab (b ) (see section . . . .) was able to neutralize the mn and iiib strains in different set-ups. this ab is the most potent neutralizing ab isolated to date, featuring neutralizing activity against % of primary isolates of hiv- tested at concentrations that could be achieved by passive immunization [ ] . to improve its affinity, the b fab was submitted to cdr walking, a procedure involving randomization of its cdr and expression of the derived libraries expressed on phages, followed by screening against on gp [ ] . sequential cdr walking of the hcdr and hcdr domains was performed and four clones were chosen for detailed analysis of their binding affinity and neutralization potency against the iiib and mn isolates. a pro glu mutation of the hcdr was identified in the clone with highest affinity, b , which bound iiib gp with an -fold improved affinity ( . nm) compared to the parental b fab. similarly, fab b was able to neutralize four isolates that were insensitive to the parental b fab. the cdr walking mutagenesis strategy was pursued in a subsequent study and a further -fold improvement of the binding affinity of b for gp was achieved, reaching pm [ ] . these studies were the first to demonstrate that recombinant fabs (devoid of the typical igg contamination residual of calpain cleavage) featured neutralizing activities similar to those of whole igg. as the fabs fragments were easier to produce and their smaller size allowed them to target binding sites that were not accessible to full-length igs, this led to the construction of many fab libraries to elucidate the immune response to hiv- and to identify therapeutic antibodies. the extremely high binding affinity of b was also applied to develop an immunotoxin which could specifically kill hiv- -infected lymphocytes [ ] . the authors engineered b scfv fused to a truncated form of pseudomonas exotoxin a. the b (fv)-pe fusion immunotoxin bound to the mn strain of gp with the same affinity as the parental fab antibody and specifically killed a gp -expressing cell line and a chronically hiv-infected lymphocytic cell line. this study provided the proof-of-concept that high affinity anti-hiv- antibodies have a dual application since they may be used for their neutralizing potency but also as carriers for antiviral compounds. most antibodies obtained through screening fab libraries against monomeric gp targeted epitopes related to the cd -binding domain of gp , pointing to it as an immunodominant epitope [ ] . to expedite the identification of ntabs directed against weakly immunogenic epitopes, a strategy called epitope-masking was applied in several studies. this biopanning approach is designed to mask a particular epitope with antibodies or ligands directed against the region of interest prior to addition of the phage library. ditzel et al. panned a fab library from an asymptomatic hiv- -infected patient on immobilized recombinant gp and identified two dominant clones targeting the cd -binding site [ ] . these two fabs were then incubated with gp to mask their respective epitopes and the panning of the library was repeated, highlighting (based on sequence similarities) four groups of fabs recognizing gp with affinities in the range of to nm. epitope mapping of one representative fab for each group showed that gp binding of three clones was influenced by the v loop and the cd -binding site and was not affected by the glycosylation status of gp . furthermore, one of these fabs (l ) featured a broad neutralization spectrum against various hiv- strains. the authors performed further epitope masking by using different selection strategies with the same fab phage library [ ] . the first strategy involved masking the cd -binding site (cd bs) epitopes either with soluble cd or with a cd bs ab. all fabs selected on scd -bound gp recognized the c region, while the fabs isolated from the cd -bs ab-captured gp were classified in four different groups: (i) fabs targeting the c region, similarly to fabs isolated on scd -bound gp ; (ii) fabs directed against the c -c -region; (iii) fabs recognizing the v loop; and (iv) fabs directed against a cd bs/v loop region, similar to the neutralizing fab isolated by ditzel et al. [ ] . multiple epitope masking was then conducted by masking the cd bs-mab-captured gp with one of the c -specific fabs selected on the scd -bound gp prior to phage addition, leading to the identification of two c /c -dependent fabs. all isolated fabs bound their targets with affinities ranging from to nm. however, these fabs targeting weakly immunogenic regions were not or poorly neutralizing. more recently, koefoed et al. investigated the anti-gp ab repertoire of the circulating gp -binding igg-bearing b cells of hiv- -infected patients by constructing phage displayed fabs libraries from unselected cells or from cells preselected with immobilized gp [ ] . panning against gp selected for a higher number of phagotopes from the preselected library. clones from the unselected library recognized the v loop, while clones from the preselected library targeted the cd bs or a cd -induced epitope encompassing the c region. these fabs displayed no significant differences with respect to epitope specificity, affinity and neutralization ability compared to fabs obtained from bone marrow libraries, and most of them were unable to neutralize hiv- . these results were in accordance with previous findings by parren et al. ( ) concluding that the majority of the circulating hiv- specific antibodies were elicited by viral debris and were therefore devoid of neutralizing activity [ ] . antibodies recognizing the amino acids - of the gp cd bs were isolated from patients suffering from the systemic lupus erythematosus autoimmune disease [ , ] . however, whether these antibodies neutralized hiv- was not known, which prompted karle et al. to quantify gp -recognizing abs in an existing scfv phage-displayed library from the pbmcs of lupus-suffering patients [ ] . biopanning selected for clones binding both gp and the - region of the gp -cd -binding site. one of these clones (jl ) neutralized r and x -tropic hiv- primary isolates from clades b, c and d with ic ranging from . to . µg/ml. a subset of gp -binding antibodies was shown to hydrolyze gp by a mechanism analogous to serine protease [ ] , as the nucleophilic region responsible for this activity was localized in the light chain [ , ] , a library of light chains prepared from three lupus patients [ ] was screened with an electrophilic analogue of gp residues - to isolate antibodies capable of binding and hydrolyzing gp [ ] . one of the light chain clones selected (skl ) cleaved a gp - -reporter substrate as well as full-length gp . engineering of abs composed of such a light chain coupled to a gp -binding heavy chain might provide abs with anti-viral proteolytic activities. the advantages of reduced antibody formats were also explored with naturally occurring smaller antibodies. in addition to conventional antibodies, camelids also produce antibodies devoid of light chains ( figure d ). these heavy-chain only antibodies (hcabs) lack the c h domain and their binding specificity is provided by the variable heavy-chain domains of hcabs (v hh or nanobodies) [ ] . the cdr regions of nanobodies are on average longer cdr than those of conventional abs and display a protruding conformation, thereby more easily binding clefts and active sites. nanobodies were successfully used for panning against various pathogens (reviewed by vanlandschoot [ ] ). forsman et al. immunized llamas with a recombinant gp of subtype b/c (cn ) [ ] . three different panning strategies against various gp resulted in the selection of three nanobodies (a , d and c ) with neutralizing activity against a limited panel of clade b and c strains (ic values ranging from . to µg/ml). these nanobodies bound gp with affinities from . to nm and inhibited its binding to cd as well as to mabs known to recognize the gp cd binding site and, last but not least, competed with each other for gp binding. in a follow-up study, koh et al. described a family-based approach to produce nanobodies similar to a and d [ ] . they used a degenerated oligonucleotide annealing to the last six codons of cdr and framework (fr) and an fr -specific primer to amplify a sublibrary of related v hh clones with properties similar to the parental a or d v hh . more than phagotopes were tested for their ability to neutralize clade b and clade c strains. all tested v hh displayed similar neutralizing activity against the clade b viruses. three different neutralization profiles (broad a -like, intermediate and narrow d -like potency) were observed against the clade c type strains and the breadth of neutralization potency appeared to be correlated to the presence of an yyd motif in the c terminus of a cdr . when the cdr ydd motif from a was introduced within d , gp binding affinity increased by -fold and neutralization of clade c strains increased by -fold. these studies were the first to demonstrate bntabs elicited in immunized animals. as v hh are stable and can be produced at a relatively low cost, they are promising hiv- inhibitors. the cd receptor recognizes gp through residues located within its v region, and engineering of this cell receptor was applied to identify cd variants with a better affinity for gp , thereby displaying hiv- inhibitory properties. in , krykbaev et al. constructed a phage-displayed library of cd v and v -v variants generated by error-prone pcr and screened it against gp [ ] . five clones with increased affinity for gp and presenting mutations within the cd v domain were identified. all of these clones inhibited hiv- entry with ic ranging from . to µg/ml. the phage display technology was used to improve the affinity of the mab - d for its v loop epitope, which had also been identified through phage display [ , ] . in , thompson et al. expressed mab - d as scfv on phages and combined its v h with λ and κ chains from a non-immunized pbl repertoire prior to panning on a peptide containing the v loop sequence [ ] . additional shuffling of the hcdr and hcdr regions was combined to hcdr -spiking‖, i.e., the introduction of random mutations, resulting in the identification of four key residues that could be mutated to improve ab affinity. a sublibrary in which all four codons were simultaneously mutated was constructed and biopanning allowed to select for one scfv ( p h ) with improved k d against the mab - d epitope. two neutralizing fabs (fab loop and fab do - ) were obtained by screening a fab library against recombinant gp reaching ic ranging from . to µg/ml [ , ] . in , ferrer and harrison screened -mer, -mer and constrained -mer rpl against gp and identified two peptides from the -mer rpl [ ] . the first sequence rinnipwseamm ( p ) inhibited cd as well as ntmab b binding while the second peptide, tspyedwqtylm ( p ) did not affect the cd interaction and rather enhanced b binding. the p peptide was further investigated and shown to inhibit binding of monomeric yu gp to both scd and b with ic values of . and . µm respectively. the p peptide also inhibited binding of these ligands to trimeric envelope glycoproteins, blocked binding of gp to the native coreceptor ccr , and specifically inhibited hiv- infection of target cells in vitro [ ] . hiv- entry is a multi-step process requiring the successive binding of gp to cd and to a coreceptor, ccr or cxcr , and triggers successive conformational changes that expose transient epitopes. targeting of these epitopes with ntabs could therefore prevent hiv- infection, as has been proven with the clinically approved fusion inhibitor enfuvirtide. to identify such receptor-induced epitopes, moulard et al. screened a fab library constructed from an hiv- infected patient (fda- ) with high ntab titers against gp -cd -ccr complexes [ ] . one fab clone, x , bound gp from several strains with a low nanomolar affinity. furthermore, binding affinity was significantly increased in the presence of cd and slightly enhanced by ccr . competition assays with a panel of antibodies targeting different hiv- epitopes revealed that x recognized an epitope located in close vicinity to the cd and coreceptor binding sites. neutralization assays with isolates from clades a, b, c, d, f and g demonstrated that x neutralized all isolates with potency comparable to that of b . x is the first bntab recognizing a receptor-induced epitope identified to date. to select for ligands for cd -induced epitopes, murine leukemia virus particles carrying the env protein of the dual-tropic . strain pre-incubated with scd were recently used to screen -mer, -mer-c and -mer rpls [ ] . one of the selected phagotopes (xd : hkqpwydywllr) displaying sequence similarities (in bold) with the n-terminal extracellular part of the ccr and cxcr coreceptors was identified, suggesting novel potential leads for tyrosine sulfation. both the xd phagotope and xd peptides strongly and specifically bound to . gp regardless of cd and xd competed with mab b for binding to a cd -induced epitope. the sulfated form of the xd peptide recognized x , r and dual-tropic strains and inhibited hiv- entry in the high micromolar range. the salp salivary protein of ixodes scapularis inhibits cd + t cells activation by binding to the cd molecule in a region that may overlap with the gp binding site. this inhibition is mediated by the c-terminal - gpngqtcaeknkcvghipgc sequence [ ] . juncadella et al. thus analyzed salp as a potential hiv- inhibitor and demonstrated that salp inhibited gp -cd interaction and subsequent cell fusion and that the gpngqtcaeknkcvghipgc peptide also interacted with gp [ ] . to identify which gp amino acids interacted with peptide - of salp , the authors screened a -mer rpl against salp and isolated a hvitplw sequence homologous to an (i/l)tpl motif of the gp c /v domain which is highly conserved across hiv- isolates. finally, they mapped the interaction site of full-length salp protein or of its - c-terminal domain were able to bind to the pcvkltplcvtlnct peptide within the gp c /v region. in addition to epitope mapping and inhibitor identification, phage display was also widely applied to elucidating the determinants of the initial response to hiv- antigens and more particularly the importance and different roles of igm and igg during the establishment of infection. indeed, all known bntabs are iggs that are somatically hypermutated and are thus more difficult to elicit. in contrast, igms are closer to germline antibodies and the identification of hiv- specific igm could be relevant for the development of vaccine immunogens. the studies listed in this section explored and emphasized the importance of the initial igm response against hiv- and viral strategies to skew it towards non-neutralizing or infection-enhancing antibodies. screening against gp with igm and igg fab repertoires constructed from a healthy donor demonstrated that only fabs isolated from igm were able to recognize gp , although they were polyreactive, displayed low affinities and no neutralizing properties [ ] . sequence analysis evidenced that selected gp -binding fabs originated from different v h germline genes. several studies reported that gp displays superantigenic properties giving it the ability to bind and stimulate non-immune b cells to secrete v h ig in vitro [ ] . interestingly, the v h antibody family is the most represented immunoglobulin gene family in healthy adults ( % of peripheral repertoire) and hiv- infection leads to altered v h production through selective depletion of the anti-hiv- v h antibodies [ ] . toran et al. further applied the phage display technology to examine and compare the human v h genes involved in igm and igg responses to gp to identify the correlates of long-term non progression. two igm and igg phage-displayed fab libraries from an hiv- -infected ltnp with high gp -specific igm and igg titers were constructed and screened [ ] . several clones were selected from the igm library (m , m , m and m ) and three clones from the igg library (s , s and s ). all igm fabs were polyreactive and had a binding affinity for gp in the micromolar range while the igg fabs were specific and bound gp with affinities in the nanomolar range, as expected. sequence analysis showed that igg fabs originated from the same germline ab. the igm fab m displayed the same v h region nucleotide substitutions as those of igg fab s and used similar d h and j h segments, suggesting that s arose from m by isotype switching. in addition, a four aminoacid difference in the hcdr sequence of m (tgqwe) and s (rggsi) was proposed to be associated with the -fold affinity increase for gp and to the higher neutralizing activity of igg fab s (id = ng/ml) than of igm m (id = µg/ml). in a follow-up study conducted two years later, the three igg fabs were submitted to reverse mutations to reconstitute the germline amino acid residues [ ] . the higher affinity and neutralizing ability of s were due to the ala arg and ala asp somatic mutations in the hcdr region of the germline gene sequence, providing clues for rational modifications of cdr in human antibodies to improve affinity and hiv- neutralization capacity. the igm to igg isotypic switch generating high affinity neutralizing or non-neutralizing antibodies is triggered by the activation of igm-producing b cells. to characterize the epitopes recognized by hiv- -specific igm and to assess the effects of these abs on hiv- infection, chen et al. constructed a fab library from blood, lymph nodes and spleen from healthy donors [ ] . the library was panned against gp (env ectodomain containing both gp and a truncated gp lacking transmembrane domain and cytoplasmic tail) of a clade b isolate and allowed for the selection of one fab clone (r h m) with a relatively high binding affinity for gp from different strains. a sublibrary derived from this clone was panned against gp from different isolates, resulting in the selection of clones (m , m a, m b, m c and m d) binding with high affinity to clade b and f gp (ec ranging from nm to nm). while these antibodies had weak neutralizing properties against x -tropic isolates, they did not inhibit and in some cases even enhanced infection with r -tropic isolates. the m ab, whose sequence is relatively similar to the germline ab, targeted highly conserved epitopes located near the cd binding site or the coreceptor binding site. the high immunogenic capacity of the conserved non-neutralizing epitopes of such antibodies could divert the immune system from actually neutralizing epitopes. the authors suggested that these newly identified mabs could be used as probes to further characterize conserved non-neutralizing or enhancing epitopes and to modify or remove them from candidate vaccine immunogens. the epitope masking strategy (section . . . .) might be applied to these epitopes to redirect the immune system to elicitation of antibodies targeting neutralizing epitopes. to select for antibodies closely resembling the germline antibodies as a candidate source of bntabs, the same authors constructed a cord-blood-derived igm library which they submitted to parallel screening against hiv- env, sars coronavirus protein binding domain (rbd) and soluble hendra virus g protein (sg) [ ] . although rbd and sg antigens provided enriched igm, the library could not be enriched in hiv env-binding phagotopes. these results are in accordance with the hypothesis that hiv- could have evolved strategies based on weak or absent binding to antibodies of the germline repertoire [ ] . presenting epitopes unsuitable for binding of somatically hypermutated antibodies would enable the virus to escape from strong immune responses. . this library was screened on the mn peptide encompassing the f epitope eldkwa, and led to the identification of the z mab [ ] . epitope mapping experiments based on synthetic peptides and recombinant proteins showed that the epitope targeted by mab z was located downstream of the f epitope and centered on the nfwdit sequence. mab z neutralized primary isolates from hiv- b, c and e subtypes. to enhance binding potency, fabs sublibraries of z variants were engineered and screened against an mper peptide and gp [ ] . the selected z e variant displayed an over -fold increase in neutralization breadth and potency compared to the parental z mab. binding experiments coupled to competition assays revealed that the z e mab bound to a waslwnwfditn minimal epitope overlapping the f epitope and that the asn and asp residues were essential for peptide recognition as well as hiv- neutralization. very recently, phage-and yeast-displayed abs libraries constructed from an hiv- -infected patient with f -like bntabs were panned against peptides containing the f epitope and against the hiv- jr-fl gp [ ] . two mabs (m and m . ) were identified and the most mutated variant (m . ) neutralized hiv- with a higher potency than m . ala substitutions indicated that both abs recognized the dkw core of the f epitope and two additional leucine residues located upstream (l( , )). fusion of viral and host membranes, the last step of hiv- entry, requires the initiated by the insertion of the gp -encoded fusion peptide into the host cell membrane and the formation of an extended prehairpin intermediate (phi). the gp n-and c-terminal heptad repeats (nhr, chr) then collapse to form a six-helix bundle ( -hb) in which the nhr form a trimeric coiled-coil, creating grooves where the chr bind. the viral and cellular membranes are thereby brought into close proximity, enabling fusion ( figure ). during phi formation, the nhr and chr do not interact and may thus be transiently targeted by compounds which prevent the formation of the six-helix bundle in a dominant-negative manner. one such compound is the synthetic chr mimic enfuvirtide (t ) [ , ] . a hydrophobic pocket located on the n-terminal peptide trimer groove of the -hb is highly conserved among hiv- sequences and plays a critical role in membrane fusion, and therefore represents a select target for inhibitors. eckert et al. used the particular approach called -mirror-image phage display‖ to identify d-peptides targeting the gp hydrophobic pocket [ ] . in this approach, a phage library of natural l peptides is screened against the mirror image of a target synthesized as d-peptide [ ] . by symmetry, the selected d-peptide phagotope sequences will bind the natural l-form of the target. the main advantage of d-peptides over l-peptide inhibitors is their resistance to natural proteases which enhances their oral bioavailability and serum half-life. in a first study, the authors screened a constrained -mer rpl against the d-peptide sequence of the gp hydrophobic pocket fused to a soluble trimeric coiled-coil (izn ). pocket-specific binders with a consensus motif cx ewxwlc were identified and inhibited cell fusion or hiv- entry into cells with an ic in the micromolar range when synthesized using d-amino acids. in a second study, the same authors constructed a sublibrary based on the consensus sequence identified in their first study [ ] which allowed the selection of sequences with a fourfold potency increase [ ] . surprisingly, the most potent peptide was a -mer with a cx ewxwlc motif which was probably selected from the -mer sublibrary because its smaller size favored a more compact hydrophobic core upon binding to the gp hydrophobic pocket. screening of second generation d-peptides from a -mer cx wxwlc library led to the selection of pocket-specific inhibitor of entry (pie) with an ic of nm. dimeric and trimeric forms of pie had respective ic values of . nm and pm. in a third study, the same authors constructed a phage library based on the pie core sequence flanked by two randomized amino acids (xxcdypewqwlcxx) and obtained phages with the h(a/p)-[pie core]-(r/k/e)l consensus sequence [ ] . a peptide (pie , hpcdypewqwlcel) exhibited a broad neutralizing spectrum and was even more efficient than t , reaching an ic of . nm, when trimerized. moreover, this third generation pie -trimer displays broadened inhibitory potency and resistance to viral variants, as escape mutants required over weeks of selection in vitro to emerge. the pie trimer is thus a promising entry inhibitor and may be used as a topical microbicide in its d conformation. in , there was no evidence of abs capable of binding the highly conserved nhr region targeted by the t inhibitor. to determine whether antibody fragments could target this determinant on the gp protein, miller et al. constructed a phage-displayed naï ve scfv library and screened it against a synthetic protein mimicking the -hb [ ] . this construct, named -helix, lacks one of the three chrs and the nhr trimer is partially exposed, presenting a single binding site for a chr mimic [ ] . the scfv library was also panned against the izn compound, a homotrimerized form of nhr amino acids fused to a coiled-coil peptide, therefore representing a -hb mimic devoid of the chr trimer [ ] . the authors identified a scfv (d ), which blocks hiv- entry and inhibits infection in a single-cycle infectivity assay. this scfv retained its properties when produced as a whole igg . the antibody was found to bind the hydrophobic pocket of the nhr trimer and ala scan experiments revealed the crucial role of residues l , w , and k located in the hydrophobic pocket for this interaction. igg d was able to neutralize at least five hiv- isolates with ic ranging from to nm, thereby demonstrating that the hydrophobic pocket of the nhr trimer is accessible for binding of hiv- inhibitors as large as iggs. the same year, another study demonstrated that antibodies binding with high affinity and specificity to heptad repeats can be isolated from synthetic fab minimalist libraries presenting tyr/ser randomization of their cdr [ ] . very recently, liu et al. took advantage of these results and constructed a minimalist fab library where the lcdr and hcdr - domains were randomized with tyr and ser [ ] . screening of this library on the -helix mimic selected for fabs with affinity and specificity values comparable to those obtained with the scfv of the previously described d ab [ , ] . huang et al. used the n (l )c polypeptide mimicking the -hb [ ] to screen -mer and -mer rpls and selected sequences bearing a hxx(n/d)pf motif [ ] . this consensus motif synthesized as peptide (jch- ) inhibited hiv- -mediated syncytia formation [ ] . fusion inhibitors were also identified by screening non-immune human fab libraries. louis et al. screened such a library [ ] against antigens comprising the trimeric coiled-coil nhr fused or not to the gp six-helix bundle (n ccg-n and nccg-gp , respectively) [ , ] . they identified fabs targeting (i) the -hb; (ii) the nhr trimeric coiled-coil or (iii) both -hb and trimeric coiled-coil [ ] . these antibodies were tested in a cell fusion inhibition assay and the two more potent mabs, belonging to the third group, featured an ic of - µg/ml. two years later, the same library was screened against nccg-gp and -hb antigens [ ] . two clones, fabs and , inhibited cell fusion while one fab clone selected against nccg-gp was also effective in infection neutralization assays. fab bound the -hb as well as stable nhr trimers, and recognized an epitope that partially overlapped the hydrophobic pocket targeted by the d ab. the same authors subsequently demonstrated that the n mut(e,g) peptide presenting mutations within the th and th aa residues of the heptad repeat [ ] increased the temporal window of viral sensitivity to fab and thereby synergistically enhanced the neutralizing activity of fab as well as of the bntabs f and e [ ] . a fab sublibrary was created by affinity maturation of the fab hcdr loop and screened against nccg-gp , selecting for three fabs (fabs , and ) with enhanced potency (average -fold decrease in ic ) and neutralization breadth [ ] . to follow-up a study demonstrating that affinity-purified iggs from rabbits immunized with n ccg n inhibited hiv- -mediated fusion [ ] , nelson et al. rescued a scfv antibody library from these animals [ ] . three n ccg n binders were selected, and one of them, k , displayed neutralizing activity against hxb . in parallel a more complex fab library was constructed from the fda- hiv- positive patient from whom z ab had previously been isolated [ ] . screening this library against n ccg n allowed for the isolation of fab dn [ ] . both scfv k and fab dn neutralized hiv- infection with a panel of viral strains with ic ranging from to nm and targeted the nhr trimeric coiled-coil, presumably close to the hydrophobic pocket. three additional gp -specific abs (m , m and m ) were obtained by screening antibody phage libraries from asymptomatic seropositive patients [ ] against gp [ ] [ ] [ ] . a recombinant gp (gp r ) isolated from an asymptomatic seropositive patient with bntabs was reported to elicit bntabs in monkeys, further demonstrating that immunogenic epitopes were exposed on this recombinant antigen [ ] . competitive antigen panning (cap) (biopanning approach designed to outcompete phagotopes binding to an immunodominant region of a multi-domain target through concomitant addition of an excess of soluble forms of this immunodominant domain) using a mixture of gp r as antigen and gp r as competitor resulted in the selection of a gp -specific m ab [ ] . m displayed broad neutralization properties and recognized a conformational epitope and bound weakly to -helix antigen but not to the trimeric nhr nor to -hb. in two other studies, the same libraries were panned against gp / from three different isolates ( . , cm and r ), which led to the identification of the m ab recognizing a conformational epitope of gp [ ] and the m ab, which binds gp , -helix and -hb but not to the nhr trimeric coiled-coil. m recognized a conserved conformational epitope and neutralized isolates from different clades with a significantly higher potency than e or z [ ] . the competitive antigen panning approach against gp / thus allows the selection of abs recognizing conformational epitopes on gp , which are not properly folded when gp is used as a target. most of the studies found in the literature that apply the phage display technology to the discovery of hiv- inhibitors target the env protein. however, reports about the identification of peptides directed against other hiv- proteins involved in viral replication as well as interfering with rna sequences have been published and are summarized in this section. vpr is involved in the nuclear import of the viral preintegration complex (pic) as well as in the induction of apoptosis after cell cycle arrest and can be packaged into virions in quantities similar to the structural proteins [ ] [ ] [ ] [ ] . vpr was also reported to be associated with numerous cellular proteins such as glucocorticoid receptors, transcription factors or the uracyl dna glycosylase (udg) [ , [ ] [ ] [ ] . to investigate whether vpr may be used as a docking protein to deliver anti-viral compounds into virions, bouhamdan et al. used a -mer rpl to determine a common motif involved in the interaction between vpr and its various ligands [ ] . screenings rounds against vpr fusion proteins pinpointed sequences sharing a wxxf motif. since udg contains a wxxf sequence, mutants were constructed and confirmed the importance of this motif for vpr binding. cotransfection experiments indicated that the wxxf motif might be used to deliver a fusion protein into the hiv- virion through a new docking strategy. in , krichevsky et al. conducted a study to elucidate the exact role of vpr and its contribution to the nuclear import process of the hiv- pic [ ] . to that aim, a semi-synthetic scfv library [ ] was screened against the n-terminal (aa - ) part of vpr (vprn) conjugated to bsa (vprn-bsa). purified scfvs fragments featuring their strong and specific binding to the vprn sequence recognized full-length vpr and inhibited vpr-mediated nuclear import, indicating that targeting vpr may lead to the development of new peptides to fight viral infection. the phage display technology has also been applied to the identification of the hiv- integrase inhibitors. in , desjobert et al. screened a -mer rpl against recombinant hiv- integrase and identified a high affinity phagotope displaying the fhnhgkq sequence [ ] . in peptide format, this sequence inhibited the strand transfer activity of in by competing with the target dna, providing the proof-of-concept that in is also a valuable target for phage display. interaction of the viral transcription activator tat with the human cyclint subunit of the positive transcription elongation factor (p-tefb) complex and the cooperative binding of this complex to the transactivation response element (tar) rna are prerequisites of hiv- transcription [ ] . screening rpls or fab libraries against tat, cyclint or tar elements using the phage display technology identified peptides impairing tat-mediated hiv- replication. the first study was conducted in , when pilkington et al. screened a fab library constructed from the ab repertoire of an hiv- -infected asymptomatic patient and selected fabs recognizing a region comprised between amino acids to of the tat protein in a conformation-dependent manner [ ] . many years later, a non-immune human scfv phage-displayed library was explored to identify peptides binding to cyclint [ ] . clones recognizing the cyclin box domain of cyclint or interacting with the tat/tar recognition motif (trm) were isolated after panning against the n-terminal amino acids of cyclint . when expressed as intrabodies (antibody or antibody fragment expressed intracellularly), one of these scfvs inhibited tat-mediated transactivation without impairing cellular basal transcription or inducing apoptosis and partially inhibited hiv- replication in cultured cells. the tar rna sequence adopts a specific structure recognized by the basic arginine rich motif (arm) of tat and thus represents a potential target for phage display screening. in , kolb and boiziau screened a -mer rpl against tar rna molecules and selected -mer and unexpectedly -mer sequences from the library [ ] . the latter were proposed to arise from incomplete enzyme restriction during the construction of the initial library. clones were further characterized in peptide format and displayed tar-specific binding. the authors suggested that the surprisingly long peptides might have been selectively retrieved from the library because they presented a conformation that shorter -mer peptides were unable to adopt. the hiv- nucleocapsid protein p (ncp ) is processed from the gag precursor and is involved in the protection and encapsidation of viral rna leading to viral assembly through interaction with a specific secondary structure of the -base long psi rna [ ] [ ] [ ] [ ] . lener et al. screened a constrained -mer rpl against ncp and selected phagotopes sharing a ppx(d/e)r consensus motif [ ] . further binding experiments suggested that the ncp -phage interactions involved amino acids to of ncp , encompassing a zinc finger domain. studies to identify inhibitors of viral packaging were also conducted. rpls were screened on the psi rna immobilized onto a streptavidin-coated surface by annealing its '-end to a biotinylated oligomer, leading to the selection of peptides characterized by an hwwpww motif [ ] . peptide variants presenting this motif were subsequently synthesized and the most efficient binder was shown to strongly reduce virus release by infected cells, suggesting that it could serve as a lead compound to develop new anti-hiv- drugs [ ] . a similar screening campaign was conducted, where the '-end of the psi rna was covalently immobilized, leaving the secondary structure intact and fully accessible [ ] . screening of a -mer rpl selected for four clones with either whxt or hssxy motifs which were assessed for specific and dose-dependent binding to psi rna. the most prevalent sequence (syqwwwhspqtl) was expressed in fusion with the maltose-binding protein and was able to compete with ncp for binding to psi rna, confirming the value of the peptide as a potential hiv- inhibiting compound. hiv- accessory proteins nef and vif have an important role in hiv- viral replication and infectivity and, as such, represent as such interesting targets for inhibitors. in , yang et al. demonstrated that vif was able to multimerize and that its -aalikpkqikpplp- domain was critical for multimerization pointing to it as an interesting target to impair vif-mediated viral replication [ ] . the authors therefore, screened a -mer rpl library against vif and selected phages sharing a common pxp motif [ ] . four of these sequences synthesized as peptides bound the c-terminus of vif with high affinity and were able to inhibit vif-vif as well as vif-hck tyrosine kinase interactions. moreover, these peptides inhibited hiv- replication in cultured cells. besides the identification of inhibitors from rpl, the discovery of abs targeting nef and vif applicable to intrabody-based therapy may represent an alternative way to impede viral replication [ ] . in a very recent study, yoshikawa et al. evaluated the effect of two different schedules of nef and vif administration for mice immunization prior to the construction of scfvs phage-displayed libraries [ ] . results demonstrated that the immunization protocol influenced the complexity of the elicited ab repertoire and thus the successful identification of abs specifically recognizing the target. a nanobody (sdab ) recognizing a conformational epitope and reacting with a high affinity (k d : nm) with nef proteins from a panel of hiv- m, n, o and p groups was isolated through phage displaying the v hh repertoire of a llama immunized with a purified recombinant nef protein (fragment - ) [ ] . when expressed as an intrabody, this anti-nef sdab inhibited important biologic functions of nef both in vitro and in vivo in cd c/hiv- nef transgenic mice. the first step in cytotoxic t lymphocytes (ctl) activation is the recognition by a t cell receptor (tcr) of the antigen-derived peptide/mhc class i complex (pmhc). to date, few studies were undertaken to examine antigen presentation at a cellular and molecular level. nunoya et al. screened pooled scfv libraries [ ] on an immunodominant hla-a* (a ) restricted ctl epitope within the nef protein (nef - ; rypltfgwcf) [ ] . the panning procedure yielded clones binding specifically to nef - /a . clones scfv and scfv were able to bind to nef - /a expressed at the cell surface and retained this specificity when expressed as reconstituted whole iggs. this recent study was the first to address the identification of monoclonal antibodies binding specifically to an immunodominant hiv- ctl epitope loaded on an hla class i molecule. reverse transcriptase is a valuable target for anti-hiv- compounds, as illustrated by the success of the multiple small compounds used in haart. in , gargano et al. panned a phage-displayed library of synthetic combinatorial human fab fragments against recombinant hiv- rt [ ] . two ab fragments that specifically inhibited the rna-dependent dna polymerase (rddp) activity of rt were identified. both fragments also inhibited the activities of avian and murine retroviral rts as well as the human dna polymerase α and prokaryotic dna polymerases. because of their lack of specificity, these abs fragments were not exploited further as anti-hiv- molecules. to develop a panel of recombinant mabs reacting with different epitopes of the rt, ohba et al. immunized mice with recombinant rt expressed in a vaccinia virus vector and constructed a phage-displayed mice fab fragments library [ ] . biopanning against recombinant rt led to the identification of two fab fragments ( f and g) able to strongly inhibit the rddp activity of hiv- rt. epitope mapping and competitive elisa showed that f and g recognized an epitope similar or closely related to the epitope targeted by the mouse mab ( c ) previously described by the same authors [ ] . two years later, a semisynthetic phage display library of human scfvs with randomized heavy and light chain cdr was screened against recombinant rt [ ] . five different scfv abs directed against rt were isolated, of which three (f- , e , b ) inhibited the rddp activity of rt; of note, (f- ) also inhibited rt dna-dependent dna polymerase (dddp) activity. synthesis of the peptides corresponding to the cdr regions of the heavy and light chains showed that the heavy chain cdr inhibited rddp activity while the light chain peptide had no effect. these hcdr peptides represent the smallest antibody fragments inhibiting the rt identified to date and demonstrated that hcdr repertoire is a potential source of bioactive molecules (see section . . . .). rev is a key regulatory protein. oligomerized rev binds to unspliced or singly spliced viral mrna and ensures its transport to the cytoplasm, thereby allowing the translation of viral gene products. despite considerable efforts, the structure of rev is poorly characterized since rev is refractory to crystallization, mainly because of its tendency to form insoluble aggregates [ ] . in the absence of structural information, the phage display technology was used by different authors to map the domains involved in the interaction of rev with its network of partners. pilkington et al. identified two rev-specific fabs from a fab library derived from the ab repertoire of an hiv- -infected asymptomatic patient [ ] . these fabs were directed against sites adjacent to the rev basic nuclear localization signal (nls) (residues - ) and to the activation domain (residues - ). two years later, jensen et al. screened a -mer rpl to identify potential rev peptidic antagonists [ ] . three groups of sequences sharing a srlxg(x) - r motif (group i), sharing a rvv(x) - rg/a motif (group ii) or featuring no sequence similarity (group iii), were obtained. three clones were selected based on their high frequency of occurrence (p and p , group i) or on their strong binding affinity for rev (p , group iii). they were synthesized as peptides and were shown to retain rev binding specificity. more recently, llama nanobody libraries from animals immunized with recombinant rev allowed the identification of rev-binding nanobodies [ ] . one of them (nb ) prevented or disrupted rev multimerization by interacting with lys and tyr of the rev n-terminal α-helix [ ] . besides inhibitor discovery, fabs were recently proposed as -crystal chaperones‖ to support crystallization of their partners by locking them in specific conformations and blocking aggregation [ ] . stahl et al. described the preparation, characterization, and crystallization of an equimolar complex formed between rev and a chimeric rabbit/human fab (sjs-r ) selected through phage display [ ] . the rev/sjs-r fab complex was successfully crystallized and the fab sjs-r was shown to recognize a conformational epitope in the n-terminal half of rev. structural characterization of the crystallized fab/rev complex is ongoing and a corresponding scfv has been engineered and may have anti-hiv- properties. to identify peptides interfering with hiv- capsid assembly, sticht et al. screened a -mer rpl against the capsid (ca) protein generated by the proteolysis of the gag precursor and identified phagotopes whose sequences could be classified in four groups [ ] . one of these sequences (cai, capsid assembly inhibitor) competed with phagotopes for binding to ca and inhibiting capsid assembly in vitro. interaction with cai was mapped to ca amino acids , with additional contacts in helix . cai did not inhibit capsid assembly in vivo, but may nevertheless serve as tool for drug screening and as a starting point for drug design based on its ca-binding properties. peptides and antibody fragments selected by means of phage display may also be used for diagnostic purposes or to assess the diversity of the immune response against hiv- -specific antigens. de haard et al. constructed a scfv library from pbls of an hiv- positive patient presenting antibodies against gp , gp and p and screened the library against gp and p [ ] . one phagotope recognizing an epitope within the f and e bntabs epitopes on gp (ab# ) with affinities in the nanomolar range was isolated. importantly, it was shown to compete with out of gp -reactive plasma samples from north-american and african hiv- positive patients, indicating that this antibody recognizes an epitope conserved in a large panel of isolates and might be suitable for diagnostic applications. since all vaccine candidates elicited antibodies reacting positively in hiv tests, khurana et al. applied the phage display technology to identify hiv- epitopes susceptible to help discriminate between successfully immunized vaccinees and seroconverters [ ] . they constructed a phage library displaying the full hiv- genome and screened it against the sera of newly seroconverted hiv- positive individuals. they identified conserved epitopes present in gp and in gag p that were not part of the vaccine used at that moment and established a new detection test, named selectest, that demonstrated over % selectivity and sensitivity for the early detection of seroconversion. hiv selectest was able to detect antibodies against these epitopes in newly infected patients as early as to weeks after infection. in parallel to the targeting of viral proteins, many efforts were undertaken to identify peptides, antibody fragments or modified ligands binding to the hiv- host proteins and impairing their interactions with the viral proteins (table ). the hiv- host receptors cd , ccr and cxcr are involved in the early steps of hiv- infection and thus represent valuable targets for the identification of antiviral peptides or neutralizing abs. moreover, these receptors display very low variability compared to the viral env proteins facilitating the identification of neutralizing antibodies. although the cd receptor plays a crucial role in the entry process, the only phage display biopanning assays reported to date targeted the chemokine receptors ccr and cxcr . however, to circumvent the difficulties of purifying and immobilizing such complex receptors on a solid support without losing their native structure, biopanning procedures had to be adapted. in this regard, screening strategies using biopanning on living cells [ , ] , proteoliposomes [ ] or peptides derived from the receptors extracellular parts [ , ] were particularly successful. the cc chemokine receptor (ccr ) is one of the two major hiv- coreceptors and binds three different endogenous chemokines ccl (rantes), ccl (mip- β) and ccl (mip- α) which were reported to prevent r -tropic hiv- entry. interestingly, inhibition of ccr binding to hiv- provides an almost complete protection against r -tropic viruses with only minor effects on the normal physiological functions of the cells [ ] . the first biopanning experiment targeting ccr was performed using receptor embedded in paramagnetic proteoliposomes [ ] . to create such proteoliposomes, magnetic beads were added to a mixture of synthetic lipids, a detergent-solubilized c -tagged ccr receptor and a capture antibody, reconstituting membrane bilayers containing pure, native and properly oriented ccr receptor. these proteoliposomes were used in biopanning experiments with a human scfv antibody library and several antibody fragments specifically binding to ccr -expressing cells were identified. the same year, steinberger et al. used the phage display technology to select and to humanize rabbit anti-ccr antibodies preventing the export of ccr to the cell surface [ ] . following rabbit immunization with a gst-nterm ccr fusion protein, the authors constructed a phage displayed fab library that was screened against the antigen initially used for the immunization. a phagotope (st ) binding strongly and specifically to the immobilized antigen as well as to ccr -positive cells was identified, expressed as a scfv and humanized by successive replacements of the rabbit light and heavy chains by their human counterparts. one humanized antibody fragment, st / , that retained the strong ccr -binding capacity of the parental st antibody was isolated from the screening of the intermediate libraries. when expressed as intrabody the st / scfv efficiently blocked the ccr expression at the cell surface. [ ] (reviewed in chevigné et al. [ ] . in this strategy, a phage library displaying randomly mutated and n-terminally extended ccl chemokine variants (xs#xssx###-ccl , where # represents either a, p, s or t) was constructed and screened on ccr -expressing cells. only intracellular phagotopes that had induced ccr receptor internalization were recovered. two ccl variants (p = lspvssqssa-ccl and p = fsplssqssa-ccl ) were identified. these variants displayed a higher selectivity for ccr and had more potent hiv- inhibitory abilities than the wild-type ccl chemokine. further characterization demonstrated that p acted as a ccr superagonist and potently induced intracellular ccr sequestration. p was less potent but significantly reduced ccr -dependent intracellular calcium signaling. in a subsequent study, p and p variants were optimized by phage display biopanning to select for variants that retained the high anti hiv- potency of p and p but reduced ccr -agonist activity [ ] . three successive generations of libraries (xxpx q#tp-ccl , qgpplmx -ccl and qgpΨ$x -ccl where Ψ represents g, l or p and $ represents g, l or m) were constructed and screened as previously described [ ] . the three most interesting candidates ( p -ccl , p -ccl and p -ccl ) produced as soluble proteins displayed highly potent antiviral activities. analogue p -ccl acted as an agonist and sequestered ccr , p -ccl induced no signaling or receptor sequestration while p -ccl induced ccr internalization without triggering g-protein signaling. altogether these data demonstrated that antiviral activities of similar molecules identified through phage display screening can rely on various mechanisms of action. shortly after the identification of the p and p variants, zhang et al. reported an alternative biopanning methodology relying on the use of small cyclic biotinylated peptides mimicking ccr extracellular loops (ecl , ecl and ecl ) for the identification of ccr -binding scfvs [ ] . a mouse phage-displayed scfv library was incubated with biotinylated-ecl peptides and ecl-binding phages were specifically recovered using streptavidin-coated beads. among the ccr -binding scfv identified, three clones (a , b and l ) selected on cyclic ecl or ecl peptides inhibited r -tropic hiv- . screening of rpl was also applied to the discovery of small ccr -blocking peptides through the targeting of receptor-expressing cells [ , ] . vyroubalova et al. screened a partially randomized -mer phage library (cdx kpcallryx -piii) using competitive elution with a ccl analogue (nny-ccl , nm) and selected a unique peptide (allrynpfyylsfsp). this peptide was further optimized through n-terminal extension, exon shuffling and biopanning. by applying successive treatments consisting of a preselection using a low amount of nny-ccl ( pm) to discard low-affinity binding phages followed by classical alkaline elution using tea and a competitive elution containing nny-ccl ( µm) they identified an extended peptide (lldstfftadallrynpfyyls-fsp) inhibiting r -tropic hiv- cell fusion with an ic of μm. in parallel, wang et al. screened a fully randomized -mer rpl using acidic elution and identified phagotopes binding specifically to ccr -expressing cells and sharing the afdwtfvpslil sequence [ ] . in peptide and phage formats this sequence blocked the binding of the anti-ccr neutralizing d mab and completely inhibited binding of the chemokine ccl to the receptor [ ] . the cxc chemokine receptor (cxcr ) is the second major hiv- coreceptor. cxcr binds only to one endogenous chemokine ligand (cxcl ) and is also expressed at the surface of numerous cancer cell types underlining its high value as therapeutic target [ , ] . despite this importance and the relative success of phage biopanning on ccr , only two recent studies reported the use of phage display to search cxcr inhibitors [ , ] . in , jahnichen et al. isolated llama-derived v hh binding specifically to cxcr and inhibiting the entry of x -tropic virus [ ] . to select v hh binding exclusively to functional and properly folded receptor, llamas were immunized with cxcr -expressing hek t cells. a phage library was subsequently constructed from the pbmcs of immunized camelids and several phage clones inhibiting the binding of labeled cxcl chemokine to the receptor were identified. in particular, two v hhs ( d and d ) showed low nanomolar affinity for the receptor and inhibited entry of x and x /r -viruses into different cxcr + cell types with ic values ranging from to nm. dimerization of d and d to form biparatopic proteins increased their antiviral properties to ic values in the picomolar range. epitope mapping revealed that the two v hh s inhibited cxcr mainly through binding to the second extracellular loop. very recently, we used a peptide corresponding to this particular extracellular loop (ecl ) as target to identify short cxcr antagonists [ ] . by screening a non-immune phage library displaying the human hcdr peptide repertoire [ ] , several small peptides binding to the ecl peptide that specifically recognized cxcr -expressing cells were identified. notably, one of these hcdr peptides (typgry) acted as a cxcr antagonist with potency in the micromolar range. in addition to targeting host receptors, a few studies reported the identification of peptides or antibody fragments directed against other host proteins such as cell surface determinants (cd) or intracellular enzymes [ , , , ] . the cd receptor, a member of the tumor necrosis factor receptor superfamily, and its cd l ligand are involved in several biological processes including cell proliferation, activation and production of cytokines and chemokines. during hiv- infection, viruses were proposed to selectively downregulate or even deplete the pool of cd l-expressing cd + t cells. in this context, antibodies binding to cd and restoring the hiv- -induced cd l downregulation might be of interest. in , ellmark et al. identified a set of anti-cd antibody fragments through biopanning of a human scfv phage library against a biotinylated cd antigen [ ] . when expressed as full-length igg one antibody (clone b ) suppressed hiv- infection by a r -tropic virus, most probably through induction of cc chemokine production [ ] . more recently, the ddx protein, a cellular rna helicase involved in rna unwinding was shown to play important roles in hiv- replication. this protein presents a unique region (alramkeng) responsible for high affinity binding to the hiv- rna. garbelli et al. carried out a biopanning experiment with a mix of linear -mer and linear and constrained -mer rpl against a peptide mimicking the ddx region interacting with hiv- . they identified a -mer peptide (sdvptqv) blocking the replication of an x -tropic virus with an ic of μm [ ] . besides therapeutic purposes, the phage display technology has also been applied for fundamental studies of host proteins. in particular, a study focusing on the roles and the diversity of the anti-cd autoimmune repertoire in the myelosuppression appearing in hiv- infected individuals was reported by rubinstein et al. [ ] . using a substractive biopanning procedure from the immune repertoire of a hiv- seropositive patient, the authors selected fab fragments binding specifically to the cd receptor. sequencing and binding analyses of these antibody fragments demonstrated the heterogeneous origin of the anti-cd autoimmune repertoire and suggested that these autoantibodies might be generated through antigen-specific driven processes. the use of phages for protease cleavage specificity profiling was first described by matthews and wells in [ ] . this -phage substrate‖ approach relies on the use of phage particles to screen for enzyme substrates instead of classical binder selection. protease cleavage profiling using phage takes advantage of the natural resistance of phage particles to proteolysis. phage particles displaying random peptides are immobilized on a solid support and submitted to proteolytic elution to specifically liberate phages presenting peptides corresponding to the protease cleavage site. the phage substrate approach allows thus to rapidly determine the cleavage profile of a given protease and provides optimized substrate candidates which can be further used as leads for the development of specific inhibitors. over the last two decades, phage substrate has been applied to a large variety of proteases including the hiv- protease (pr) [ , ] . the hiv- pr is a homodimeric aspartic protease responsible for nine critical cleavage steps within both the structural (gag) and the non-structural (gag/pol) polyproteins. the hiv- protease recognizes substrate residues encompassing p to p ' positions (schechter and berger's nomenclature), with the primary determinants from positions p to p ' positions [ , ] . interestingly, alignment of the nine natural substrate cleavage sites of the hiv- pr shows a high sequence diversity suggesting a broad proteolytic specificity. in , beck et al. reported the use of hexapeptide phage library to unravel the hiv- pr specificity and develop new protease inhibitors (table ) [ ] . this library was constructed by fusing the mab -e epitope upstream of the randomized sequences. phages were first incubated with the hiv- pr and uncleaved phages were removed by addition of pansorbin cells. biopanning selected for highly diverse sequences consistent with the suggested broad substrate specificity of the hiv- protease. however, none of the selected peptides corresponded to the hiv- polyproteins cleavage sites. nonetheless, several phage-displayed peptides were very efficiently cleaved by the hiv- protease. in peptide format, most of them displayed a k m value lower than the one determined for a peptide mimicking the natural substrate at the matrix/capsid junction (irkil↓fldg) ( table , italic). the most potent selected substrate (gsgif↓letsl) was cleaved times more efficiently and had a k m value of μm i.e., times lower than the natural substrate (k m = μm). interestingly, the gsgif↓letsl substrate displayed only two residues (in bold) of the optimal cleavage site model designed based on the most frequently selected residues for each position (sgvy↓fvts) ( table ) . table . phage substrate analysis of the hiv- protease cleavage specificity. arrow denotes cleavage sites in the natural or the selected substrates sequences. underlined substrates correspond to sequences used to derived inhibitors. italic sequences correspond to natural substrate (matrix/capsid junction) used as reference for the assessment of the cleavage efficiently. Ψ: amid-reduced bound. table . hiv- protease specificity model. the model was build based on the most efficiently cleaved phage substrates. underscript numbers represent the frequency of appearance of the amino acid at a given position. sequences corresponding to the four most potent substrates were synthesized as peptidic transition state analogues and presented inhibitory activity in the nanomolar range (table , underlined sequences). in , the same authors applied the phage substrate approach to compare the relative specificities of the human (hiv- ) and feline (fiv) immunodeficiency virus prs [ ] . hexapeptides specifically processed by each of the two proteases as well as peptides cleaved by both enzymes were identified. further mutational analysis of synthetic peptides derived from a sequence processed with the same efficiency by the two proteases (ksgvf↓vvng) was performed to assess the influence of amino acid substitutions on the catalytic process of each pr (table , underlined sequence). they showed that substitutions for a val at position p or p ' increased the cleavage by the hiv- protease whereas the introduction of a val at position p was more favorable to fiv protease activity. in particular, the gsgvfΨ(ch nh)vvngl inhibitor identified in the first study was active against both prs with different potencies and replacement of its amide group by a hydroxyethylene group resulted in a peptide with equivalent inhibitory activity towards both the hiv- and the fiv proteases. in parallel to epitope mapping, inhibitor discovery and enzyme profiling applications, bacteriophage particles were exploited as carriers for the development of anti-hiv- vaccines. for this particular purpose, phages are not used as affinity selection tools but rather to display antigens to the immune system, aiming to elicit specific neutralizing antibodies or/and efficient cytotoxic t-cell responses. indeed, phages can also be considered as biologically inert particles characterized by a dense and repetitive organization capable of displaying a wide range of exogenous proteins at a precise valency and in a controlled manner. moreover, phages were shown to be naturally immuno-stimulatory [ , ] and are particularly affordable, easy and rapid to produce and to administer in many animal models [ ] . altogether these characteristics make bacteriophages a suitable and valuable carrier for hiv- vaccine development. anti-hiv- vaccination trials to elicit humoral and cellular responses against various hiv- proteins such as the envelope proteins gp and gp as well as the rt and the p proteins [ ] [ ] [ ] [ ] [ ] were conducted with a large diversity of bacteriophages including m , t , ms and lambda phages [ , [ ] [ ] [ ] [ ] ( table ) . numerous phage-vaccination trials using mimotopes identified on anti-hiv- antibodies were reported (see tables - ). all these attempts aimed mainly at eliciting antibodies directed against the genuine epitope of an existing antibody. besides these descriptions, a few studies were exclusively dedicated to the use of phage particles as vehicles to display fragments or complete hiv- proteins to the immune system to prime new immune responses. the pioneer attempt was reported by minenkova et al. in [ ] . immunization of rabbits with filamentous m phage particles displaying a small peptide (gedrw) derived from the p gag protein in fusion with piii minor coat protein elicited the production of specific iggs reacting with the natural p antigen as well as with the gag precursor protein. shortly afterwards, two studies reported that mice immunization with fd phages displaying the gp v loop fused to the piii protein induced high titers of antibodies cross-reacting with v loops of different strains and featuring neutralizing activity [ , ] . the major coat protein pviii of the fd phage ( copies per particles) was also investigated as scaffold protein for vaccination. immunization with phages displaying a pviii-fused peptide corresponding to residues - of the hiv- rt resulted in the induction of a specific cytotoxic t-cell response (ctl) against the displayed peptide. priming nevertheless required a co-immunization with a t-helper epitope from the same protein (kdswtvndiqklvgk) provided by either the same or a separate phage particle [ ] . more recently attempts to prime ctl response were conducted with m phages displaying a mixture of thousands of variants of ctl epitopes (rgpgxax or xgxgxaxvxi) derived from the gp v loop (residues - ; rgpgrafvti) presented in an immunoglobulin v h domain scaffold [ ] . mice immunization provided potent and broad epitope-specific long lasting response ( months) and effector memory t cells were induced. moreover, recent studies demonstrated that mice immunized with these variable epitope libraries are capable of neutralizing half of the subtype b viral isolates used for challenge, including hiv- isolates which are known to be resistant to neutralization by several potent monoclonal antibodies [ ] . although they are versatile and easy to manipulate, filamentous phages present limitations for vaccination, and namely low display level and the limited size of the foreign peptides that can be incorporated without interfering with the natural functions of the phage coat proteins. therefore, alternative bacteriophage models were explored. t phage and its two accessory capsid-decorating proteins soc and hoc were demonstrated to be particularly efficient for high copy display of various large hiv- antigens [ , , ] . in contrast to piii and pviii proteins, soc and hoc present the major advantage of being dispensable for phage assembly yet of being able to strongly bind to the outer surface of the capsid, if added subsequently [ ] . soc and hoc self-associate to the capsid at a display level of and copies per capsid, a far higher density than that of filamentous minor coat protein ( to copies). hoc protein provides high-density display of single as well as multiple antigens and immunization of mice with the p -hoc-t elicits strong humoral and cellular responses. moreover, fusion of different antigens to soc and hoc offer the possibility to develop multicomponent hiv- vaccine particles [ ] t phage displaying v loop of the gp protein fused to soc protein was reported to elicit antibodies capable of recognizing the native antigen [ ] . further study demonstrated that hoc protein can also be used to display various hiv- antigens including the p , nef or a trimeric peptide derived from the c-terminus of gp [ ] . more recently, lambda phage and its decorating capsid protein were used for dense display of glycosylated mammalian cell-derived trimers of gp protein [ ] . lambda phage provided a display level of copies of gp trimer per particle, a -fold higher display than observed on native hiv- virions ( ± spikes per virion) [ ] . rabbit immunization trials were rather disappointing and higher antibody titers were elicited in animals receiving soluble oligomeric gp . these results were proposed to be due to the sequential immunization process and to the strong and immunodominant response against phage capsid proteins, which is most probably boosted upon sequential immunization, and could lead to a decrease of the humoral immune response to the displayed antigen. virus like particles (vlp) derived from the ms bacteriophage coat protein displaying the v loop of gp and devoid of phage genome were also reported [ ] . although characterized by a low display level ( copies per vlp) these particles were nevertheless able to elicit high titers of specific antibodies when injected in mice and provided neutralizing activity at / sera dilution. more recently, pp phage-derived vlps displaying the v loop of gp were used to immunize mice providing high-titer antibody response [ ] . aids was first described in [ ] , and two years later hiv- , its causative agent, was isolated [ ] . the phage display technology was first published almost concomitantly in [ ] . both protagonists lived parallel lives until , when burton et al. identified a human fab recognizing the cd -binding site of hiv- gp through phage display [ ] . this first antibody, b , turned out to be one of the rare bntabs characterized to date, and many studies are still ongoing to set up scaffolds presenting its epitope in a conformation capable of eliciting abs with b -like hiv- inhibiting properties. this article was the starting point of a dense literature exploiting the different applications of the phage display technology to gain as much knowledge as possible on the hiv- infection process. the complex hide-and-seek game between hiv and the host immune system and the absence of an efficient vaccine candidate emphasizes the need to better comprehend the hiv- -related immune response and to identify antiviral molecules. hiv- is to the best of our knowledge the only pathogen to which all four applications of the phage display technology described in this review-i.e., epitope mapping, inhibitor discovery, substrate presentation and carrier phage-have been applied. parallel improvements in the technology on one hand, and crucial discoveries in the hiv- field on the other hand, most likely account for such a tight interplay. the phage display technology applied to hiv- has provided precious knowledge concerning the regions recognized by the ab repertoire of immune patients, and allowed to identify immunodominant regions. it has also allowed to map the epitopes of numerous monoclonal antibodies directed against viral and host proteins, and, most importantly, discontinuous epitopes. alternative phage display biopanning substractive procedures such as epitope masking or competitive antigen panning also precisely highlighted non-immunodominant regions of the hiv- antigen, which had remained occult using classical biopanning approaches. although the antibodies identified through epitope-masking are mostly devoid of neutralizing activity, this strategy led to the identification of fabs binding with high affinity to specific targets. taking advantage of this affinity, approaches aiming at engineering bispecific antibodies, coupling these inhibitors to toxins, or multimerizing them to increase their binding avidity could lead to the development of antiviral compounds or more efficient vaccine candidates. although highly informative, immunization trials performed with mimotopes/phagotopes selected through phage display remained altogether rather disappointing as no long-lasting neutralizing response was reported, and provided yet further proof that antigenicity does not necessarily imply immunogenicity [ ] . among the bottlenecks in the field of hiv- vaccine research and development are the weak immunogenic properties of the identified mimotopes or native antigens. whether the difficulty of eliciting and/or isolating strong or broad neutralizing antibodies from hiv- -infected patients, be they normal progressors or ltnps, is a caveat of phage display or a real reflect of the restricted humoral response in fighting hiv, or to both, remains to be established. nevertheless, these discrepancies are not exclusive of hiv- mimotopes and were observed with other antigens. from a fundamental prospect, the use of phage-displayed igm libraries to model abs close to the germline abs allowed to elegantly expore the poorly characterized determinants of the initial antiviral immune response. phage display also contributed to a better knowledge on the structure of the diverse hiv- proteins as well as a gain of insight on non-structural proteins involved in replication mechanisms. a highlight example would be the phage substrate approach which allowed the precise characterization of the hiv- protease cleavage specificity and thereby provided valuable inhibitor candidates. unraveling viral replication steps or protein interactions led to exploit phage display to engineer various types of inhibitors targeting either viral or host proteins. in parallel, phage display allowed the identification of various types of inhibitors targeting either viral or host proteins. nevertheless, only a few strong antiviral candidates with broad neutralizing activities and/or potencies in the nanomolar or even picomolar range were identified with the phage display technology while most of them presented inhibiting activities that ranged at the best in the micromolar range and could not be further exploited. some of the most potent inhibitors originated from fab libraries derived from asymptomatic hiv- -infected patients whose reactivity against hiv- was previously assessed (b , x , z ) or from immunized camelids (v hh anti cxcr ). other inhibitors were identified from semi-synthetic (ligand analogues of ccl ) or randomized peptide libraries (d-peptides: pie ) and their affinity was improved through secondary libraries. these inhibitors were selected against different targets (env proteins, coreceptors) by biopanning carried out using different types of support (immobilized proteins, cells, peptides), illustrating the power and versatility of the phage display technology [ , , ] (figure ). inhibitors blocking key steps in the entry process were identified using the phage display technology. these inhibitors target: the cd binding site (fab b and z ), the coreceptors ccr (ccl variants) or cxcr (v hh d and d ), the cd -induced epitope of gp (fab x ) or the heptad repeat region of gp (peptide pie ). despite the variable success of the phage display technology per se in developing a direct antiviral agent or immune response, this technology continues to contribute precious and otherwise inaccessible developments in the field of hiv- research, mainly owing to the ability of phages to display a high density of either single antigens or libraries of antigens/mimotopes. to this regard, phages are being extensively explored to increase immune responsiveness. the latest developments include exploiting the phage as dna vaccine carrier or hybrid phage and pursuing the symbiotic interplay between phage display and hiv- . pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) running a tightrope: regulatory challenges in the development of antiretrovirals potent suppression of hiv- replication in humans by t- , a peptide inhibitor of gp -mediated virus entry maraviroc (uk- , ), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor ccr with broad-spectrum anti-human immunodeficiency virus type activity -dihydroxypyrimidine carboxamides and n-alkyl- -hydroxypyrimidinone carboxamides are potent, selective hiv integrase inhibitors with good pharmacokinetic profiles in preclinical species plasma hiv rna decline and emergence of drug resistance mutations among patients with multiple virologic failures receiving resistance testing-guided haart an overview of the determinants of ccr and cxcr co-receptor function the human immunodeficiency virus type envelope spike of primary viruses can suppress antibody access to variable regions continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers the antibody response against hiv- . cold spring harb. perspect filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface lambda-display: a powerful tool for antigen discovery antibody-selectable filamentous fd phage vectors: affinity purification of target genes human immunodeficiency virus type long-term non-progressors: the viral, genetic and immunological basis for disease non-progression production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type envelope glycoprotein identification of hiv vaccine candidate peptides by screening random phage epitope libraries anti-human immunodeficiency virus type human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries defining critical residues in the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies a human monoclonal antibody to a complex epitope in the v region of gp of human immunodeficiency virus type has broad reactivity within and outside clade b epitope mapping of anti-hiv and anti-hcv monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library permissive residues within the minimal epitopes of neutralizing monoclonal antibodies to the v loop of hiv- neutralization of divergent human immunodeficiency virus type variants and primary isolates by iam- - f , an anti-gp human monoclonal antibody human immunodeficiency virus type -neutralizing monoclonal antibody f is multispecific for sequences flanking the dkw core epitope constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-hiv- mab f helical epitopes determined by low-stringency antibody screening of a combinatorial peptide library phage-displayed mimotopes recognizing a biologically active anti-hiv- gp murine monoclonal antibody identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type antibody b probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage immunogenicity of hiv type gp cd binding site phage mimotopes the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv- peptides selected from a phage display library with an hiv-neutralizing antibody elicit antibodies to hiv gp in rabbits, but not to the same epitope structural and functional characterization of an epitope in the conserved c-terminal region of hiv- gp a peptide inhibitor of hiv- neutralizing antibody g is not a structural mimic of the natural carbohydrate epitope on gp antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a -amino acid sequence of the viral envelope, gp principal neutralizing domain of the human immunodeficiency virus type envelope protein human monoclonal antibody that recognizes the v region of human immunodeficiency virus gp and neutralizes the human t-lymphotropic virus type iiimn strain a conserved neutralizing epitope on gp of human immunodeficiency virus type humoral immune response to immunocomplexed hiv envelope glycoprotein a large array of human monoclonal antibodies to type human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals recombinant human fab fragments neutralize human type immunodeficiency virus in vitro computational prediction of the cross-reactive neutralizing epitope corresponding to the [corrected] monclonal [corrected] antibody b specific for hiv- gp a conserved hiv gp glycoprotein structure involved in chemokine receptor binding exploration of antigenic variation in gp from clades a through f of human immunodeficiency virus type by using monoclonal antibodies characterization of conserved human immunodeficiency virus type gp neutralization epitopes exposed upon gp -cd binding the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of α → mannose residues on the outer face of gp human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type the mannose-dependent epitope for neutralizing antibody g on human immunodeficiency virus type glycoprotein gp selection of hiv-specific immunogenic epitopes by screening random peptide libraries with hiv- -positive sera protection of rhesus macaques against disease progression from pathogenic shiv- . pd by vaccination with phage-displayed hiv- epitopes dissection of the humoral immune response toward an immunodominant epitope of hiv: a model for the analysis of antibody diversity in hiv+ individuals collection of phage-peptide probes for hiv- immunodominant loop-epitope mimotopes selected with antibodies from hiv- -neutralizing long-term non-progressor plasma inducing cross-clade neutralizing antibodies against hiv- by immunofocusing evolution of antibody landscape and viral envelope escape in an hiv- crf _ag infected patient with e -like antibodies unravelling the antigenic landscape of the hiv- subtype a envelope of an individual with broad cross-neutralizing antibodies using phage display peptide libraries mapping of hiv- gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system the protein data bank u. d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins molecularly cloned shiv- ipd n : a highly replication-competent, mucosally transmissible r simian-human immunodeficiency virus encoding hiv clade c env requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on ccr monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor ccr : implications for the tertiary structure of the receptor and for an n-terminal binding site for hiv- gp inhibition of m-tropic hiv- infection by the fd phage-gene protein with mip- α-binding activity identification of a linear peptide recognized by monoclonal antibody d capable of generating ccr -specific antibodies with human immunodeficiency virus-neutralizing activity identification of a lfa- region involved in the hiv- -induced syncytia formation through phage-display technology interaction of chemokine receptor ccr with its ligands: multiple domains for hiv- gp binding and a single domain for chemokine binding ccr levels and expression pattern correlate with infectability by macrophage-tropic hiv- , in vitro human immunodeficiency virus (hiv) infection in cd + t lymphocytes genetically deficient in lfa- : lfa- is required for hiv-mediated cell fusion but not for viral transmission molecular profile of an antibody response to hiv- as probed by combinatorial libraries specific killing of hiv-infected lymphocytes by a recombinant immunotoxin directed against the hiv- envelope glycoprotein cdr walking mutagenesis for the affinity maturation of a potent human anti-hiv- antibody into the picomolar range in vitro evolution of a neutralizing human antibody to human immunodeficiency virus type to enhance affinity and broaden strain cross-reactivity neutralizing recombinant human antibodies to a conformational v -and cd -binding site-sensitive epitope of hiv- gp isolated by using an epitope-masking procedure antibodies to the superantigenic site of hiv- gp : hydrolytic and binding activities of the light chain subunit cross-clade hiv- neutralization by an antibody fragment from a lupus phage display library generation of a family-specific phage library of llama single chain antibody fragments that neutralize hiv- llama antibody fragments with cross-subtype human immunodeficiency virus type (hiv- )-neutralizing properties and high affinity for hiv- gp jones, i. mutant cd molecules with improved binding to hiv envelope protein gp selected by phage display affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity mapping the protein surface of human immunodeficiency virus type gp using human monoclonal antibodies from phage display libraries peptide ligands to human immunodeficiency virus type gp identified from phage display libraries broadly cross-reactive hiv- -neutralizing human monoclonal fab selected for binding to gp -cd -ccr complexes peptide ligands selected with cd -induced epitopes on native dualtropic hiv- envelope proteins mimic extracellular coreceptor domains and bind to hiv- gp independently of coreceptor usage broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type glycoprotein gp an affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type gp recognizes an epitope between those of f and e potent d-peptide inhibitors of hiv- entry design of a potent d-peptide hiv- entry inhibitor with a strong barrier to resistance inhibiting hiv- entry: discovery of d-peptide inhibitors that target the gp coiled-coil pocket a human monoclonal antibody neutralizes diverse hiv- isolates by binding a critical gp epitope design of potent inhibitors of hiv- entry from the gp n-peptide region synthetic fab fragments that bind the hiv- gp heptad repeat regions identification of the hiv- gp core-binding motif-hxxnpf the mechanism by which molecules containing the hiv gp core-binding motif hxxnpf inhibit hiv- envelope glycoprotein-mediated syncytium formation characterization and hiv- fusion inhibitory properties of monoclonal fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of hiv- gp a monoclonal fab derived from a human nonimmune phage library reveals a new epitope on gp and neutralizes diverse human immunodeficiency virus type strains affinity maturation by targeted diversification of the cdr-h loop of a monoclonal fab derived from a synthetic naive human antibody library and directed against the internal trimeric coiled-coil of gp yields a set of fabs with improved hiv- neutralization potency and breadth sequestering of the prehairpin intermediate of gp by peptide n mut(e,g) potentiates the human immunodeficiency virus type neutralizing activity of monoclonal antibodies directed against the n-terminal helical repeat of gp antibody elicited against the gp n-heptad repeat (nhr) coiled-coil can neutralize hiv- with modest potency but non-neutralizing antibodies also bind to nhr mimetics cross-reactive human immunodeficiency virus type -neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp and lacks reactivity against self-antigens cross-reactive hiv- neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp ) isolated from a patient (r ) with broadly hiv- neutralizing antibodies selection of a novel gp -specific hiv- neutralizing human antibody by competitive antigen panning diversity of hiv- vpr interactions involves usage of the wxxf motif of host cell proteins antibody fragments selected by phage display against the nuclear localization signal of the hiv- vpr protein inhibit nuclear import in permeabilized and intact cultured cells identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type integrase recombinant human fab antibody fragments to hiv- rev and tat regulatory proteins: direct selection from a combinatorial phage display library inhibition of tat-mediated transactivation and hiv- replication by human anti-hcyclint intrabodies selection by phage display of peptides targeting the hiv- tar element use of a constrain phage displayed-peptide library for the isolation of peptides binding to hiv- nucleocapsid protein (ncp ) identification of peptide ligands for target rna structures derived from the hiv- packaging signal ψ by screening phage-displayed peptide libraries inhibition of hiv- by a peptide ligand of the genomic rna packaging signal ψ selection and characterization of peptides specifically binding to hiv- ψ (ψ) rna modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against hiv- nef and vif inhibition of the nef regulatory protein of hiv- by a single-domain antibody short communication: generation of recombinant monoclonal antibodies against an immunodominant hla-a* -restricted hiv type ctl epitope the multimerization of human immunodeficiency virus type i vif protein: a requirement for vif function in the viral life cycle potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type (hiv- ) vif proteins an immunodominant neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type reverse transcriptase revealed by phage display antibodies a novel neutralization epitope on the -thumb‖ subdomain of human immunodeficiency virus type reverse transcriptase revealed by a monoclonal antibody recombinant human antibodies against the reverse transcriptase of human immunodeficiency virus type- hiv- rev nuclear export signal binding peptides isolated by phage display measuring cooperative rev protein-protein interactions on rev responsive rna by fluorescence resonance energy transfer an intrabody based on a llama single-domain antibody targeting the n-terminal α-helical multimerization domain of hiv- rev prevents viral production generation and characterization of a chimeric rabbit/human fab for co-crystallization of hiv- rev a peptide inhibitor of hiv- assembly in vitro potential drug targets on the hiv- envelope glycoproteins, gp and gp small-molecule hiv- gp inhibitors to prevent hiv- entry: an emerging opportunity for drug development molecular characterization of the circulating anti-hiv- gp -specific b cell repertoire using antibody phage display libraries generated from pre-selected hiv- gp binding pbls relevance of the antibody response against human immunodeficiency virus type envelope to vaccine design binding of glycoprotein and peptides from the hiv- envelope by autoantibodies in mice with experimentally induced systemic lupus erythematosus and in patients with the disease prospects for immunotherapeutic proteolytic antibodies phosphonate ester probes for proteolytic antibodies naturally occurring proteolytic antibodies: selective immunoglobulin m-catalyzed hydrolysis of hiv gp theory of proteolytic antibody occurrence site-directed mutagenesis of proteolytic antibody light chain naturally occurring antibodies devoid of light chains nanobodies(r): new ammunition to battle viruses mode of action for linear peptide inhibitors of hiv- gp interactions cutting edge: cd is the receptor for the tick saliva immunosuppressor, salp the ixodes scapularis salivary protein, salp , prevents the association of hiv- gp and cd natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type gp epitopes a vh clonal deficit in human immunodeficiency virus-positive individuals reflects a b-cell maturational arrest selective variations in vivo of vh and vh gene family expression in peripheral b cell igm, igd and igg during hiv infection molecular analysis of hiv- gp antibody response using isotype igm and igg phage display libraries from a long-term non-progressor hiv- -infected individual improvement in affinity and hiv- neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by hiv- infection cross-reactive human igm-derived monoclonal antibodies that bind to hiv- envelope glycoproteins characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: implications for mechanisms of immune evasion and design of vaccine immunogens cross-reactive hiv- -neutralizing human monoclonal antibodies identified from a patient with f -like antibodies enfuvirtide: the first therapy to inhibit the entry of hiv- into host cd lymphocytes identification of d-peptide ligands through mirror-image phage display protein design of an hiv- entry inhibitor molecular recognition by a binary code structural basis for hiv- neutralization by a gp fusion intermediate-directed antibody subdomain folding and biological activity of the core structure from human immunodeficiency virus type gp : implications for viral membrane fusion fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides covalent trimers of the internal n-terminal trimeric coiled-coil of gp and antibodies directed against them are potent inhibitors of hiv envelope-mediated cell fusion design and properties of n(ccg)-gp , a chimeric gp molecule with nanomolar hiv fusion inhibitory activity design of a novel peptide inhibitor of hiv fusion that disrupts the internal trimeric coiled-coil of gp neutralizing and infection-enhancing antibody responses to human immunodeficiency virus type in long-term nonprogressors the human immunodeficiency virus type vpr transactivator: cooperation with promoter-bound activator domains and binding to tfiib human immunodeficiency virus type vpr induces apoptosis following cell cycle arrest human immunodeficiency virus vpr product is a virion-associated regulatory protein identification of hiv- vpr product and function human immunodeficiency virus type vpr protein binds to the uracil dna glycosylase dna repair enzyme the glucocorticoid receptor type ii complex is a target of the hiv- vpr gene product interaction of virion protein vpr of human immunodeficiency virus type with cellular transcription factor sp and trans-activation of viral long terminal repeat antibody fragments from a -single pot‖ phage display library as immunochemical reagents hiv- tat interacts with cyclin t to direct the p-tefb ctd kinase complex to tar rna a mimic of hiv- nucleocapsid protein impairs reverse transcription and displays antiviral activity evidence of interactions between the nucleocapsid protein ncp and the reverse transcriptase of hiv- hiv- : fifteen proteins and an rna basic residues in human immunodeficiency virus type nucleocapsid promote virion assembly via interaction with rna human fc epsilon rialpha-specific human single-chain fv (scfv) antibody with antagonistic activity toward ige/fc epsilon rialpha-binding human recombinant antibody fragments neutralizing human immunodeficiency virus type reverse transcriptase provide an experimental basis for the structural classification of the dna polymerase family human immunodeficiency virus type regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich -bubble‖ located within the rev-responsive region of viral mrnas selection of human anti-human immunodeficiency virus type envelope single-chain antibodies from a peripheral blood cell-based phage repertoire human immunodeficiency virus (hiv) vaccine trials: a novel assay for differential diagnosis of hiv infections in the face of vaccine-generated antibodies identification of peptide ligands to the chemokine receptor ccr and their maturation by gene shuffling selection of cc chemokine receptor -binding peptide from a phage display peptide library paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, ccr selection of active scfv to g-protein-coupled receptor ccr using surface antigen-mimicking peptides selection of a cxcr antagonist from a human heavy chain cdr -derived phage library the role of a mutant ccr allele in hiv- transmission and disease progression generation and characterization of a recombinant human ccr -specific antibody. a phage display approach for rabbit antibody humanization human immunodeficiency virus type entry inhibitors selected on living cells from a library of phage chemokines highly potent, fully recombinant anti-hiv chemokines: reengineering a low-cost microbicide cxcr nanobodies (vhh-based single variable domains) potently inhibit chemotaxis and hiv- replication and mobilize stem cells identification of a strongly activating human anti-cd antibody that suppresses hiv type infection modulation of the cd -cd ligand interaction using human anti-cd single-chain antibody fragments obtained from the n-coder phage display library a motif unique to the human dead-box protein ddx is important for nucleic acid binding, atp hydrolysis, rna/dna unwinding and hiv- replication engineering and screening the n-terminus of chemokines for drug discovery the therapeutic potential of cxcr antagonists in the treatment of hiv infection, cancer metastasis and rheumatoid arthritis targeting cxcr in hiv cell-entry inhibition non-immunized natural human heavy chain cdr repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope anti-cd + fabs generated against hematopoietic stem cells in hiv-derived combinatorial immunoglobulin library suggest antigen-selected autoantibodies sensitivity of hiv type primary isolates to human anti-cd antibody-mediated suppression is related to coreceptor use substrate phage: selection of protease substrates by monovalent phage display molecular basis for the relative substrate specificity of human immunodeficiency virus type and feline immunodeficiency virus proteases identification of efficiently cleaved substrates for hiv- protease using a phage display library and use in inhibitor development on the size of the active site in proteases. i. papain kinetic and modeling studies of s -s ' subsites of hiv proteinases interferon production by t coliphage bacterial viruses as human vaccines? bacteriophage lambda is a highly stable dna vaccine delivery vehicle design of specific immunogens using filamentous phage as the carrier phage display of peptide epitopes from hiv- elicits strong cytolytic responses structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. the v loop of hiv- gp antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of hiv- gp phage display of functional, full-length human and viral membrane proteins engineering a peptide epitope display system on filamentous bacteriophage phage t soc and hoc display of biologically active, full-length proteins on the viral capsid immunogenic display of diverse peptides on virus-like particles of rna phage ms dense display of hiv- envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to hiv- env phage display of intact domains at high copy number: a system based on soc, the small outer capsid protein of bacteriophage t variable epitope library-based vaccines: shooting moving targets assembly of human immunodeficiency virus (hiv) antigens on bacteriophage t : a novel in vitro approach to construct multicomponent hiv vaccines immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus l epitope, on virus-like particles of the rna bacteriophage pp variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type -neutralizing antibody response the two dispensable structural proteins (soc and hoc) of the t phage capsid; their purification and properties, isolation and characterization of the defective mutants, and their binding with the defective heads in vitro distribution and three-dimensional structure of aids virus envelope spikes antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations this manuscript is supported by the -centre de recherche public-santé‖, luxembourg (grants gpcr and eumimo). the authors are grateful to danielle perez-bercoff for thorough reading and helpful discussions. the authors also want to thank carole devaux, virginie fievez, marie-eve dumez and martyna szpakowska for critically reading the manuscript. key: cord- - y ex d authors: mcdade, thomas w.; sancilio, amelia title: beyond serosurveys: human biology and the measurement of sars‐cov‐ antibodies date: - - journal: am j hum biol doi: . /ajhb. sha: doc_id: cord_uid: y ex d nan coronavirus disease (covid- ) has emerged as a deadly clinical disease. the virus that causes covid- , severe acute respiratory syndrome coronavirus (sars-cov- ), is readily transmitted in the community, where it is having devastating social and economic impacts. yet our understanding of sars and covid- is derived primarily from studying the most severe cases in clinical and hospital settings. a complementary, field-based approach is desperately needed, and human biologists are well-positioned to make important contributions to our understanding of which individuals, and communities, are most vulnerable and why. much has been said about shortcomings in the roll out ofsars-cov- testing and how it has frustrated efforts to identify cases and isolate individuals who are shedding virus. less has been said about the opportunities that testing provides for a wide range of research applications. in this commentary, we describe antibody testing and how human biologists can use it to inform our understanding of the pandemic, and to address questions of longstanding interest regarding the causes and consequences of human biological variation. | testing for sars-cov- : current and prior exposure nucleic acid-based (ie, polymerase chain reaction, pcr) tests of naso-pharyngeal swabs and/or saliva can detect the presence of virus in the acute stage of infection. these tests are important for clinical diagnosis, and if deployed more widely can be used to identify viral spread in the community. however, shortages of swabs, personal protective equipment (ppe), transport media, and accurate testing platforms have led to a rationing of tests. as a result, priority has been given to testing suspected cases of covid- , with limited application outside the clinical context through the first wave of the pandemic. it is also becoming apparent that false negative results may be more common than originally thought, as viral rna production in the naso-pharynx is transient and subject to sampling variability. serological testing is a complementary approach that detects the presence of antibodies against sars-cov- in blood samples from exposed individuals (world health organization, ). as the immune system mounts a response to infection, b lymphocytes produce antibodies against viral proteins which bind, and in some cases, neutralize the virus. the isotype immunoglobulin m (igm) is the first antibody to appear in circulation following initial exposure to an antigen. it is a large pentamer that is detectable to days after infection, but its expression is transient and concentrations decrease in the weeks following exposure (zhao et al., ) . igg production is slower to come online, but antibodies of this isotype remain detectable for months, and often years, after infection (tan et al., ; xiao, gao, & zhang, ) . based on these dynamics, antibody testing can be applied clinically to diagnose a current or very recent infection, and epidemiologically as a surveillance tool. for example, in some cases individuals present with symptoms of covid- but test negative with pcr because the virus has been cleared, viral shedding is not occurring at the time of sampling, and/or technical errors lead to a false negative result. if sufficient time has passed since the initial infection, the presence of igm antibodies against sars-cov- antigens can be used to confirm a clinical case of covid- . the time course of igg production makes testing less relevant for diagnosis of acute infection, but since levels of anti-sars igg antibodies remain elevated long after infection, igg testing can be used to identify "cases" after the fact. as described below, there are several ways these tests can inform research and policy related to there are currently two predominant approaches to antibody testing: enzyme linked immunosorbent assay (elisa), and lateral flow immunoassay (lfia). in elisa, viral antigen is fixed to the bottom of a microtiter plate well, diluted serum or plasma is added, and antibodies specific to the viral antigen, if present, are "captured" in the well. the addition of anti-human igg or igm antibody with a label (eg, horseradish peroxidase) generates a signal proportional to the concentration of captured antibody, which is quantified in a spectrophotometer. elisa protocols for sars-cov- igm and igg antibodies for use with serum or plasma are now established (amanat et al., ) . however, the requirement for serum/plasma is a significant constraint, particularly in the context of the current pandemic. under the best of circumstances, venipuncture is difficult to implement outside the clinical setting due to the logistics of drawing, transporting, and processing venous blood. these challenges are compounded when people are told to stay at home, and when phlebotomists and ppe are in short supply because cases of covid- are surging. lateral flow immunoassay tests have the potential to overcome these obstacles in that they typically require only a few drops of capillary whole blood, collected from a simple finger stick. as such, they can be readily implemented in nonclinical, community-based settings with the potential to reach larger numbers of people. in lfia, the antigen-antibody dynamics of elisa are applied in a cartridge format: blood (and often diluent) is placed in a small well, and as it diffuses through the cartridge antibodies are labeled and captured, with a test line emerging to indicate a positive result. an advantage of lfia is that it is a "point-of-care" test, with results available in to minutes. however, these tests are qualitative rather than quantitative, and even though they use only a few drops of finger stick blood, they are difficult to self-administer and usually require a trained health care worker to implement. in addition, recent analyses have raised substantial concerns regarding the accuracy of lfia tests for sars-cov- igg antibodies (adams et al., ). there is a middle ground in dried blood spot (dbs) sampling, which combines the convenience of blood collection in the community with the quantification that is possible in the lab (mcdade, ; mcdade, williams, & snodgrass, ) . a sterile lancet is used to prick the finger, and up to five drops of whole blood are collected on filter paper. once the sample dries, the cards can be closed, stacked, and transported to the lab without a cold chain. most analytes remain stable in dbs for days, if not weeks or months, providing flexibility in blood collection protocols. human biologists are accustomed to conducting research outside the clinic or lab, and dbs sampling has been an important part of our toolkit for more than years (worthman & stallings, ) . recently, we validated an elisa for sars-cov- igg antibodies in dbs that provides results that correlate highly with serum (r = . ) . the dbs approach has several advantages that make it particularly well-suited to address important gaps in the current covid- testing landscape. first, individuals can self-sample in the home. although some samples may be inadequate for analysis, prior applications have demonstrated the feasibility of having, participants collect their own dbs sample (roberts et al., ) . second, samples can be returned in the mail without special handling (the cdc and us postal service consider dbs specimens nonregulated, exempt materials) (centers for disease control and prevention, ). third, since dbs samples are analyzed in the lab, we can apply more accurate and quantitative protocols than is possible with lfia. in developing a low-cost elisa for sars-cov- antibodies, our hope is that others can draw on the longstanding tradition of methodological innovation in human biology to promote community-based research on covid- . the burden of covid- is not shared equally. for example, older persons are at higher risk for more serious complications and death, while rates of infection appear low for children and risk of mortality is even lower (center for disease control and prevention, ). worldwide, minority and vulnerable populations have been disproportionately impacted by the covid- pandemic. in the uk, though people from ethnic minorities are younger on average than the white british population, death rates are higher (kirby, ) . in the us, african americans comprise % of covid- hospitalizations (kirby, ) . in the city of chicago, as of june the infection rate for latinx residents was cases per , compared with per white residents. mortality risk of covid- was . times higher for african-americans in comparison with whites (chicago department of public health, ). of course, these data paint an incomplete picture of the actual distribution of the virus since they are based on pcr tests for active infections in clinical settings. by identifying mild and asymptomatic cases, antibody testing can provide a more accurate and comprehensive record of the social and geographic spread of the virus. these data are important for informing estimates of the seroprevalence of infection and case fatality rates, for identifying subgroups of individuals more susceptible to infection, and for evaluating the effectiveness of various policy efforts (eg, social distancing, closing of schools and businesses) in mitigating transmission in the community. these are important first order questions, the answers to which can be used to inform public health responses to future outbreaks. as human biologists we can contribute to this effort, but we can also dig deeper. we can complement the public health emphasis on surveillance, and the clinical emphasis on diagnosis and treatment, with research that illuminates the contextual, interpersonal, and individual factors that explain patterns of exposure and response to infection. we can draw on biosocial/biocultural frameworks to develop a more holistic picture of individual variation in vulnerability to infection by integrating biological, sociocultural, and environmental data. a key strength of this perspective is the emphasis on simultaneously defining and measuring causal pathways at multiple levels, which can highlight proximate as well as more distal causes of inequities in exposure, infection, and death. for example, are higher rates of covid- mortality among african-americans a product of increased exposure to sars-cov- , or increased vulnerability to disease following exposure? not everyone is afforded the same opportunity to shelter-in-place. workers designated as "essential," and those who cannot afford to stay home even when rates of community transmission are high, are at increased risk for exposure (as are the other members of their household and social networks). furthermore, food deserts, inadequate health care, limited opportunities for physical activity, and stress all contribute to hypertension and diabetes-conditions that predispose to covid- mortality. as discrimination, concentrated disadvantage, and other forms of structural racism increase burdens of chronic degenerative disease among african-americans in the us, they may also contribute to inequities in covid- mortality. antibody testing can be used to cast light on the inequitable distribution of viral exposure and the factors that contribute to higher levels of transmission in disadvantaged communities. human biologists are also well-positioned to consider a life course perspective on variation in outcomes in response to sars-cov- infection. why are older people more vulnerable, while children are largely spared? why do infections tend to be mild in pregnancy, in contrast to the influenza pandemic when mortality was particularly high for pregnant women (taubenberger & morens, ) ? developmental plasticity, ecological sensitivity, and the finite nature of resources are key concepts from evolutionary life history theory that may generate important insights. for example, the immune system is a central component of maintenance effort, and the defenses that provide protection against covid- are costly to develop and activate (mcdade, ) . one might therefore hypothesize that the response to infection is shaped by the availability of nutritional resources, particularly resources during sensitive periods of immune development in infancy. similarly, microbial exposures early in development may calibrate investments in innate vs specific immunity, with implications for the regulation-or dysregulation-of inflammation in adulthood (mcdade, georgiev, & kuzawa, ) . a theoretically grounded, hypothesis driven life history approach may help us identify how, and why, individuals differ in the magnitude and effectiveness of immune responsiveness to sars-cov- infection. quantifying the antibody response to infection provides a direct measure of humoral immunity, and additional indicators of immune activity (eg, markers of inflammation, cell mediated responses) can further characterize the magnitude and direction of response. we can also reach across generations to consider the potential long-term implications of the pandemic. even though pregnant women do not appear to be at elevated risk of infection, subtle long term effects on individuals born during the influenza epidemic are well-documented (almond, ) , and recent research showing how maternal adversity can shape placental architecture and nutrient transfer point toward the possibility of intergenerational impacts of infection (miller et al., ) . in addition, it is not just mothers that we should consider: the experience of fathers may be transmitted across generations as well, through epigenetic modifications to the germline that are inherited along with gene sequence (ryan & kuzawa, ) . we can also reach back in time, to consider how adaptations to environmental pressures may influence responses to infection in the present. for example, recent research with highaltitude populations in regions of tibet, bolivia, and ecuador suggests that physiological responses that promote survival in hypoxic environments may also serve to decrease susceptibility to sars-cov- infection (arias-reyes et al., ) . these are all questions that can be answered, at least in part, with measures of antibody response to identity individuals who have been exposed. human biologists are uniquely positioned to make important contributions to our understanding of covid- , and methods that facilitate research in community-based settings globally will be central to that effort. antibody testing is a necessary surveillance tool, but we can also apply it in the service of advancing our understanding of human biological variation more broadly. in doing so we accept an obligation to challenge misleading claims regarding the significance of a "positive" antibody test. at this point it is not known if high levels of sars-cov- igg antibodies confer immunity against future infection, and talk of antibody badges or passports is premature. we also need to be mindful of the potential for seroprevalence data to stigmatize members of the community, and to politicize debates regarding the costs and benefits of initiatives designed to mitigate viral transmission. the current pandemic underscores the social nature of human biology, and a contextualized, community-based approach is an essential complement to current clinical and public health research paradigms. author contributions thomas w. mcdade: conceptualization; writing-original draft; writing-review and editing. amelia sancilio: writing-original draft; writing-review and editing. thomas w. mcdade https://orcid.org/ - - - x amelia sancilio https://orcid.org/ - - - is the influenza pandemic over? longterm effects of in utero evaluation of antibody testing for sars-cov- using elisa and lateral flow immunoassays does the pathogenesis of sar-cov- virus decrease at high-altitude? shipping guidelines for dried-blood spot specimens latest data evidence mounts on the disproportionate effect of covid- on ethnic minorities. the lancet respiratory medicine life history theory and the immune system: steps toward a human ecological immunology development and validation of assay protocols for use with dried blood spot samples trade-offs between acquired and innate immune defenses in humans enzyme immunoassay for sars-cov- antibodies in dried blood spot samples: a minimally-invasive approach to facilitate community-and population-based screening what a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research mothers' childhood hardship forecasts adverse pregnancy outcomes: role of inflammatory, lifestyle, and psychosocial pathways selfcollected dried blood spots as a tool for measuring ovarian reserve in young female cancer survivors germline epigenetic inheritance: challenges and opportunities for linking human paternal experience with offspring biology and health viral kinetics and antibody responses in patients with covid- influenza: the mother of all pandemics population-based age-stratified seroepidemiological investigation protocol for covid- virus infection hormone measures in finger-prick blood spot samples: new field methods for reproductive endocrinology profile of specific antibodies to sars-cov- : the first report antibody responses to sars-cov- in patients of novel coronavirus disease beyond serosurveys: human biology and the measurement of sars-cov- antibodies key: cord- -l q n ri authors: foss, stian; watkinson, ruth; sandlie, inger; james, leo c; andersen, jan terje title: trim : a cytosolic fc receptor with broad antibody isotype specificity date: - - journal: immunol rev doi: . /imr. sha: doc_id: cord_uid: l q n ri antibodies are key molecules in the fight against infections. although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. this postentry viral defense mechanism requires binding of the antibody to a cytosolic fc receptor named tripartite motif containing (trim ). in contrast to other fc receptors, trim shows remarkably broad isotype specificity as it does not only bind igg but also igm and iga. when viral pathogens coated with these antibody isotypes enter the cytosol, trim is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. in addition, inflammatory signaling is induced. as such, trim acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. here, we summarize our current understanding of how trim orchestrates humoral immunity in the cytosolic environment. antibodies are a crucial part of the immune response toward invading pathogens such as viruses, and the induction of so-called neutralizing antibodies is a primary goal of vaccination ( ) . in addition, there is great interest in the development of broadly neutralizing antibodies specific for major human pathogens such as human immunodeficiency virus (hiv) and influenza virus ( , ) . neutralization of viruses by antibodies is predicted to depend on high-affinity binding to specific epitopes of surface-exposed viral proteins that are required for binding to target cell receptors ( ) . such antibodies are thought to function according to the occupancy model that requires binding of a critical number of antibodies to a viral particle in such a way that most or all neutralizing epitopes are occupied ( ) . this may occur independently, or in concert with other antibody-mediated effector functions such as antibody-dependent cellular phagocytosis or antibody-dependent cellular cytotoxicity ( , ) . these effector functions are induced upon binding of antibody-virus immune complexes to classical fc c receptors (fccrs) expressed on the surface of hematopoietic cells such as natural killer (nk) cells, macrophages, and dendritic cells, which results in clearance and induction of t-cell responses ( ) . neutralizing antibodies also prevent infection in concert with non-classical fc receptors such as the neonatal fc receptor (fcrn) and the polymeric ig receptor (pigr) ( , ) . while fcrn mediates bidirectional transport of igg across mucosal epithelial surfaces ( ) ( ) ( ) ( ) ( ) , pigr mediates unidirectional transcytosis of iga and igm from the tissue into the luminal space ( ) . transcellular transport of neutralizing but also non-neutralizing antibody has been shown to facilitate protection against viral infection ( , ( ) ( ) ( ) ( ) ( ) ( ) . although neutralizing antibodies are produced during antiviral responses, the majority of the antibodies in the polyclonal response have no neutralizing activity by the classical definition ( , ) . this is due to the fact that they bind internal viral epitopes released from infected lysed cells, or viral surface proteins that are not involved in viral attachment and entry into host cells ( ) . in addition, viruses are known to display immune-dominant non-neutralizing epitopes that can bias the polyclonal response toward a nonneutralizing phenotype ( ) ( ) ( ) ( ) . as such, neutralization of viruses by antibodies has until recently been assumed to be a solely extracellular or intravesicular event. however, in recent years, it has become clear that the antiviral function of antibodies also extends into the cytosolic compartment of cells ( ) ( ) ( ) . this additional level of protection is orchestrated by the interferon (ifn)-inducible cytosolic fc receptor tripartite motif containing- (trim ). engagement of trim results in rapid postentry elimination of antibody-virus via recruitment of the proteasomal machinery ( , ) , in a mechanism termed antibody-dependent cellular neutralization (adin). concurrently, inflammatory signaling is also induced ( ) . therefore, antibodies that have failed to block entry of a virus particle into the cell and that is not intercepted by antibody-mediated effector functions operating in the extracellular environment may still be protective in the cytosolic compartment, even though they are, by the classical definition, non-neutralizing. instead, the cell takes advantage of trim to set up one last line of antiviral defense. trim can be distinguished from other fc receptors in two ways. firstly, trim shows remarkably broad antibody specificity as it can activate both of its functions upon binding to igg, igm as well as iga ( , ) , while other fc receptors display more restricted antibody isotype and subtype specificities ( ) ( ) ( ) . secondly, trim is broadly expressed by cells of most histogenic linages ( ) , while expression of classical fccrs is mainly restricted to hematopoietic cells ( ) . this suggests that a susceptible pathogen may be targeted by trim independently of the site of infection and local distribution of antibody isotypes. the trim -igg interaction was first described in a yeast two-hybrid screen in a study that investigated the role of trim as an autoantigen in immune disorders such as systemic lupus erythematosus and sj€ ogren's syndrome, in which it is referred to as ro or ss-a ( ) . in subsequent studies, trim was immunoprecipitated independently of antibody specificity, and the binding site for trim was postulated to be localized to the c h -c h interface of fc as it was found to compete with binding of staphylococcus protein a and streptococcus protein g ( , ) . furthermore, the corresponding binding site on trim was found localized to the c-terminal pryspry domain as truncation of this domain resulted in loss of binding. although the interaction between trim and igg was initially thought to be irrelevant due to the topologically distinct localization of the two proteins, a specific role in antiviral defense was more recently described ( ) . trim is an fc receptor that is structurally unrelated to all other classes of fc receptors ( , ) . it is part of the trim family which consists of over members in humans ( ) , with a diverse set of cellular roles including antiviral defense ( , ) . one of the most studied members is trim a, which mediates restriction of simian immunodeficiency virus via an antibody-independent mechanism ( ) . trim shares the same structural architecture as other trim proteins and consists of an n-terminal ring domain with e ubiquitin ligase activity, a b-box, and a central coiled-coil domain that is referred to as rbcc ( ) . it is, however, the c-terminal domain of trim proteins that determines ligand specificity and function, and in half of all known trim proteins this is a so-called pryspry domain. the pryspry domain of trim contains the antibody binding site, and is a globular fold comprising a b-sandwich of two antiparallel b-sheets connected by flexible loops ( ) , and is a fusion of pry and spry elements which are of distinct evolutionary origin ( ) . furthermore, trim proteins are known to form dimers or higher order structures via their coiled-coil domains and both heteromeric and homomeric trims have been described ( ) . crystallographic data of the trim coiled-coil have revealed that it has an antiparallel helical structure that places the n-terminal ring domains at opposite sides of the dimeric structure, while the c-terminal pryspry domains are positioned at the center ( ) . although a crystal structure of full-length trim has yet to be solved, the presence of the coiled-coil suggests that trim adopts a similar structural arrangement that would place its two pryspry domains in close proximity to each other. consistent with this, full-length trim has been shown to exist as a dimer in solution and form stable : complexes with human igg ( ) . thus, the two pryspry domains of a dimeric trim molecule may bind simultaneously to one igg fc ( ) . this symmetrical mode of binding will allow trim to rapidly intercept incoming antibodies ( , ) . a detailed understanding of the trim -igg interaction has been obtained from solving a co-crystal structure between the c-terminal pryspry domain of human trim and an fc fragment derived from human igg ( ) . the complex reveals a : stoichiometry where a pryspry domain binds to the interface between the c h and c h found on each side of the fc. as such, the binding site for trim is distinct from that of the classical fccrs and c q in the lower hinge and c h domain ( - ), but overlaps with the binding site for fcrn ( , ) as well as bacterial and viral fc receptors ( ) ( ) ( ) . in contrast to binding to fccrs, neither trim nor fcrn binding to igg is affected by removal of n -linked glycans of the c h domains ( ) . the core interaction site is formed between the spry element and the c h domain of igg fc ( , ) . here, the protruding and conserved fc loop encompassing residues - is inserted into a deep hydrophobic pocket on the spry surface where the apex fc residues h , n , and h (hnh motif) form a central hydrogen bond network surrounded by a hydrophobic shield of aromatic side chains that are engaged in aromatic stacking interactions. specifically, h fc and n fc interact with d spry located at the base of the spry binding pocket via hydrogen bonds, while h fc also interacts with w spry via aromatic stacking, which may also involve w spry . furthermore, h fc forms a hydrogen bond with d spry and stacks with f spry . in addition, i fc of the c h loop encompassing residues - is involved in a hydrophobic interaction with w pry . an overview of the co-crystal complex and the interaction network is presented in fig. . in contrast to the strict ph dependence of the fcrn-igg interaction, trim binds fc ph independently, but is sensitive to high salt concentrations ( ) . the interaction has been shown to be highly conserved in mammals, and displays wide cross-species reactivity as both human and mouse trim bind igg from a range of mammals ( ) . this is supported by inspection of the co-crystal structure between the mouse trim pryspry domain and an fc fragment derived from mouse igg a, which shows that the mouse and human mode of binding are highly similar ( ) . the binding affinity between human igg and the monomeric human pryspry domain has been measured to nm using isothermal titration calorimetry, while the murine interaction has an affinity of nm, where the human interaction occurs with more negative enthalpy ( , ) . however, the fact that full-length trim is a dimeric molecule has direct implications for its functional affinity to antibodies. the two pryspry domains of a dimeric trim molecule may bind simultaneously to one igg fc as suggested by the crystal structures ( ) . simultaneous engagement of both heavy chains likely explains the significantly increased interaction of dimeric trim , yielding an affinity of . nm for the igg-trim interaction as measured by fluorescence anisotropy ( ) . notably, this represents an increase of more than -fold relative to monomeric affinity, which makes trim the highest affinity fc receptor in humans. while fc receptors are highly selective in regard to isotype and subtype binding, trim not only binds igg but also igm and iga ( , ) . interestingly, the binding site for trim on iga and igm overlaps with that of fc a receptor i (fcari), fca/l receptor, pigr, as well as several pathogen-produced fc receptors ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the binding affinities of igm and iga toward monomeric human pryspry are considerably weaker, and have been measured to be and lm, respectively ( , ) . however, despite relatively weak monomeric binding, the dimeric nature of full-length trim suggests that the functional affinities for igm and iga are much stronger ( ) . assuming that the increase over monomeric affinity upon binding of full-length trim to igg is similar for igm and iga, the binding strength may be in the sub-micrometer range during physiological conditions ( ) . the weak monomeric binding of igm and iga is very likely due to the sequence variation in the stretch of amino acids corresponding to the c h -loop - of igg ( ) . specifically, the apex hnh motif of igg is pnr in igm and pla in iga. however, binding to trim is still accommodated, as there is structural conservation of the fc loop among the three isotypes. this has been addressed by molecular modeling showing that the structural loops in igg and igm superimpose closely at the secondary structure level allowing them to insert into the hydrophobic pryspry binding pocket ( ) . although the individual mutations h a, n a, n d, and h a in a murine igg antibody ( ) have been shown to abolish trim recognition, the specific replacements in igm and iga are predicted to form specific interactions with pryspry residues ( ) . in igm, conservation of the central n is thought to be crucial, as its hydrogen bonding potential with d spry is likely maintained, while replacement of h fc with proline will abolish its hydrogen bonding potential with d spry and its ability to interact with two fc residues simultane-ously. however, the proline residue may still interact with w spry and w spry . even though proline-tryptophan pairing is not a true aromatic stacking, it generates binding energy that is roughly double that of a hydrogen bond ( ) . furthermore, the replacement of h fc with arginine in igm abolishes its aromatic stacking potential with f spry , but the arginine may still bind d spry , given a favorable side chain conformation. in iga, p corresponding to h in igg is thought to have a similar role as in igm. the presence of l , the equivalent of n in igg, cannot interact with d spry , but is instead thought to form hydrophobic contacts with l s pry and l spry , while a fc is predicted not to contribute in binding ( ) . structural predictions of the interaction between igm and iga are shown in fig. . during infection with a non-enveloped virus, antibodies that are attached to the viral capsid are also carried into the cytosolic compartment of target cells where they are recognized by trim ( , ) (fig. ) . subsequently, trim engagement targets the virus for proteasomal degradation before it has had time to replicate and spread. this was first demonstrated using human adenovirus type (adv ) carrying the green fluorescent protein reporter gene as a model pathogen, incubated with anti-adv polyclonal serum from goat ( ) . upon infection of hela cells, protection from infection was found to correlate with the level of trim expression, as depletion of trim gave high infection levels while upregulation of trim by ifn-a stimulation increased adin ( ) . following endocytosis via the coxsackie and adenovirus receptor (car), adv in complex with non-neutralizing antibodies lyses the endosomal membrane and enters the cytosol where it is intercepted by trim . engagement of trim results in autoubiquitination in a manner that is dependent upon the e activity of its ring domain ( ) . ubiquitination leads to recruitment of the proteasomal machinery and rapid degradation of the viral complex, in a process that is perturbed by the proteasomal inhibitor, epoxomicin. the degradation process has been demonstrated to be strictly dependent on the aaa atpase v /vcp ( ) . vcp is a multi-domain protein complex that interacts directly with multi-ubiquitin chains via its n-terminal domain and is capable of extracting proteins from larger complexes as well as unfolding them ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . therefore, a general role for vcp in a preproteasomal degradation step has been suggested ( ) , and vcp was recently found to be recruited to stalled proteasomes ( ) . considering that the intact adv capsid measures nm in diameter ( ) , and that the diameter of the proteasomal barrel is only nm ( , ), such a predisassembly step is intuitively required ( ) . it is likely the challenging nature of the viral capsid rather than an intrinsic feature of adin that results in vcp dependence, as trim mediates the proteasomal turnover of ectopically expressed igg fc independently of vcp ( ) . the fast rate of adin is crucial as it must occur before adv reaches the nucleus for initiation of replication. this has been demonstrated using a fate-of-capsid assay, in which cellular levels of the major capsid protein, hexon, were degraded within h of infection ( ) . the adin mechanism is strictly dependent on ubiquitination, in which trim itself is the substrate for attachment of ubiquitin ( , ) . the ubiquitin machinery involves four enzymes: the ubiquitin activating (e ), the ubiquitin conjugating (e ), the substrate specifying ubiquitin ligase (e ), as well as the ubiquitin erasing deubiquitinase (dub), of which e is responsible for the ubiquitin chain linkage topology ( ) ( ) ( ) . the e enzyme ube w was recently shown to mediate attachment of k -linked monoubiquitin to trim ( ) . this makes trim accessible as a substrate for the e enzyme pair ube n/ube v , resulting in extension of k linked ubiquitin chain in vitro. furthermore, it was demonstrated that both ube w and ube n were required for attachment of both k and k -linked ubiquitin chains to trim in cells, in which the attachment of k -linked ubiquitin chains is thought to result in targeting to the proteasome ( ) . as trim undergo autoubiquitination, this could allow it to be effective against a diverse range of pathogens. this hypothesis is supported by the fact that antibody-coated beads transfected into cells become positive for ubiquitin, suggesting that any cytosolic particles bound by antibodies are potential targets for trim -mediated adin. during the initial stages of the antibody response, igm is the main antibody isotype produced. igm forms pentamers through association with the common joining chain (j-chain). iga is the main isotype at mucosal barriers and the second most prevalent isotype in serum at - mg/ml compared to mg/ml for igg. two subclasses of iga exist, iga and iga , and each is expressed as monomeric (miga), dimeric (diga), and secretory (s-iga) forms ( ) . in serum, miga is the prevalent form while s-iga dominates at mucosal sites ( , ) . immune complexes containing miga interact with fcari, which mediates phagocytosis, cellular cytotoxicity, antigen presentation, and cytokine release in professional cells, equivalent to the classical fccrs for igg ( ) . iga and igm bind the pigr, which mediates transport from the tissue into the lumen of body cavities, and upon release they are associated with the pigrderived secretory component (sc) to form s-iga and s-igm ( ) . interestingly, the sc-binding site on iga overlaps with the binding site for trim ( ), although sc is thought to associate with only one heavy chain in each iga monomer ( ) . this may explain the weak affinity between s-iga and the pryspry domain ( ) . during infection with adv , both igm-and iga-bound antibodies are recognized by trim and direct elimination of the virus via adin. this has been demonstrated with human igm and iga isolated from serum, and as observed for igg, ifn-a stimulation increases neutralization in a trim -dependent manner for both isotypes ( , ) . despite reduced affinity for s-iga, virus-specific s-iga from human serum was found to activate adin, as cellular depletion of trim partially restored viral infection. although the presence of the sc appears to inhibit trim function, data suggest that the disulfide bonds between iga and the sc may be broken upon exposure to the reducing environment of the cytosol, allowing attachment by trim ( ) . interestingly, in a recent study, it was demonstrated that s-iga forms trimers and tetramers in nasal secretions of healthy adults that contributed to enhanced neutralization potency ( ) . such higher forms of s-iga may likely reach the cytosol together with trim -susceptible pathogens. an intriguing feature of the adin mechanism is that only a few antibodies per virus are required for it to be effective as adin activity closely correlates with the expression level of trim rather than the extent of opsonization of the virus by a non-entry blocking antibody ( ) . using a murine monoclonal igg antibody ( c ) specific for the major capsid protein of adv, hexon, a non-entry neutralizing epitope, together with manipulation of cellular trim levels using ifn-a or trim shrna, allowed investigation of how antibody, trim and virus contribute to the efficiency of neutralization. in ifn-a-stimulated cells that expressed high levels of trim , as few as . antibody molecules per virus were found to be sufficient for neutralization, while five times more antibody was required in unstimulated cells ( ) . furthermore, adin was found to increase in a linearly incremental manner, demonstrating that each antibody associated with the virus contributes equally to its elimination upon binding to trim ( ) . this is in contrast to the occupancy model of entry neutralization, in which efficient prevention of infection can occur only after a critical number of antibodies occupy specific viral epitopes that are involved in cellular attachment and entry. consequently, this results in a lag phase in neutralization curves when infection levels are plotted against antibody concentration until the critical number of antibodies required to block entry has been reached ( ). this finding was supported by data demonstrating that polyclonal serumcontaining antibodies specific for adv required a higher antibody-virus stoichiometry for neutralization to occur when cellular trim was depleted, indicating a shift from cytosolic adin to entry blocking ( ) . thus, when the polyclonal response contains few entry neutralizing antibodies, adin may be responsible for the majority of the neutralization activity. low antibody-virus stoichiometry may also result in inefficient fccr-mediated effector functions by immune cells as efficient phagocytosis requires the formation of immune complexes and cross-binding to cell-surface fccrs. in addition, as trim also engages igm and iga, it is likely to contribute to early protection, and at the gate of entry of most viral pathogens, the mucosal barrier. this however, does not mean that trim -mediated protection becomes irrelevant in the later stages of infection when the amounts of entry neutralizing antibodies with high affinity are likely to increase. infection by several viruses has been shown to occur even at saturating levels of entry neutralizing antibodies ( ) . the virus particles that still infect their target cells under these conditions are known as the persistent fraction. in the case of adv , the magnitude of the persistent fraction has been shown to correlate with the expression level of trim , as a considerable increase in the persistent infection level was measured upon trim depletion ( ) . adin is also saturated at high viral loads, supporting the notion that trim levels are deterministic and suggesting that trim could be overcome by pathogens during high levels of viremia. as the efficacy of adin is also dependent on other cellular components such as vcp and the proteasome, the expression levels and rate of enzymatic turnover of these components are also likely to affect the ability of trim to induce adin ( ) . while extracellular detection of antibody-virus immune complexes by cell-surface-expressed fccrs results in phosphorylation of their intracellular immunoreceptor tyrosinebased activation motif, triggering phagocytosis and immune activation ( ) , known mechanisms for detection of pathogens in the intracellular environment occur independently of antibodies and are instead dependent upon recognition of pathogen-associated molecular patterns. this is achieved via pathogen recognition receptors (prrs) such as toll-like receptors ( ) , and cytosolic receptors for viral nucleic acid such as retinoic acid-inducible gene- (rig- ) and melanoma-differentiation associated protein (mda ) ( ). the immune system can also be activated by tissue damage as a result of pathogen replication via the mislocalization of selfmolecules. such host derived factors are known as damageassociated molecular patterns (damps) ( ) . even though the concentration of igg in human serum is as high as mg/ml, it is excluded from the cytosolic compartment of cells during normal physiological circumstances. however, during infection, when antibodies of the igg, igm, and iga isotypes enter the cytosol bound to a pathogen, they act like a damp upon trim recognition ( ) . importantly, in addition to adin, engagement of trim induces an antiviral state by activating inflammatory responses. unanchored k -linked ubiquitin is an immune second messenger that activates the transcription factor pathways nf-jb, ap- , irf , irf , and irf , via the enzyme complexes tak -tab -tab ( , ) and ikka-ikkb-nemo ( , ) , and inhibition of these complexes prevents trim -induced signaling ( ) . whenever a ubiquitin-modified substrate is targeted to the proteasome, there are three dubs in the s regulatory particle, usp /ubp , uch /uchl , and rpn /poh , that remove ubiquitin before substrate degradation ( ) . it was recently demonstrated that cells depleted of poh were unable to activate nf-jb signaling in response to adv -igg complexes, suggesting that poh cleaves off trim k -linked ubiquitin chains, which thereby activate an innate signaling response ( ) . this assure that trim only induces a signaling response if a virus antibody complex is targeted for proteasomal destruction, a mechanism that prevents adin from antagonizing signaling by rapid degradation of the virus, which thus synchronizes the activation of the two effector functions ( ) . activation of innate signaling pathways by trim induces rapid production of pro-inflammatory cytokines and chemokines, which has been shown to occur in both primary human lung fibroblasts and monocytes. specifically, the cytokines il- , il- , and ifn-c, as well as the chemokines cxcl and ccl were detected in response to igg-coated adv . furthermore, it was shown that inflammatory signaling was induced solely by trim upon detection of adv , and independently of known fccrs and prrs, as chemical inhibition of myd , trif, and the tyrosine kinase syk or knockdown of cytosolic rig- and mda did not impact signaling ( ) . activation of trim sensing places the cell in an antiviral state. this involves increased expression of major histocompatibility complex class i (mhc i), as well as the activating mhc i-like nk g d ligands in non-hematopoietic cells ( ) . as trim engagement results in rapid degradation of viral proteins, as well as inflammatory signaling that upregulates the expression of mhc i, it is possible that peptides derived from the virus could be transported via the transporter associated with antigen processing (tap) and loaded onto mhc i or nkg d molecules. this would then make the cell a target for the cytotoxic effects of nk cells and cd + t cells; however, this has not yet been tested experimentally. in principle, the cell could 'cure' itself from infection by adin rather than being killed by effector lymphocytes. induction of cell killing by lymphocytes requires clustering of mhc i molecules and t-cell receptors ( ) , and it may be speculated that the level of infection could be a factor that determines whether or not the cell would clear the virus solely via adin, or if t-cell help is required. an important question is how the induction of signaling via trim is regulated. the immune system has evolved specific mechanisms to prevent excessive inflammation and such regulation must also be active in the context of trim . for the classical fccrs, this is achieved by regulation of the expression levels of activating receptors and the inhibitory fccriib ( ) . as trim detection of cytosolic antibodies results in synchronized activation of both an effector and a signaling response, there must be strict regulation to avoid excessive inflammation at low pathogenic load. this may be a result of different requirements for activation of downstream ubiquitin-dependent processes, which must be related to how trim detects incoming antibodyvirus complexes, and is potentially also self-limiting because the signaling stimulus (i.e. intracellular antibody) is also rapidly degraded by the effector mechanism ( ). the full spectrum of pathogens that activates adin and trim signaling has yet to be determined. however, as both the effector and sensory functions of trim are strictly dependent on the presence of antibodies in the cytosol, the mechanism by which the pathogen enters the cell is crucial ( ) . regarding viral infections, the main targets for trim are non-enveloped viruses, as enveloped viruses shed their outer membrane coat upon fusion with the plasma or endosomal membrane and deliver their nucleocapsids into the cytosol. therefore, antibodies will be left behind at the cell membrane or inside endosomal vesicles. in line with this, the enveloped respiratory syncytial virus (rsv) is not detected by trim when added to cells in complex with anti-rsv antibodies ( ) . some non-enveloped viruses may also avoid trim detection due to their route of infection. for example, some rhinoviruses of the family picornaviridae are thought to inject their genome through a pore in the endosomal membrane, thereby leaving their nucleocapsid and any attached antibody behind inside the endosome ( ) . in the case of adv, its interaction with the cell-surface-expressed car triggers endocytosis ( ) . the virus then escapes endosomes and enters the cytosol by lysing the endosomal membrane, a process that relies on partial un-coating of the virus and exposure of an internal capsid protein that induces membrane damage ( ) . once inside the cytosol, the sub-viral particle uses microtubule-associated motor proteins to reach the nuclear membrane ( ) . subsequently, it docks to the nuclear pore complex through which the viral genome enters the nucleus ( ) . therefore, any attached antibody is readily accessible for trim recognition. viruses may also differ in their susceptibility to trim detection due to their behavior once they reach the cytosolic compartment ( , ) . although it is likely that some viruses have evolved to evade trim or even take advantage of trim as part of an un-coating mechanism, the rapid detection at a stage prior to synthesis of new viral proteins, as well as bridging by antibody, may make trim detection particularly difficult to antagonize ( ) . indeed, neutralization of several non-enveloped viruses by few antibody molecules has been described for adv, papillomaviruses, and poliovirus ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . importantly, trim sensing is not restricted to virus infection as it can activate antibody-dependent nf-jb signaling in response to the intracellular bacterium salmonella enterica ( ) , which traffics between intracellular vesicles and the cytosol ( , ) . such trim -dependent immune signaling has been shown to be even more potent upon infection with a salmonella mutant (dsifa) that fails to maintain vesicle integrity, resulting in a greater proportion of bacteria in the cytosol ( ) . although bacteria are presumably too large to be degraded by the proteasome, and adin elimination of adv has been shown to happen independently of autophagy ( ), a possible link between trim and the formation of autophagosomes in the context of salmonella has been suggested ( ) . the fact that evolution has maintained strong binding between trim and antibodies across species points to its importance in host defense. humoral immunity during the course of an infection occurs by a variety of different mechanisms, and the relative contribution of each will depend on the nature of the pathogen as well as the polyclonal antibody response ( , , ) . therefore, to address whether or not trim makes a significant contribution to systemic protection in each case, this must be studied in the context of immune serum raised naturally against the pathogen. this has so far been investigated both in vitro and in vivo using the wildtype mouse adenovirus type- (mav- ) ( , ) which causes hemorrhagic encephalitis in mice. to recover from acute mav- infection, mice need a robust antibody response ( ) ( ) ( ) . the ability of anti-mav- serum to neutralize mav- was assessed in cells derived from wildtype or trim knockout mouse embryonic fibroblasts, and a significant proportion of neutralization was trim dependent ( ) . furthermore, upregulation of trim expression by ifn stimulation increased the adin potency of both pooled serum and immune serum collected from individual mice. the importance of trim for mav- protection in vivo was studied by comparing wildtype trim +/+ , trim heterozygote +/À , and trim knockout À/À c bl/ mice ( ) . upon challenge with mav- , it was shown that naive mice lacking trim had a higher viral load and increased mortality within the first week of infection. as these experiments were performed in naive animals, protection was presumably a result of interaction between anti-mav- igm and trim . in passive transfer experiments using antisera against mav- , it was demonstrated that survival closely correlated with trim expression, as transfer of serum was fully protective in wildtype, but not in trim -deficient, mice. heterozygous animals expressing intermediate levels of trim showed a haplo-insufficiency phenotype, with comparable mortality to mice homozygous null for trim , but an average brain viral load that was intermediate between wildtype and knockout animals at the end point of the experiment. moreover, in agreement with in vitro findings, ifn induction correlated with increased trim expression and reduced infection, highlighting the importance of trim for efficient adin activity. early anti-serum and diluted late anti-serum was more dependent on trim for protection compared to non-diluted late anti-serum ( ) . this indicates that when other antibodymediated mechanisms such as entry neutralization are incompletely protective, trim functions become increasingly important as they are able to operate under conditions of few antibodies per virus ( , ) . in addition, the use of a fully replicative wildtype virus in these studies demonstrates that trim is effective during a spreading infection ( , ) . as pathogens enter the body through mucosal surfaces, it raises an intriguing question as to whether trim is activated in polarized epithelial cells. iga and igm bind pigr, which mediates transport from the basolateral to the apical surface, and detection of viruses intracellularly may direct their transcytosis to the apical surface ( ) . in regard to igg, fcrn mediates bidirectional transport of monomeric igg or igg-immune complexes across polarized cellular layers ( ) ( ) ( ) ( ) ( ) . fcrn has even been shown to facilitate neutralization in a specific case where a ph-dependent igg antibody against influenza only neutralizes when the antibody-containing endosome undergoing fcrn transport fuses with a virus-containing acidic endosome ( ) . however, unpicking the antiviral contribution of immunoglobulin transport receptors like fcrn in vivo is complicated by their role in determining antibody serum half-life and biodistribution. an anti-hiv- igg engineered for improved fcrn binding was shown to have increased serum half-life, enhanced mucosal localization and superior protection against intrarectal infection with simian-hiv ( ) , but the mechanistic correlation between these properties was not determined. while there are many examples of viruses hijacking antibody transport receptors to facilitate infection, whether or not fcrn and pigr can directly prevent infection, or if there is crosstalk between these endosomal receptors and trim as they intercept viruses bound by antibody, remains to be addressed. during an infection, trim detects antibodies that are carried by pathogens into the cytosol. once antibodies are detected, trim mediates adin, a highly efficient viral elimination mechanism and concomitantly activates proinflammatory signaling resulting in a protective response. although the complete spectrum of pathogens that are susceptible has yet to be determined, the fast rate of detection and the adaptor function of antibodies are believed to make antagonism on the part of the virus particularly difficult. it is therefore likely that diverse non-enveloped viruses are targets for trim . trim has been shown to make a significant contribution to systemic protection, and thereby provide important immune defense alongside extracellular mechanisms. in addition, the ability of trim to interact with three antibody isotypes is a unique feature among known fc receptors. this, together with the broad expression profile of trim , allows it to function during all stages of an immune response at diverse sites. although the antibody binding properties and antiviral functions of trim have been well established, there are several questions regarding its biology that remain unanswered. of particular interest is how the effector and signaling responses are regulated in order to avoid excessive inflammation. as both arms of trim function depend on a complex sequential ubiquitination process, which results in synchronized activation, regulation may depend on different activation thresholds for downstream ubiquitindependent processes. it will also be important to address how trim synergizes with other immune responses. for instance, adin may contribute to cross-presentation of viral peptides on mhc class i molecules, resulting in the recruitment of cytotoxic lymphocytes to the infected cell. while trim is promiscuous in its antibody binding, the functional implications of this are largely unexplored as are the isotypespecific responses that might be elicited. for instance, while trim binds all four subtypes of human igg, it is not known whether their use during infection results in different responses. moreover, as the four igg subclasses differ significantly in their ability to interact with classical fc receptors, trim activity could be altered indirectly. the main gateway for viral pathogens into the body is via mucosal surfaces. this raises intriguing questions as to whether or not trim makes a significant contribution to protection at these body sites, and whether it functionally intersects with the transepithelial transport of antibodies by fcrn and the pigr. recently, it was demonstrated that complement factor c attached to different non-enveloped viruses can be carried inside the cytosol during infection ( ) . here, c detection resulted in proteasomal targeting of the virus and induction of inflammatory signaling in a process dependent upon mitochondrial antiviral-signaling protein (mavs) ( ) . as such, it could be speculated that other intracellular receptors specific for other immune serum proteins with antiviral functions have yet to be discovered. antibodies, viruses and vaccines broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses broadly neutralizing antiviral antibodies neutralization of animal viruses the antiviral activity of antibodies in vitro and in vivo broadly neutralizing hemagglutinin stalk-specific antibodies require fcgammar interactions for protection against influenza virus in vivo broadly neutralizing anti-hiv- antibodies require fc effector functions for in vivo activity fcgamma receptors as regulators of immune responses secretory iga: designed for antimicrobial defense immunoglobulin transport across polarized epithelial cells bidirectional fcrn-dependent igg transport in a polarized human intestinal epithelial cell line transcytosis and catabolism of antibody receptor-mediated immunoglobulin g transport across mucosal barriers in adult life: functional expression of fcrn in the mammalian lung fcrn-mediated antibody transport across epithelial cells revealed by electron tomography the recycling and transcytotic pathways for igg transport by fcrn are distinct and display an inherent polarity enhanced neonatal fc receptor function improves protection against primate shiv infection transfer of igg in the female genital tract by mhc class i-related neonatal fc receptor (fcrn) confers protective immunity to vaginal infection defense-in-depth by mucosally administered anti-hiv dimeric iga and systemic igg mabs: complete protection of rhesus monkeys from mucosal shiv challenge maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal fc receptor-mediated transcytosis intracellular neutralization of viral infection in polarized epithelial cells by neonatal fc receptor (fcrn)-mediated igg transport protective effect of rotavirus vp -specific iga monoclonal antibodies that lack neutralizing activity early high-affinity neutralizing anti-viral igg responses without further overall improvements of affinity the role of antibody concentration and avidity in antiviral protection antiviral antibody responses: the two extremes of a wide spectrum antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid neutralizing antibodies to adenovirus serotype vaccine vectors are directed primarily against the adenovirus hexon protein location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies hiv- evades antibodymediated neutralization through conformational masking of receptor-binding sites trim -dependent intracellular antibody neutralization of virus infection intracellular antibody immunity cellular self-defense: how cell-autonomous immunity protects against pathogens antibodies mediate intracellular immunity through tripartite motifcontaining (trim ) aaa atpase p /vcp is essential for trim -mediated virus neutralization intracellular antibodybound pathogens stimulate immune signaling via the fc receptor trim translocalized iga mediates neutralization and stimulates innate immunity inside infected cells fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond human antibody-fc receptor interactions illuminated by crystal structures specificity and affinity of human fcgamma receptors and their polymorphic variants for human igg subclasses the tripartite motif family identifies cell compartments protein-protein interactions between native ro and immunoglobulin g heavy chain the mw ro/ss-a autoantigen in sjogren's syndrome/systemic lupus erythematosus (ro ) is an interferongamma inducible tripartite motif protein associated with membrane proximal structures trim is a trimeric protein that binds igg fc via the b . domain structural basis for prysprymediated tripartite motif (trim) protein function trim is an igg receptor that is structurally, thermodynamically, and kinetically conserved trim proteins as ring finger e ubiquitin ligases trim family proteins: retroviral restriction and antiviral defence trimmunity: the roles of the trim e -ubiquitin ligase family in innate antiviral immunity anti-retroviral activity of trim alpha relationship between spry and b . protein domains. evolution of a component of immune defence? trim family: pleiotropy and diversification through homomultimer and heteromultimer formation the tripartite motif coiled-coil is an elongated antiparallel hairpin dimer the structure of a human type iii fcgamma receptor in complex with fc the . -a crystal structure of the human igg fc fragment-fc gammariii complex structure of fcgammari in complex with fc reveals the importance of glycan recognition for high-affinity igg binding mapping of the c q binding site on rituxan, a chimeric antibody with a human igg fc structural basis of phdependent antibody binding by the neonatal fc receptor structural insights into neonatal fc receptor-based recycling mechanisms crystallographic refinement and atomic models of a human fc fragment and its complex with fragment b of protein a from staphylococcus aureus at . -and . -a resolution model for the complex between protein g and an antibody fc fragment in solution crystal structure of the hsv- fc receptor bound to fc reveals a mechanism for antibody bipolar bridging structural requirements for the interaction of human iga with the human polymeric ig receptor the nonplanar secretory iga and near planar secretory iga solution structures rationalize their different mucosal immune responses identification of residues in the ch /ch domain interface of iga essential for interaction with the human fcalpha receptor (fcalphar) cd localization of the binding site for the monocyte immunoglobulin (ig) a-fc receptor (cd ) to the domain boundary between calpha and calpha in human iga the human iga-fc alpha receptor interaction and its blockade by streptococcal igabinding proteins streptococcal iga-binding proteins bind in the calpha -calpha interdomain region and inhibit binding of iga to human cd structural requirements for the interaction of human igm and iga with the human fcalpha/mu receptor sites in the ch domain of human iga that influence sensitivity to bacterial iga proteases regulation of virus neutralization and the persistent fraction by trim another role of proline: stabilization interactions in proteins and protein complexes concerning proline and tryptophane the aaa-atpase cdc /p regulates spindle disassembly at the end of mitosis cdc p is required for the cell cycle commitment point at start via degradation of the g -cdk inhibitor far p the ubiquitin-selective chaperone cdc- /p links myosin assembly to human myopathy emerging functions of the vcp/p aaa-atpase in the ubiquitin system distinct aaa-atpase p complexes function in discrete steps of nuclear assembly cdc /p promotes reformation of the nucleus by extracting the kinase aurora b from chromatin structure of the aaa atpase p valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation a conserved unfoldase activity for the p aaa-atpase in proteasomal degradation stalled proteasomes are directly relieved by p recruitment crystal structure of human adenovirus at . a resolution the s proteasome: assembly and function of a destructive machine structure, assembly and homeostatic regulation of the s proteasome sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim the e ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines noncanonical mms -encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for dna repair the mechanism of linkage-specific ubiquitin chain elongation by a single-subunit e the function of immunoglobulin a in immunity the immune geography of iga induction and function the structure and function of human iga iga fc receptors relationship of the quaternary structure of human secretory iga to neutralization of influenza virus occupancy and mechanism in antibody-mediated neutralization of animal viruses toll-like receptors and innate immunity viral evasion and subversion of pattern-recognition receptor signalling complexity of danger: the diverse nature of damage-associated molecular patterns trim is an innate immune sensor for the retrovirus capsid lattice direct activation of protein kinases by unanchored polyubiquitin chains activation of ikk by tnfalpha requires sitespecific ubiquitination of rip and polyubiquitin binding by nemo sensing of lys -linked polyubiquitination by nemo is a key event in nf-kappab activation trimming of ubiquitin chains by proteasomeassociated deubiquitinating enzymes mechanisms for t cell receptor triggering intracellular antibody-mediated immunity and the role of trim uncoating of human rhinoviruses drifting motions of the adenovirus receptor car and immobile integrins initiate virus uncoating and membrane lytic protein exposure adenovirus protein vi mediates membrane disruption following capsid disassembly dynein-and microtubulemediated translocation of adenovirus serotype occurs after endosomal lysis the role of the nuclear pore complex in adenovirus dna entry site of human rhinovirus rna uncoating revealed by fluorescent in situ hybridization opening of size-selective pores in endosomes during human rhinovirus serotype in vivo uncoating monitored by singleorganelle flow analysis two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at a resolution functional basis of poliovirus neutralization determined with monospecific neutralizing antibodies bivalent attachment of antibody onto poliovirus leads to conformational alteration and neutralization neutralizing antibody blocks adenovirus infection by arresting microtubuledependent cytoplasmic transport the interaction of neutralized poliovirus with hela cells. i. adsorption antipeptide antisera define neutralizing epitopes on the adenovirus hexon postentry neutralization of adenovirus type by an antihexon antibody neutralization of poliovirus by polyclonal antibodies requires binding of a single igg molecule per virion interaction between hela cells and adenovirus type virions neutralized by different antisera growth and killing of a salmonella enterica serovar typhimurium sifa mutant strain in the cytosol of different host cell lines autophagy recognizes intracellular salmonella enterica serovar typhimurium in damaged vacuoles antibody-and trim -dependent intracellular restriction of salmonella enterica simultaneous neutralization and innate immune detection of a replicating virus by trim intracellular antibody receptor trim prevents fatal viral infection fatal disseminated mouse adenovirus type infection in mice lacking b cells or bruton's tyrosine kinase mouse adenovirus type infection of natural killer cell-deficient mice mouse adenovirus type- replication is restricted to vascular endothelium in the cns of susceptible strains of mice intracellular sensing of complement c activates cell autonomous immunity this work was supported in part by the research council of norway through its center of excellence funding scheme (project number ). j. t. a. was supported by the research council of norway (grant no. / key: cord- - oe nj j authors: wang, hua; shen, guoli; yu, ruqin title: aspects of recent development of immunosensors date: - - journal: electrochemical sensors, biosensors and their biomedical applications doi: . /b - - . - sha: doc_id: cord_uid: oe nj j this chapter focuses on the recent developments in the field of immunosensors. immunosensors incorporate the specific immunochemical reaction with the modern transducers including electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fluorescence, luminescence, refractive index), and microgravimetric transducers. these immunosensor devices with dramatic improvements in the sensitivity and selectivity possess the abilities to investigate the reaction dynamics of antibody–antigen binding and the potential to revolutionize conventional immunoassay techniques. with the rapid development of immunological reagents and detection equipments, immunosensors have allowed an increasing range of analytes to be identified and quantified and in particular, simple-to-use, inexpensive, and reliable immunosensing systems have been developed for areas such as outpatient monitoring, large screening programs, and remote environmental surveillance. immunosensors with lowered detection limits and increased sensitivities have been developed in various fields, particularly in clinical analysis. a noticeable development trend is also observed in the development of immunosensors combining with other techniques such as flow injection analysis (fia) or capillary electrophoretic (ce) analysis, which complement and improve the present immunoassay methods. belov et al. have proposed a novel immunophenotyping method for leukemias which uses a cluster of differentiation antibody microarray, and a microarray of enzyme-linked immunosorbent assay has been developed for autoimmune diagnosis of systematic rheumatic disease. development of microfluidic immunosensor systems for proteomics and drug discovery have also been reported in recent years where the microfluidic system integrates multiple processes in a single device to improve analytical performance by reducing the reagent consumption and the analysis time, and increasing reliability and sensitivity through automation. immunosensors are affi nity ligand-based biosensing devices that involve the coupling of immunochemical reactions to appropriate transducers. in recent decades, immunosensors have received rapid development and wide applications with various detection formats [ ] [ ] . the general working principle of the immunosensors is based on the fact that the specifi c immunochemical recognition of antibodies (antigens) immobilized on a transducer to antigens (antibodies) in the sample media can produce analytical signals dynamically varying with the concentrations of analytes of interest. here, the highly specifi c reaction between the variable regions of an antibody and the epitopes of an antigen involves different types of bonding, basically hydrophobic and electrostatic interactions, van der waals force, and hydrogen bonding. the antigenantibody reaction is reversible and, owing to the relative weakness of the forces holding the antibody and antigen together, the complex formed would dissociate in dependence upon the reaction environment (e.g. ph and ion strength). the strength of the binding of an antibody to an antigen could be characterized by its affi nity constant (k), which is of the order between ϫ and ϫ l mol Ϫ . the high affi nity and specifi city of this antigen-antibody binding reaction defi nes the unique immunosensor characteristics. the general immunosensor design consists of three individual parts in close contact: a biological recognition element, a physicochemical transducer, and an electronic part. antibodies or antibody derivatives (antigens or haptens) usually serve as the biological recognition elements, which are either integrated within or intimately associated with a physicochemical transducer. this recognition reaction defi nes the high selectivity and sensitivity of the transducer device. the electronic part is used to amplify and digitalize the physicochemical output signal from the transducer devices such as electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fl uorescence, luminescence, refractive index), and microgravimetric devices. gizeli and lowe [ ] suggested that an ideal immunosensor design should possess the following specifi cations: the ability to detect and quantify the antigens (antibodies), the capacity to transform the binding event without externally added reagents, the ability to repeat the measurement on the same device, and the capacity to detect the specifi c binding of the antigens (antibodies) in real samples. all of these specifi cations have been the main issues to pursue in developing immunosensors applied in various fi elds. as an important branch of immunoassay techniques, immunosensors possess all essential performance characteristics of immunoassays. they show high selectivity, sensitivity, reversibility and effi cient reagent usage. at the same time, the immunosensors are generally simple to operate, and easy to realize automation, digitization, and miniaturization. they may bypass some inherent problems of traditional analytical methods. therefore, immunosensors have been the subject of expanding interest in the immunochemical studies with enormous potential in clinical diagnosis [ ] [ ] ] , environmental analysis [ ] [ ] , and biological process monitoring [ ] . as for the medical diagnosis of some diseases, herein considerable efforts have been devoted to the development of precise, rapid, sensitive, and selective immunosensors by measurement of the markers or pathogenic microorganisms responsible for the diseases, such as proteins, enzymes, viruses, bacteria, and hormones [ , [ ] [ ] . chagas' disease, an american trypanosomiasis caused by the hemofl agellate trypanosoma cruzi, is an example. an amperometric immunosensor has been recently proposed to probe the presence of antibodies against t. cruzi in blood donors, and to follow the antibody decay during treatment of chagasic patients with the available drugs [ ] . yuan et al. reported a novel potentiometric immunosensor for detection of hepatitis b surface antigen by immobilizing hepatitis b surface antibody on a platinum electrode [ ] . a piezoelectric immunosensor was developed for the on-line detection of severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) in sputum in the gas phase. compared to other sars detection techniques, this method can rapidly test sars-cov at low cost [ ] . moreover, the determination of some tumor markers plays an important role in diagnosing, screening, and determining the prognosis of a cancer disease. such tumor markers to be detected are often found in abnormally high amounts in the blood, urine, or tissue of patients with certain types of cancers. the examples include carcinoembryonic antigen (cea), carbohydrate antigen - (ca - ), carcinoma antigen (ca ), alpha-fetoprotein (afp), prostate specifi c antigen (psa), ca - and human chorionic gonadotropin (hcg) [ ] [ ] [ ] . wilson proposed an electrochemical immunosensor for the simultaneous detection of two tumor markers of cea and afp [ ] . an increasing number of immunosensors have been utilized to analyze a series of biochemical targets for diagnosing infectious diseases, although there are still problems concerning the assay of analytes in real sample matrixes [ ] . since immunosensors usually measure the signals resulting from the specifi c immunoreactions between the analytes and the antibodies or antigens immobilized, it is clear that the immobilization procedures of the antibodies (antigens) on the surfaces of base transducers should play an important role in the construction of immunosensors. numerous immobilization procedures have been employed for diverse immunosensors, such as electrostatic adsorption, entrapment, cross-linking, and covalent bonding procedures. they may be appropriately divided into two kinds of non-covalent interactionbased and covalent interaction-based immobilization procedures. this type of immobilization of immunoactive entities is based on the non-covalent interactions between the antibody or antigen molecules and the transducer substrates, and usually refers to hydrophobic interaction, electrostatic interaction, van der waals force, and hydrogen bonding. one notices that besides pure physical adsorption, some weak chemical interactions are also involved here. the non-covalent interactions may vary from the different substrates of transducers. for a non-polarity sensing substrate, the antibody or antigen molecules can be adsorbed through the hydrophobic interaction and van der waals force. wenmeyer et al. attached anti-digoxin antibodies at the surfaces of polystyrene microtubes by direct adsorption interaction, achieving the determination of digoxin with a detection limit of pg ml Ϫ [ ] . while for the charged substrates, the non-covalent interactions are mainly associated with the electrostatic interactions. the most typical layer-by-layer technique of self-assembly has attracted considerable attention in biomolecular immobilizations [ ] [ ] [ ] [ ] [ ] . caruso and coworkers assembled polyallylamine hydrochloride/polystyrene sulfonate layers on the self-assembled monolayer of mercaptopropionic acid, providing a charged polyelectrolyte layer on the transducer surface [ ] . the biomolecules of avidin and anti-immunoglobulin (igg) antibodies were then well immobilized through electrostatic interaction. a novel biosensing interfacial design strategy has been developed for immobilizing the antibodies onto the positively charged surfaces of plasma-polymerized fi lm (ppf) via electrostatic interaction through a polyelectrolyte-mediated layer [ ] . the immunosensors so prepared exhibited excellent response sensitivity due to the low disturbance of the electrostatic adsorption immobilization to the activity of antibody. the ppf surfaces can be regenerated repetitively by changing the ph of the buffer solutions to remove the polyelectrolyte-mediated layer. moreover, antibodies or antigens may be physically entrapped into the fi lms of organic high polymers or inorganic materials (e.g. sol-gel, graphite powder) with stereo meshy structures. of these entrapment immobilizations, the sol-gel-based immobilizations have recently attracted much attention due to their ability to encapsulate biomolecules at low temperature, as well as the physical tenability, optical transparency, mechanical rigidity, and low chemical reactivity [ ] [ ] . most applications of the sol-gel-based immobilizations have been primarily directed to the optical immunosensors [ , , ] and the electrochemical immunosensors [ , [ ] [ ] [ ] [ ] . martínez-fàbregas et al. proposed a polishable entrapment immobilization based on rigid biocomposite materials consisting of graphite powder, rabbit igg, and methacrylate (or epoxy resins) [ ] . the surface of the immunosensor can be regenerated by simply polishing to obtain a fresh layer of immunocomposite ready for next immunoassay. the aforementioned physical interaction-based immobilization procedures are demonstrated to be operated simply and rapidly. however, their immobilization stability might be infl uenced by the bulk metal surfaces and environmental factors such as temperature, ph, and ion strength of solution, resulting in a loss of bioactivity or denaturation of the proteins. moreover, the gradual elution of proteins physically adsorbed may occur during the analytical performances, which may in turn bring about some problems associated with loss of detection sensitivity and low reproducibility of the sensors. in recent years, nanomaterials (e.g. noble metals, magnetic oxides, and carbon nanoparticles or nanotubes) with unique physical and chemical properties have been successfully applied to modify immunosensing interfaces to achieve greatly improved immobilization of antibodies or antigens [ ] [ ] [ ] . some pioneering works have shown that the assembly of the gold nanoparticle layer on an electrode would lead to substantially increased electrode surface areas available for direct adsorption of biological entities, thus offering the possibility of the great enhancement of analytical sensitivity [ , [ ] [ ] [ ] [ ] [ ] . for example, a new immobilization procedure of antibodies for capacitive immunosensor has been recently proposed using thiol compound and gold nanoparticles [ ] . it was here demonstrated that the proposed immobilization procedure could retain the high biological activity of immobilized entities and provide favorable sensing performances. moreover, magnetic nanoparticles as special carriers for immobilizing biomolecules have also been the current hot subject of a series of investigations for the construction of different immunosensors [ ] [ ] [ ] . the easy localization of magnetic beads was used to generate a sensing layer at the surface of a piezoelectric sensor, where the magnetic beads bearing antibodies were immobilized with the help of a permanent magnet at the surface of the crystal [ ] . more recently, an amperometric immunosensor has been developed by employing a kind of core-shell magnetic nanoparticle of (cdfe o øsio ) to immobilize antibody onto the electrode surface with a magnet fi eld [ ] . additionally, magnetic beads may be applied to label or attach antibodies (antigens) for the magneto-detection of the immune complex based on the perturbation of a magnetic fi eld, which could be quantifi ed using a suitable electronic device [ ] . compared with the conventional immobilization methods, these magnetism-driven immobilization procedures may have some merits such as simple manipulation, easy biomolecule modifi cation, low cost, and repeatable regeneration. the covalent interaction procedures, typically the cross-linking methods, are the most popular immobilization manipulation for fabricating various immunosensors. due to the lack of an amount of active covalently binding sites at some transducer substrates (e.g. metals, semiconductor, or optical fi bers), the precoatings of the base transducers with thin fi lms are generally necessary for covalently binding the antibodies or antigens by using the functional reagents such as glutaraldehyde, carbodiimide succinimide ester, maleinimide, and periodate. many traditional coating materials, such as polyethyleneimine [ ] [ ] , (γ-aminopropyl) trimethoxysilane [ ] [ ] , and copolymer of hydroxyethyl-and methyl-methacrylate [ ] , are often used as the mediate layers for immunoactive molecule immobilization. in recent decades, however, some new coating or functionalized fi lm techniques (materials) have been introduced into this fi eld. self-assembled monolayers (sams) offer promising functionalized fi lms for the immobilization of antibodies or antigens [ ] [ ] . since sulfur donor atoms strongly coordinate on noble metal substrates (e.g. au, ag, and pt), various sulfur-containing molecules such as disulfi des (r-ss-r), sulfi des (r-s-r), and thiols can form various functionalized sams of highly organized and compact construction. the applications of the sam technique in the immobilization of biomolecules have been widely documented [ ] [ ] [ ] . knoll and coworkers presented a versatile biotin-functionalized sam, on which the biotinylated antibodies can be readily immobilized through an avidin mediator [ ] . mixed sams composed of long-chain thiols with carboxylic and hydroxyl groups are also used to attain a specifi c and stable affi nity interface of immunosensors [ ] [ ] . langmuir-blodgett (lb) fi lms are other useful alternatives to traditional mediate layers [ ] [ ] [ ] . lb fi lms, which are usually prepared by transferring a monolayer on a solid substrate, have great potential in helping to control the orientation and surface density of the antibodies. hirata et al. [ ] successfully prepared the lipid-tagged antibody/phospholipid monolayers with high immobilization properties using the lb technique. vikholm et al. demonstrated the incorporation of lipid-tagged single-chain antibodies into lipid monolayers obtaining desirable retention of antibody activities [ ] [ ] . moreover, recent years witness a newly emerged ultra-thin polymer fi lm, plasma-polymerized fi lm (ppf), which is reported with successful applications in various immunosensor designs [ , [ ] [ ] [ ] . ppfs, which are generally prepared by using glow discharge or plasma of organic vapors, are extremely thin, homogeneous, mechanically and chemically stable, with strong adhesion to the substrates. karube's group fi rst reported the application of ppf to qcm immunosensors [ ] . they verifi ed that the resultant sensors were more reproducible from batch to batch, and might have lower noise and higher sensitivity than sensors using some conventional organic coatings (e.g. polyethylenimine). this kind of functionalized fi lm may offer promising alternatives in interfacial design of immunosensors of various transducers. in recent years, various nanomaterials are found to be skillfully applied in combination with the covalent interaction-based immobilization procedures for immunosensors. carbon nanotubes (cnts), for example, have been recognized as the quintessential nano-sized materials since their discovery in [ ] . these nanotubes are now chemically functionalized for the immobilization of biological entities for different biosensors, i.e. electrochemical devices [ ] [ ] [ ] . pantarotto et al. successfully bound a model pentapeptide and a virus epitope of foot-and-mouth disease onto single-walled cnts [ ] . they found that the cnts-loaded peptide might retain the structural integrity to be well recognized by monoclonal or polyclonal antibodies, indicating the potential applications for diagnostic purposes and vaccine delivery. a silica nanoparticles-based immobilization strategy was also proposed by wang et al. for direct immunosensing determination of toxoplasma gondii-specifi c igg [ ] . herein, the preparation strategy could allow for antigens covalently bound with higher loading amount and better retained immunoactivity compared to the commonly applied crosslinking methods. the aforementioned covalent interaction-based procedures may usually allow for the immunoactive proteins immobilized with high stability and repeatability, and the robust covalent bonds may favor the low noise of detection. nevertheless, problems associated with these covalent bond immobilizations are the decrease of binding capacity of antibodies (antigens) in the immobilization process. such a phenomenon may be presumably contributed to the partial loss of the immunoactive sites and the random orientation of antibody molecules bound on the transducer surfaces. in addition, cross-linking can produce a three-dimensional multilayer matrix that creates diffusion barriers and transport limitations, resulting in long immunoreaction time and low sensitivity [ ] . it is established that the oriented immobilization of antibodies has low infl uence on their immunological activities to a certain degree [ ] [ ] [ ] [ ] [ ] , which antigen binding capacity was demonstrated with a factor of - higher than that of antibodies randomly immobilized [ ] . therefore, special interest has been given to the development of the orientation-controlled immobilization techniques for antibodies, i.e. mostly through proteins a or g to specifi cally bind the antibody fc fragment, or by directly binding the chemical groups at antibody fc region [ , [ ] [ ] [ ] . lee et al. utilized the self-assembled layer of thiol group-modifi ed protein a for the oriented immobilization of antibodies [ ] . an increased binding capacity was further observed. as another illustrative instance, a protein a-based orientationcontrolled immobilization strategy for antibodies was proposed for the fabrication of a qcm immunosensor using nanometer-sized gold particles and amine-terminated ppf [ ] . moreover, in recent years, there has emerged another oriented immobilization methodology for antibodies through their native thiol (-sh) groups, which were liberated after the splitting of the intact igg into two antibody fragments [ ] [ ] . karyakin et al. reported a site-oriented immobilization strategy of antibodies on the gold electrode surfaces by use of native sulfi de groups of igg fragments obtained by reduction of intact igg [ ] . they found that antibodies immobilized by this procedure showed an antigen binding capacity - times higher than that of non-specifi cally adsorbed intact ones traditionally used. in general, an ideal immobilization should have the following characteristics: (i) a suffi cient loading amount of active antigens or antibodies at the transducer surface; (ii) the immobilized antigens or antibodies staying stable during the measurement process; (iii) the immobilization process having no infl uence to the sensing behavior of the transducer; and (v) the ability of sensor regeneration. an effective dissociation of the antigen and regeneration of antibody, i.e. by using gly-hcl buffer (ph . ), for cost effectiveness is of practical interest in real immunosensor applications [ ] . there are mainly three types of transducers used in immunosensors: electrochemical, optical, and microgravimetric transducers. the immunosensors may operate either as direct immunosensors or as indirect ones. for direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. the direct immunosensors detect the analytes in real time. for indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. these indirect detections used to need several washing and separation steps and are sometimes called immunoassays. compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specifi c adsorption. the majority of known immunosensor devices belong to the group of electrochemical immunosensors. electrochemical immunosensors may possess several advantages, for example high sensitivity, low cost, and portable design. the principle of their operation is based on the electrochemical detection of the labeled immunoagents or markers such as enzymes, metal ions, or other electroactive compounds, thus providing an opportunity to analyze complex multicomponent mixtures for diagnosing diseases or monitoring the status of patients [ ] . the kinds of detection transducer for electrochemical immunosensors can be mainly subdivided into potentiometric, conductometric, capacitive, impeditive, and amperometric (metal and graphite electrodes) devices. potentiometric transducers now belong to the most mature transducers with numerous commercial products. for potentiometric transducers, a local equilibrium is established at the transducer interface at near-zero current fl ow, where the change in electrode or membrane potential is logarithmically proportional to the specifi c ion activity. the relationship of logarithmical proportionality constitutes the fundamental principle of all potentiometric transducers such as the ion-selective electrodes (ise). the groups of biosensors are characterized as simple in preparation, robust in operation, and moderately selective in analytical performance [ ] [ ] [ ] [ ] [ ] [ ] . janata fi rst proposed a potentiometric transducer for immunosensing and named it "immuno-electrode" [ ] , using the immuno-electrode to detect concanavalin a through covalent attachment to the surface of a pvc membrane deposited on a platinum electrode. the incorporation of ises, ph electrodes or gas-sensing electrodes into potentiometric immunosensors to improve their assay sensitivity has been extensively investigated by rechnitz and coworkers, i.e. for immunochemical measurements of digoxin and human igg [ ] [ ] . d'orazlo et al. reported the indirect measurements of immunoagents using ion-selective electrodes [ ] . a potentiometric immunosensor based on a molecularly imprinted polymer was prepared as a detecting element in micro total analysis systems with the intent of providing easy clinical analysis [ ] . moreover, the ion-selective fi eld-effect transistor (isfet) as a semiconductor device is generally constructed by substituting an ion-sensing membrane for the metal gate of a fi eld-effect transistor (fet) [ ] . the isfet is able to respond to the surface potential change resulting from the specifi c immunochemical reaction between the immobilized antibodies and the free antigens. the ph-sensitive isfets, as the most widely used sensor of this type, are fabricated with a large range of possible insulators (i.e. sio , si n , and al o ) and enzyme labels (i.e. urease, peroxidase, and glucose oxidase) [ ] [ ] . nevertheless, only a few examples of isfet-based immunosensors could be found in the literature [ ] [ ] [ ] . for example, zayats et al. report the impedance measurements on an isfet device that can be used to detect antigen-antibody interactions on the gate surface [ ] . in the meantime, they performed complementary surface plasmon resonance (spr; see above) experiments to illustrate that the isfet impedance measurements and the spr reveal comparable sensitivities. conductometric transducers, as the oldest electrochemical devices, seem not to enjoy wide applications due to their poor selectivity. for example, yagiuda et al. proposed a conductometric immunosensor for the determination of methamphetamine (ma) in urine [ ] . the decrease in the conductivity between a pair of platinum electrodes might result from the direct attachment of ma onto the anti-ma antibodies immobilized on the electrode surface. the system was claimed to be a useful detection technique of ma in comparison with a gas chromatography-mass spectrometry method. capacitance and impedance transducers with high sensitivity are widely employed for various immunosensing assays [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the capacitance sensors are essentially based on the principle that the electrolyte capacitance of an electrode depends on the thickness and dielectric behavior of the dielectric layer on the electrode surface and the solid/solution interface. dijksma et al. designed an immunosensor for the direct detection of interferon-γ at the attomolar level by using the ac impedance approach [ ] . the immobilization processes of antibodies (antigens) play an important role in these immunosensors, and the sensitivity of a capacitive immunosensor increases with the decreasing thickness of the insulating layer. shen and coworkers fabricated a heterostructure of au/o-aminobenzenethiol layer/covalent-coupling antibody/electrode for the direct detection of the antibody-antigen interaction by capacitance measurements [ ] . a capacitive immunoassay based on antibody-embedded ultra-thin alumina sol-gel fi lms (∼ to nm) was reported and used for direct determination of antigens with a detection limit as low as ϳ ng ml Ϫ [ ] . fernandez-sanchez et al. reported a successful integration of the lateral fl ow immunoassay format and impedance detection for prostate-specifi c antigen of tumor marker, where the electrochemical transducer was coated with a ph-sensitive polymer layer [ ] . although capacitance and impedance immunosensors can directly be utilized to investigate the antibody-antigen interaction without the need of other reagents and a separation step, their analytical sensitivity is limited in clinical applications [ ] . in order to amplify the capacitance or impedance response to immunoreaction for the sensitive detection of various clinical markers, different labels have been used including enzymes, fl uorophores, and metal chelates [ ] [ ] . ruan et al. developed an immunosensor based on enzyme-stimulated precipitation for the detection of escherichia coli o :h using an electrochemical impedance spectroscopy [ ] . another illustrative example was the sensitized immunosensor proposed by chen et al. [ ] . in their study, a receptor protein was directly adsorbed on a porous nanostructure gold fi lm to perform a sandwich immunoreaction with the precipitation of insoluble product on the electrode. the impedance signals so amplifi ed showed good linearity with the content of igg in the range . - ng ml Ϫ with a detection limit of . ng ml Ϫ . a new strategy of signal amplifi cation was also introduced for highly sensitive impedance measurements using biotin-labeled protein-streptavidin network complex [ ] . amperometric immunosensors, as the most popular immunosensing formats, are based on the measurement of the currents resulting from the electrochemical oxidation or reduction of electroactive species at a certain constant voltage. this kind of immunosensor usually uses a complex three-electrode measuring system consisting of a working electrode (e.g. gold, glassy carbon, or carbon paste), a reference electrode (e.g. ag/agcl), and a conducting auxiliary electrode (e.g. platinum). since most antibodies and antigens are not electrochemically active, there are only a few applications available for direct amperometric sensing. therefore, most amperometric immunosensors are indirect ones which can detect mainly the redox currents associated with electroactive or catalytic labels [ - , , - ] . aizawa et al. fi rst developed an amperometric immunosensor for the determination of human chorionic gonadotropin using an amperometric oxygen electrode [ ] . among the labels used, enzymes are the most popular ones in different types of immunoassays, such as horseradish peroxidase (hrp) or glucose oxidase. an immunosensor was designed for determining isopentenyl adenosine based on the electro-polymerization of polypyrrole and poly(m-phenylenediamine) entrapped with hrp on the glassy carbon electrode [ ] . a design strategy of reagentless immunosensor was reported for the detection of carcinoma antigen- antibodies by direct hrp-labeled electrochemistry [ ] . due to the high sensitivity inherent in these transducers by enzymatic catalysis, amperometric immunosensors can obtain a much higher sensitivity than the classical elisa. for the immunosensors used in clinical applications, their surfaces should be capable of renewal. yu et al. developed a renewable amperometric immunosensor for the determination of schistosoma japonium (sj) antibody by using the paraffi n graphite-sj antigen biocomposite paste electrodes which might be regenerated by polishing the surface [ ] . ionescu and his collaborators have developed two similar amperometric immunosensors for cholera antitoxin immunoglobulins, where the cholera toxin biorecognition entities were bound to a biotinylated polypyrrole fi lm or pyrrole-biotin and lactitobionamide electropolymerized copolymer [ ] [ ] . moreover, nano-sized particles or sol-gel matrixes have also been increasingly employed in the design of amperometric immunosensors with enhanced analytical performance [ , [ ] [ ] . for example, an electrochemical immunosensor has been developed for probing complement iii (c ) by use of nanogold particle monolayer as the sensing interface [ ] . with the coupling of sol-gel and screen-printing technologies, a sensitive thick fi lm immunosensor was fabricated by dispersion of rabbit immunoglobulin g, graphite powder, and a binder in the solgel solution [ ] . a new hrp-labeled amperometric immunosensor for determination of chorionic gonadotrophin in human serum was constructed by immobilizing hcg within titania sol-gel on a glassy carbon electrode [ ] . anodic stripping voltammetry as an electrochemical assay technique has been well adopted for sensitive measurements of heavy metals such as copper and silver, which may also offer an attractive way of sensitive immunosensor development [ ] [ ] . an immunosensor was designed by coupling immunoassay with the square wave anodic stripping voltammetry technique involving copper ion-labeled antigen in the competitive immunoreaction [ ] . this immunosensor might allow rapid, accurate, and inexpensive detection of gibberellin acid with a concentration as low as µg ml Ϫ . chu et al. designed a silverenhanced colloidal gold metalloimmunoassay for the determination of schistosoma japonicum antibody (sjab) in rabbit serum [ ] . in their study, after the immunoreaction of sjab target with immobilized sj antigens, colloidal gold-labeled secondary antibody was introduced to favor the silver enhancement process. an acidic solution was further used to dissolute silver metal atoms, followed by the sensitive determination of dissolved silver ions using anodic stripping voltammetry. in addition, many immunoreaction signal-amplifi ed methods or processes have also been adopted for the development of sensitive amperometric immunosensors. willner's group reported an amplifi ed immunosensing scheme of chronopotentiometry and faradaic impedance spectroscopy by way of a bio-catalyzed precipitation of the insoluble product onto the gold electrode [ ] . they also designed a variation of this scheme with signal amplifi cation by employing liposomes labeled with biotin and hrp as a probe to amplify the sensing of antigen-antibody interactions [ ] . in this case, the electrode with the antigen-antibody complex was exposed to the biotinylated anti-igg antibody, and further the biotin-labeled hrp-liposomes through an avidin bridge to achieve the biocatalyzed precipitation of an insoluble product on the conductive support. since almost all optical phenomena at sensing surfaces (e.g. adsorption, fl uorescence, luminescence, scatter or refractive index, etc.) can be used for biochemical sensing designs, optical immunosensors are considered as one of the most promising alternatives to the traditional immunoassays in clinic diagnosis and environmental analysis. in recent years, there has been an increased trend in the use of optical transduction techniques in immunosensor technologies due to the advantages of applying visible radiation, non-destructive operation mode, and the rapid signal generation and reading [ , [ ] [ ] . the optical immunosensors may be divided into two types of approaches: direct optical immunosensors and indirect immunosensors depending upon the use of labeled signaling molecules. surface plasmon resonance (spr) as a direct and reliable optical transducer is commonly based on the evanescent wave, in which a thin gold layer is generally deposited on a prism serving as an optically rarer medium [ ] [ ] . not requiring additional labels and separation steps, the direct spr immunosensors have been proven to be powerful analytical tools for rapid real-time monitoring the immunological targets. schofi eld and dimmock developed a spr system in combination with the fl ow system for detection of infl uenza virus by use of carboxylated dextran polymer matrix to couple monoclonal antibody of hc [ ] . in order to validate the feasibility of spr immunosensor as a tool for diagnosing type i diabetes, choi et al. modifi ed mixed sams onto the optical substrate achieving the immuno-response detection for monoclonal antibodies of anti-glutamic acid decarboxylase [ ] . moreover, the fatty acidbinding protein assay has an application potential in clinical analysis for diagnosis of myocardial infarction. a direct optical immunosensor based on spr was developed for detecting the human heart-type fatty acid binding protein with a detection limit of ng ml Ϫ [ ] . highly sensitive spr-based immunosensors using self-assembled protein g have also been successfully applied for the detection of microbes such as salmonella typhimurium and legionella pneumophila [ ] [ ] . more importantly, several instrument systems using spr technology have been commercially available, such as the biacore ™ system from pharmacia biosensor, the iasys ™ system from affi nity sensors, and so on. nevertheless, at present, there are still some unsolved problems for these spr devices, such as non-specifi c adsorption and poor analytical sensitivity to analytes of low molecular weight. fluorescence immunosensors, as the total internal refl ection fl uorescence devices, continue to prove themselves as another promising type of sensitive and selective optical immunoassay technique, in which labels are sometimes used [ ] . when the fl uorescence-labeled antibodies or antigens are attached to the transducer surface and enter the evanescent fi eld, the incident light will excite fl uorescent molecules producing a fl uorescent evanescent wave signal to be detected. the optic-fi ber immunosensor system by fl uorescence enhancement or quenching is separation-free, reagentless and applicable to the determination of various proteins by antigen-antibody reactions [ ] [ ] [ ] [ ] [ ] . maragos et al. described the development of a fl uorescence polarizationbased competition immunoassay for fumonisins in maize using fumonisin-specifi c monoclonal antibodies [ ] . a fl uorescence-based immunosensor array for simultaneous determination of multiple clinical analytes was developed by rowe et al. [ ] . in their study, the patterned array of recognition elements was immobilized onto the planar waveguide to "capture" the analytes from the samples to be quantifi ed by means of fl uorescent detector molecules. moreover, in recent years, quantum dots as the most suitable fl uorescence labels have received increasing applications for developing fl uorescence immunosensors due to their high fl uorescence quantum yield and sensitivity to environmental changes upon binding proteins. aoyagi et al. proposed a reagentless, regenerable, and portable optic immunosensor for the ultra-sensitive detection of a model sample of igg based on changes in fl uorescent intensity of fl uorescent quantum dot-labeled protein a [ ] . an antibody for leukemia cell recognition was attached to the luminophore-doped nanoparticle through silica chemistry, yielding an optical microscopy imaging technique for the identifi cation of leukemia cells [ ] . experimental results in this report showed that the new technique using the antibodycoated luminophore nanoparticles could allow leukemia cells to be easily and clearly identifi ed with high effi ciency. chemiluminescence sensors have also been extensively applied in routine clinical analysis as well as biomedical research due to the advantages of no radioactive wastes, simple instrumentation, low detection limit, and wide dynamic range [ , [ ] [ ] [ ] [ ] [ ] . a chemiluminescent immunosensor for carbohydrate antigen - (ca - ) was described by lin et al., with ca - immobilized on the cross-linked chitosan membrane [ ] . the decrease of the immunosensor chemiluminescent signal was proportional to the ca - concentration in the range . - u ml Ϫ , with the detection limit of . u ml Ϫ . pandian et al. developed an automated chemiluminometric immunoassay for the measurement of hcg [ ] . it was demonstrated that the immunoassay might facilitate exploration of hcg utility for down syndrome screening, early pregnancy detection, and differentiation of invasive from non-invasive trophoblastic disease. an optical microbiosensor has been newly designed for the diagnosis of hepatitis c virus (hcv) by using a novel photo-immobilization methodology based on a photo-activable electro-generated polymer fi lm [ ] . herein, the immunosensor using optical fi ber photochemically modifi ed was tested for the determination of anti-e protein antibodies through chemiluminescence reaction. another published study presented the use of electrogenerated luminol chemiluminescence in a homogeneous immunosensor, where digoxin was labeled with luminol through a luminol-bsa-digoxin conjugate [ ] . the prepared chemiluminescence immunosensor in a competitive format was shown allowing for the detection of free digoxin with the concentration as low as . µg l Ϫ . microgravimetric immunosensors may incorporate high sensitivity of piezoelectric response and high specifi city of antibody-antigen immunoreaction. the detection principle of these devices is generally based on adsorbate recognition where the selective binding may cause the changes in mass loading and interfacial properties (i.e. viscoelasticity and surface roughness), which can be recognized by a corresponding shift in the oscillation frequency [ ] [ ] [ ] [ ] . outstanding features of these sensors include low cost, simple usage, high sensitivity, and real-time output. microgravimetric immunosensors have two kinds of sensing formats, gas phase and solution phase sensing. the sensitivity to the mass change in air on the transducer surface is about hz ng Ϫ for a bulk acoustic wave device with mhz of fundamental frequency, which can be described by the sauerbrey equation [ ] . the microgravimetric transducer is thus mainly known as the quartz crystal microbalance (qcm). microgravimetric immunosensors in solution phase sensing were used for the quantifi cation of a number of biological targets [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nüsslein's group reported a qcm assay for bacteria using a cell-selective polymer fi lm, with desirably low detection limit and no need for prior sample treatment [ ] . wang and coworkers initially developed an integrated qcm immunosensor array composed of four kinds of leukemic lineageassociated probes to explore the differentiated leukocyte antigens for immunophenotyping of acute leukemia [ ] . in their study, the probes (crystals) of the array were immobilized separately with fab fragments of leukemic lineage-associated monoclonal antibodies (markers). the developed immunosensor array was demonstrated to be able to rapidly identify normal cells from leukemic blasts and defi ne the leukemic blasts within certain phenotypic groups (lineages). recently, a qcm immunosensor using protein a for antibody immobilization has been described for the detection of salmonella typhimurium in chicken meat sample by simultaneous measurements of the resonant frequency and motional resistance [ ] . based on the modifi cation of mixed sams on gold electrodes for covalently binding antigens, another piezoelectric immunosensor has been recently developed to detect antisperm antibody [ ] . the analytical results for evaluating several clinical specimens by the developed method were found to be in satisfactory agreement with those given by the classical elisa. despite many salient successes, the use of qcm-based immunosensors for trace biological target detection is still challenged by its relatively low intrinsic sensitivity. kim et al. incorporated the immunomagnetic separation with the qcm-based impedance technique achieving a new immunoassay for quantifying salmonella typhimurium with very high sensitivity of cell detection [ ] . herein, antibodies immobilized on magnetic particles were delivered into the sample medium to capture the targets. the resultant immunocomplex was further magnetically collected onto the piezoelectric crystal to be quantifi ed with impedance spectroscopy. through the enzyme-catalyzed formation of a precipitate on the qcm surface, a mass-amplifi ed microgravimetric immunosensor was proposed in combination with a sandwich enzyme-linked immunoassay [ ] . su and his coworkers successfully used qcm for detection of dengue virus [ ] . the authors immobilized two monoclonal antibodies on the crystal that act specifi cally against the dengue virus envelope protein and non-structural protein. the sensitivity reported for the fabricated piezoelectric immuno-chip was -fold greater than the conventional sandwich elisa method. a highly sensitive microgravimetric biosensor has been developed incorporating noble metal particle-amplifi ed sandwiched immunoassay and silver enhancement reaction [ ] . upon the formation of the sandwiched immunocomplex, the sensor surfaces were coated with gold nanoparticles serving as the nucleation sites to catalyze silver ion reduction. the silver metal deposition would result in a large change in frequency responses, achieving approximately two orders of magnitude improvement in human igg quantifi cation. moreover, there is another important type of microgravimetric immunosensor which is based on the immunological agglutination events. the agglutination immunoreaction of antibody-bearing suspensoids such as polymers, microbeads, and naoparticles may induce a corresponding change in the solution parameters (i.e. density and viscosity) and the interfacial properties of the crystal monitored by the qcm device [ ] [ ] [ ] [ ] . in contrast to the common conventional piezoelectric assays, the qcm sensing format offers a unique advantage in that the immobilization of antibodies or antigens on the crystal is not necessary. the kind of qcm-sensing methods are widely recognized to be simple, sensitive, and feasible for detecting relevant targets responsible for many clinical diseases [ ] [ ] [ ] [ ] [ ] [ ] . kurosawa et al. fi rst developed an agglutination-based piezoelectric immunoassay using antibody-bearing latex, termed as the latex piezoelectric immunoassay (lpeia), for detecting c-reactive protein [ ] . recently, it has been demonstrated that the lpeia could be greatly improved by using gold nanoparticles as replacements for latex particles, resulting in a novel agglutination-based piezoelectric immunoassay for directly detecting anti-t. gondii immunoglobulins in infected rabbit sera and bloods [ ] . in recent years, considerable efforts have been devoted to the development of cantilever-based immunosensors with unique enantio-selective antibodies [ ] [ ] . these devices are mainly used for quality and process control, and diagnostic biosensing for medical analysis. they may have fast responses and high sensitivity and are suitable for mass production. lee et al. fabricated a piezoelectric nanomechanical cantilever by a novel electrical measurement. they found that this technique might allow for the labelfree detection of a prostate-specifi c antigen (psa) with a detection sensitivity as low as pg ml Ϫ [ ] . a microfabricated cantilever was utilized to perform the direct (label-free) stereo-selective detection of trace amounts of an important class of chiral analytes, the r-amino acids, based on immunomechanical responses involving nanoscale bending of the cantilever. the major advantages of the microcantilever sensors over more traditional scale transducers such as the qcm reside in the superior sensitivity to minute quantities of analytes and the ability to micro-fabricate compact arrays of cantilevers to facilitate simultaneous and high throughput measurements [ ] . moreover, mass-sensitive magnetoelastic immunosensors are exploited to design extraordinarily versatile and useful sensor platforms [ ] . magnetoelastic sensors are well established and benefi t from mass sensitivity compared to that of a surface acoustic wave (saw) sensor. however, they may cost much less and are much smaller in size than saw devices. ruan et al. proposed a mass-sensitive magnetoelastic immunosensor based on the immobilization of affi nity-purifi ed antibodies on the surface of a micrometer-scale magnetoelastic cantilever achieving the highly sensitive detection of escherichia coli o :h [ ] . in addition, imaging ellipsometry (ie) has also been developed as a new kind of immunosensor, i.e. for the detection of pathogens of yersinia enterocolitica [ ] . as another example, a label-free multi-sensing immunosensor based on the combination of ie and the protein chip was reported to be able to detect multiple analytes simultaneously, and even to monitor multiple biological interaction processes in situ and in real-time conditions [ ] . immunosensors incorporate the specifi c immunochemical reaction with the modern transducers including electrochemical (potentiometric, conductometric, capacitative, impedance, amperometric), optical (fl uorescence, luminescence, refractive index), and microgravimetric transducers, etc. [ ] . these immunosensor devices with dramatic improvements in the sensitivity and selectivity possess the abilities to investigate the reaction dynamics of antibody-antigen binding and the potential to revolutionize conventional immunoassay techniques. with the rapid development of immunological reagents and detection equipments, immunosensors have allowed an increasing range of analytes to be identifi ed and quantifi ed. in particular, simple-to-use, inexpensive and reliable immunosensing systems have been developed to bring immunoassay technology to much more diverse areas, such as outpatient monitoring, large screening programs, and remote environmental surveillance [ ] . however, there are still some unsolved problems associated with the immobilization of immunoactive entities, nonspecifi c adsorption from sample backgrounds (e.g. blood, serum, plasma, urine, and saliva) and practical applications of various transducer devices. the current development of new immunosensors should aim at solving the problems of clinical analysis in medicine and of chemical analysis in the food industry and biotechnology. the development trends of immunosensors are likely to be primarily driven by the requirements of analytical practice on the improvement in sensitivity, selectivity, rapidity, and especially effi ciency of assays (i.e. immunosensing array or microfl uidic system). immunosensors with lowered detection limits and increased sensitivities have been developed in various fi elds, particularly in clinical analysis. for example, the sandwich immunoassay using enzyme-functionalized liposomes as the catalytic label is proposed to obtain the substantially improved assay sensitivity, as validated in the immunoassay of cholera toxin [ ] . meanwhile, as the latest paradigm of development topic, nanomaterials with unique chemical and physical properties should continue to be exploited to offer important possibilities for new immunosensor designs [ ] . a noticeable development trend is also observed in the development of immunosensors combining with other techniques such as fl ow injection analysis (fia) or capillary electrophoretic (ce) analysis, to complement and improve the present immunoassay methods [ ] [ ] . moreover, the miniaturization and automation of immunosensing devices should be another important intention of development to facilitate the signifi cantly shortened analysis time and simplifi ed analytical procedure (i.e. one-step analysis). of note, protein and antibody array technologies are envisaged to have potential for biomedical and diagnostic applications in recent years [ ] [ ] [ ] [ ] [ ] . belov et al. have proposed a novel immunophenotyping method for leukemias using a cluster of differentiation antibody microarray [ ] . a microarray of enzyme-linked immunosorbent assay has been developed for autoimmune diagnosis of systematic rheumatic disease, where the high titers of antinuclear antibodies against various nuclear proteins and nucleoprotein complexes might be detected with high throughput [ ] . at the same time, the screen-printing techniques may also appear to be the most promising technology for immunosensor array to be commercialized on a large scale and widely applied in clinical diagnosis. moreover, there have been increasing reports focusing on the development of microfl uidic immunosensor systems for proteomics and drug discovery in recent years [ ] . microfl uidic system integrating multiple processes in a single device generally seeks to improve analytical performance by reducing the reagent consumption and the analysis time, and increasing reliability and sensitivity through automation. the micro total analysis systems (µtas) are already under development and should represent the future of high throughput immuno-tests [ ] . in addition, with the development of protein engineering technology and molecular biology techniques, more fl exible antibodies suitable for immunosensing applications may be expected. for example, the recombinant or fusion approach is powerful in the production of antibodies and antibody derivates. use of various new generations of antibodies should lead to the enhancement of activity and stability of the immobilized bio-species and even the improvement of the regeneration and sensitivity of the immunosensors. as an inspiringly illustrative instance, aptamers are beginning to emerge as a class of synthetic oligonucleotides or molecules that rival antibodies in both therapeutic and diagnostic applications [ ] [ ] [ ] . baldrich and coworkers fi rst demonstrated the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay, the displacement enzyme-linked aptamer assay, using enzymelabeled target as a suboptimal displaceable molecule [ ] . to sum up, immunosensors are now becoming one of the most widely used analytical techniques, embracing a vast repertoire of analytes that are detected by a diverse range of transducer devices. the enormous potential of immunosensors in clinical diagnosis, environmental analysis, and biological process monitoring has been widely accepted and increasing efforts have been devoted to these fi elds. in particular, with the continual development of transducer technology, laser technology, nano-sized material technology, and antibody engineering technology, immunosensors based on the application of these technologies should be inevitably powerful tools in increasingly wide analytical areas [ ] . aboul-enein, immunosensors in clinical analysis immunosensor principles and applications to clinical chemistry immunosensors for pesticide determination in natural waters immunochemical techniques for environmental analysis: i. immunosensors single-and dual-fractal analysis of hybridization binding kinetics: biosensor application biosensors: fundamentals and applications immunosensors: technology and opportunities in laboratory medicine immunosensor for the diagnosis of chagas' disease ultrasensitive potentiometric immunosensor based on sa and oca techniques for 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matrix suitable for the fabrication of the enzyme electrode by electrodeposition morphology-dependent electrochemistry of cytochrome c at au colloid-modifi ed sno electrodes a novel biosensing interfacial design produced by assembling nano-au particles on amine-terminated plasma-polymerized fi lms application of impedance spectroscopy for monitoring colloid au-enhanced antibody immobilization and antibodyantigen reactions a reusable capacitive immunosensor with a novel immobilization procedure based on , -hexanedithiol and nano-au self-assembled layers piezoelectric immunosensor based on magnetic nanoparticles with simple immobilization procedures core-shell magnetic nanoparticles applied for immobilization of antibody on carbon paste electrode and amperometric immunosensing the use of coated paramagnetic particles as a physical label in a magneto-immunoassay development of a piezoelectric immunosensor for the detection of human erythrocytes detection of viruses and bacteria with piezoimmunosensors piezoelectric immunosensor for the detection of candida albicans microbes piezoelectric crystal biosensor system for detection of escherichia coli piezoelectric immunosensor for the detection of immunoglobulin m formation and structure of self-assembled monolayers self-assembled monolayers for biosensors: a tutorial review quartz-crystal microbalance immunosensor for schistsoma-japonicum-infected rabbit serum biotin-functionalized self-assembled monolayers on gold-surface-plasmon optical studies of specifi c recognition reactions a piezoelectric immunoassay based on selfassembled monolayers of cystamine and polystyrene sulfonate for determination of schistosoma japonicum antibodies molecular recognition between genetically engineered streptavidin and surface-bound biotin enhanced performance of an affi nity biosensor interface based on mixed self-assembled monolayers of thiols on gold microscopic characterization of langmuir-blodgett fi lms incorporating biosynthetically lipid-tagged antibody layer formation of a lipid-tagged single-chain antibody and the interaction with antigen incorporation of lipid-tagged single-chain antibodies into lipid monolayers and the interaction with antigen a novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized fi lms for immunosensors a plasmapolymerized fi lm for surface plasmon resonance immunosensing a reusable piezo-immunosensor with amplifi ed sensitivity for ceruloplasmin based on plasma-polymerized fi lm helical microtubules of graphitic carbon carbon nanotube/tefl on composite electrochemical sensors and biosensors electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides novel immunoassay for toxoplasma gondii-specifi c immunoglobulin g using a silica nanoparticle-based biomolecular immobilization method self-assembled monolayers as the coating in a quartz piezoelectric crystal immunosensor to detect salmonella in aqueous solution fabrication of self-assembled protein a monolayer and its application as an immunosensor a protein a-based orientation-controlled immobilization strategy for antibodies using nanometer-sized gold particles and plasma-polymerized fi lm atomic force spectroscopybased study of antibody pesticide interactions for characterization of immunosensor surface in situ quartz crystal microbalance monitoring of fabЈ-fragment binding to linker lipids in a phosphatidylcholine monolayer matrix: application to immunosensors oriented immobilization of antibodies and its applications in immunoassays and immunosensors use of protein a as an immunological reagent and its application using fl ow injection: a review comparative study of igg binding to proteins g and a: nonequilibrium kinetic and binding constant determination with the acoustic waveguide device site-directed immobilization of glycoproteins on hydrazidecontaining solid supports oriented immobilization of antibodies onto the gold surfaces via their native thiol groups nanogold particle-enhanced oriented adsorption of antibody fragments for immunosensing platforms regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies immunosensors in biology and medicine: analytical capabilities, problems, and prospects potentiometric sensors: aspects of the recent development potentiometric microbiological assay of gentamicin, streptomycin, and neomycin with a carbon dioxide gas-sensing electrode potentiometric enzyme immunoassay for digoxin using polystyrene beads homogeneous potentiometric enzyme immunoassay for human immunoglobulin g ion electrode measurements of complement and antibody levels using marker-loaded sheep red blood cell ghosts potentiometric immunosensor using artifi cial antibody based on molecularly imprinted polymers protein detection with a novel isfet-based zeta potential analyzer ion sensitive fi eld effect transducer-based biosensors improvement of urease based biosensor characteristics using additional layers of charged polymers an isfetbased immunosensor for the detection of β-bungarotoxin a new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by ph-sensitive fi eld-effect transistor probing antigen-antibody binding processes by impedance measurements on ion-sensitive fi eld-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine development of an electrochemical immunosensor for direct detection of interferon-γ at the attomolar level capacitive immunosensor for transferrin based on an o-aminobenzenthiol oligomer layer ultrathin alumina sol-gel-derived fi lms: allowing direct detection of the liver fi brosis markers by capacitance measurement disposable noncompetitive immunosensor for free and total prostate-specifi c antigen based on capacitance measurement direct detection of immunospecies by capacitance measurements capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol fi lms on gold electrodes electrochemical sensors based on impedance measurement of enzyme-catalyzed polymer dissolution: theory and applications real-time monitoring of immunochemical interactions with a tantalum capacitance fl ow-through cell direct detection of biomolecules by electrochemical impedance measurements study of immunoglobulin g thin layers obtained by the langmuir-blodgett method: application to immunosensors on-line monitoring of monoclonal antibody production with regenerable fl ow-injection immuno systems a capacitative immunosensor measurement system with a lock-in amplifi er and potentiostatic control by software a label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon immunobiosensor chips for detection of escherichia colio :h using electrochemical impedance spectroscopy impedance immunosensor based on receptor protein adsorbed directly on porous gold fi lm amplifi cation of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy enzyme immunosenser: iii. amperometric determination of human cherienic gonadotropin by membrane-bound antibody application of redox enzymes for probing the antigen-antibody association at monolayer interfaces: development of amperometric immunosensor electrodes amperometric immunosensor based on polypyrrole/poly(m-phenylenediamine) multilayer on glassy carbon electrode for cytokinin n -(d -isopentenyl) adenosine assay reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen- amperometric immunosensors based on protein a coupled polyaniline-perfl uorosulfonated ionomer composite electrodes application of photoisomerizable antigenic monolayer electrodes as reversible amperometric immunosensors amperometric immunosensor for direct detection based upon functional lipid vesicles immobilized on nanowell array electrode electronic transduction of photostimulated binding interactions at photoisomerizable monolayer electrodes: novel approaches for optobioelectronic systems and reversible immunosensor devices an indirect perfl uorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin an amperometric immunosensor based on nafi onmodifi ed electrode for the determination of schistosoma japonicum antibody development of amperometric and microgravimetric immunosensors and reversible immunosensors using antigen and photoisomerizable antigen monolayer electrodes comparison between the performances of amperometric immunosensors for cholera antitoxin based on three enzyme markers construction of amperometric immunosensors based on the electrogeneration of a permeable biotinylated polypyrrole fi lm amperometric immunosensor for probing complement iii (c ) based on immobilizing c antibody to a nano-au monolayer supported by sol-gelderived carbon ceramic electrode reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase immunosensor for rapid detection of gibberellin acid in the rice grain silver-enhanced colloidal gold metalloimmunoassay for schistosoma japonicum antibody detection chronopotentiometry and faradaic impedance spectroscopy as methods for signal transduction in immunosensors liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplifi cation of antigen-antibody or oligonucleotide-dna sensing processes by the precipitation of an insoluble product on electrodes fiber-optic chemical sensors and biosensors fiber-optic chemical sensors and biosensors surface plasmon resonance sensors: a review surface plasmon resonance-based immunoassays determination of affi nities of a panel of iggs and fabs for whole enveloped (infl uenza a) virions using surface plasmon resonance enhanced performance of a surface plasmon resonance immunosensor for detecting ab-gad antibody based on the modifi ed self-assembled monolayers sensing fatty acid binding protein with planar and fi ber-optical surface plasmon resonance spectroscopy devices surface plasmon resonance immunosensor for the detection of salmonella typhimurium immunosensor for detection of legionella pneumophila using surface plasmon resonance applications of fluorescence in immunoassay fluorescence polarization as a means for determination of fumonisins in maize reagentless and regenerable immunosensor for monitoring of immunoglobulin g based on non-separation immunoassay an array immunosensor for simultaneous detection of clinical analytes development of fl uorescence change-based, reagent-less optic immunosensor conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers clinical applications of chemiluminescence chemiluminescent immunosensor for ca - based on antigen immobilization on a cross-linked chitosan membrane fully automated chemiluminometric assay for hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen) optical fi ber immunosensor based on a poly(pyrrole-benzophenone) fi lm for the detection of antibodies to viral antigen homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin use of a quartz vibrator for weighing thin layers on a microbalance piezoelectric immunosensors -theory and applications piezoelectric quartz crystal biosensors characterization of a quartz crystal microbalance with simultaneous mass and liquid loading specifi c recognition of bacteria by surface-templated polymer fi lms immunophenotyping of acute leukemia using an integrated piezoelectric immunosensor array improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection detection of antisperm antibody in human serum using a piezoelectric immunosensor based on mixed self-assembled monolayers impedance characterization of a piezoelectric immunosensor. part ii: salmonella typhimurium detection using magnetic enhancement amplifi ed mass immunosorbent assay with a quartz crystal microbalance development of immunochips for the detection of dengue viral antigens au nanoparticle-and silver-enhancement reaction-amplifi ed microgravimetric biosensor latex piezoelectric immunoassay detection of agglutination of antibody-bearing latex using a piezoelectric quartz crystal a piezoelectric immunoagglutination assay for toxoplasma gondii antibodies using gold nanoparticles detection of antistreptolysin o antibody: application of an initial rate method of latex piezoelectric immunoassay improvement of latex piezoelectric immunoassay: detection of rheumatoid factor latex piezoelectric immunoassay: effect of interfacial properties polymer agglutination-based piezoelectric immunoassay for the determination of human serum albumin immunoassay of prostatespecifi c antigen (psa) using resonant frequency shift of piezoelectric nanomechanical microcantilever enantioselective sensors based on antibody-mediated nanomechanics invited paper: wireless magnetoelastic resonance sensors: a critical review magnetoelastic immunosensors: amplifi ed mass immunosorbent assay for detection of escherichia coli o :h immunosensor for detection of yersinia enterocolitica based on imaging ellipsometry a label-free multisensing immunosensor based on imaging ellipsometry electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and gm -functionalized liposomes prussian blue modifi ed amperometric fia biosensor: one-step immunoassay for α-fetoprotein capillary electrophoretic enzyme immunoassay with electrochemical detection using a noncompetitive format protein and antibody arrays and their medical applications immunophenotyping of leukemias using a cluster of differentiation antibody microarray interdigitated array microelectrode-based electrochemical impedance immunosensor for detection of escherichia coli o :h antibody arrays for high-throughput screening of antibody-antigen interactions a microarray enzyme-linked immunosorbent assay for autoimmune diagnostics microfl uidic immunosensor systems micro total analysis system (µ-tas) in biotechnology analytical applications of aptamers aptamers: an emerging class of molecules that rival antibodies in diagnostics displacement enzyme linked aptamer assay key: cord- - mxcssjj authors: ishay, yuval; kessler, asa; schwarts, asaf; ilan, yaron title: antibody response to sars‐co‐v‐ , diagnostic and therapeutic implications date: - - journal: hepatol commun doi: . /hep . sha: doc_id: cord_uid: mxcssjj the immune response against sars‐cov‐ is comprised of both cellular and humoral arms. while current diagnostic methods are mainly based on pcr, they suffer from insensitivity. therefore, antibody‐based serological tests are being developed to achieve higher sensitivity and specificity. current efforts in treating sars‐cov‐ infection include blocking of viral entry into the host cells, prohibiting viral replication and survival in the host cells, or reducing the exaggerated host immune response. administration of convalescent plasma containing anti‐viral antibodies was proposed to improve the outcome in severe cases. in this paper, we review some of the aspects associated with the development of antibodies against sars‐cov‐ and their potential use for improved diagnosis and therapy. sars-cov- is an infectious rna virus responsible for causing the covid- disease ( ) . while current diagnostic methods for covid- diagnosis are mainly based on pcr, they suffer from insensitivity. widespread reports of both false positive tests and false negative tests have been reported. therefore, serological tests are being developed to identify patients suffering from covid- , and to assist in identifying subjects who have been diseased and may now be immune to re-infection or to severe disease. the host immune response mounted towards the virus contributes to disease severity. the immune response towards sars-cov- is comprised of both the cellular and humoral arms. current evidence points to the severe manifestation of covid- disease as being driven by inappropriate hyperactivation of the immune system, associated cytokine storm, and end organ damage ( , ) . current efforts for the treatment of covid- include blocking of viral entry into the host cells, prohibiting viral replication and survival in the host cells, or reducing the exaggerated host immune response. however, these strategies have shown limited efficacy ( ) . administration of convalescent plasma was proposed to improve patient outcomes in severe cases. in this paper, we review some of the aspects associated with the development of antibodies against sars-cov- , their biology, potential uses, expected advantage, and disadvantages. sars-cov- is an enveloped, single-stranded rna virus. the viral genome encodes four structural proteins including the spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as other non-structural proteins. the s protein of the sars-cov- consists of two subunits, s and s . acting as a homotrimer, the heavily glycosylated s protein binds its cellular receptor, angiotensin converting enzyme (ace ), present on the pneumocytes and enterocytes, via the c-terminal domain of the s subunit, in the receptor binding domain (rbd) region ( , ) . extending from the viral membrane, the s protein extends outward from the virion. while the s subunit extends furthest beyond from the virus membrane, the inner s subunits consists of a mostly helical structure, leading towards the viral membrane. the interaction of the s -ace receptor leads to conformational changes in the helical s subunit. the next event in viral binding and entry includes cleavage of the s /s protein subunits by cellular proteases. this proteolytic activity may be performed via furin protease, a feature not unique to the sars-cov- among the coronaviruses, but absent in sars-cov ( ) . the cleaving protease, dictating the exact exposed viral amino acid sequence, also determines the pattern of viralcell fusion ( , ) . the release of newly constructed virions, and the later activities of these new virions are also dependent on specific protease activity ( ) . among the sites enumerated in this description, several appear as attractive targets for biologically active antibodies. of note, while new data is continuously and vigorously obtained, specifically regarding sars-cov- , much of the functional data regarding coronavirus activity and mechanisms come from the research on sars-cov and mers-cov. this appears particularly poignant where homologies in the structure and function between these viruses are sought. while sequence and biological similarities are common, major differences exist, influencing virus function and antibody biology. these range from matters such as cleavage by similar proteases, though sars-cov- shows unique furin sensitivity, to receptor binding, where it shares the affinity towards ace with sars-cov, through highly conserved rbd residues( ). the final event of protective and effective antibody production is the differentiation of b-cells into plasma cells, a change accompanied by robust antibody production. a fraction of these cells will differentiate into memory bcells, allowing for an early antibody response upon re-infection, and have been demonstrated after sars-cov infection ( ) . presumably, the "first contact" of the sars-cov- with the immune system occurs upon introduction of viable viral particles into the airways. the very first responding part of the immune system may be the epithelial cells themselves, both acting as antigen presenting cells (apcs) ( ) and internally expressing antiviral proteins, specifically type-i interferons ( ) . type-i interferon signaling is usually initiated via toll like receptors (tlrs). variance in the vulnerability to the virus, namely men being more vulnerable, has been attributed partially to a superior tlr signaling in women, possibly resulting in the enhanced antibody production ( ) . notably, tlr functions in b-cells as well,and may contribute to enhanced function and differentiation of plasma cells ( ) . following initial contact with epithelium, innate immune cells come in contact with the virus and with infected cells. the superficial intraepithelial dendritic cells (dcs) in the lungs adjacent to the airways are required for antibody production( ). after antigen encounter, they will move to the regional lymph nodes and help trigger robust antibody production by activation of cd "follicular helper" t-cells, supporting b-cell function ( ) . some this article is protected by copyright. all rights reserved dc functions, including type-i interferon secretion in response to viral stimulation, is also dependent on tlr signaling ( ) . while existing research is focused on the endogenic immune response to sars-cov- , and its possible beneficial manipulations, isolation of neutralizing antibodies (nabs) from the infected persons or laboratory manufacturing of these antibodies is another subject of intense interest. monoclonal antibodies (mabs) with some neutralizing activities were demonstrated to occur in the infected human sera ( ) . nabs may be defined in various ways; commonly as the antibody concentration required to prevent or decrease the infectivity ( ) . the most attractive antibodies are those targeting the s protein, whether in the rbd or other regions, including the s /s proteolytic cleavage site ( ) . it is plausible that the antibodies targeting these sites will block the essential viral functions including viral-antigen binding (expected from s -rbd antibodies) and/or interfere with s protein-mediated viral fusion or cell entry ( ) ( ) ( ) . multiple specific regions in sars-cov- show high homology to the sars-cov virus, suggesting potential b and t cell epitopes for sars-cov- ( ) . a set of b cell and t cell epitopes were derived from the s and n proteins which, excluding notable differences, are generally conserved between sars-cov and sars-cov- . the lack of mutations in these identified epitopes allows assessment of possible sars-cov- immune targets ( ) . this study showed no mutations occurred between sars-cov and sars-cov- in these sequences, confirming the possibility of antibody cross reactivity and humoral immunity. in spite of this high homology, cross-reactivity of sars-cov antibody is limited between two viral s proteins ( , ) . murine polyclonal sars-cov antibodies directed against the s protein inhibited sars-cov- entry into cells, indicating that the cross-nabs targeting conserved s epitopes can be produced ( ) . s -targeting mabs from immunized transgenic mice expressing human ig variable heavy and light chains can neutralize sars-cov- and sars-cov infections ( , ) . in a previously mentioned trial, sars-cov- rbd-specific mabs were generated, among which, only two clones showed significant blocking of viral entry, associated with a high competitive capacity against ace receptor binding ( ) . similar results were observed in the studies using sera from recovered sars and covid- patients, where limited crossneutralization occurred, suggesting that cross nabs are either incompletely reactive or insufficient for disease prevention ( ) . prior to and concurrently with the isolation of specific antibodies, sars-cov s -specific serum from convalescent sars patients or from animals was proposed to cross-neutralize the sars-cov- infection by reducing s proteinmediated sars-cov- entry ( ) . cross reactivity of the antibodies from sars-cov- patients against the s proteins, but not against the rbd of sars-cov and mers-cov, has been documented. the roles played by rbd in the invasion of sars-cov- into host cells make it a potential target for nabs. blocking of binding between the rbd and its respective receptor may restrict the conformational change of s, or hamper the s -mediated membrane fusion, thereby inhibiting the viral infection of host cells ( ) . the human nabs s . and m were isolated from sars-covinfected patients. they neutralize sars-cov infection by interacting with the rbd and by blocking the binding between viral rbd and ace receptor ( ) . the sars-cov rbd-specific human nabs, cr binds sars-cov- rbd with high affinity and recognizes an epitope on the rbd that does not overlap with the ace -binding site ( ) . the s . and s . mabs can neutralize the infectious clones of sars-cov, and protect the mice against four different homologous and heterologous sars-cov strains ( , ) . of note, such mabs produced in the chimeric mouse cells and originating from sars-cov patients were shown to neutralize sars-cov- virus particles by an ace -independent mechanism, which probably has to do with s protein fusion or proteolysis and preventing viral fusion ( ) . while these studies hold both promise and interest, isolation and analysis of neutralizing antibodies remains a difficult task. a majority of patients recovered from covid- showed high titers of sars-cov- s -specific igg antibodies when tested by enzyme-linked immunosorbent assay (elisa) ( ) . however, only out of these patients manifested effective blockade of sars-cov- rbd binding to hace when tested in vitro ( ) . the transient and dynamic conformational states of the s protein have been suggested to provide a narrow window for an exposure of the immunogenic epitopes of rbd to b lymphocytes ( ) . early and transient peak levels of anti-s antibody response were associated with a less favorable outcome for the patients, compared with a more delayed and sustained response ( ) . the phage display method, allowing rapid and wide display of proteins directly correlated to their associated genes, can detect nabs against sars-cov from both naïve and immune antibody libraries, capable of blocking the binding of s domain, thereby showing virus neutralization and prophylaxis capability either in vitro or in the animal models ( , , ) . another method, possibly allowing the production and utilization of existing nabs, may include the use of epstein-barr virus (ebv) transformation of human b cells to improve the isolation of nabs from the memory b cells harvested from the sars-cov infected patients ( ) . transgenic mice with human immunoglobulin genes are being developed to produce nabs against sars-cov by antigen immunization, which are effective for virus prophylaxis in animal models ( , ) . cloning of human mabs using samples from covid- -recovered patients whose sera showed hace receptor binding inhibition has been reported ( ) . following antibody cloning, three pairs of igg variable heavy chain and light chain inserted expression plasmids were expressed and named as mab- b , mab- d , and mab- b . all three mabs bind to the rbd protein. mab- b and mab- d blocked sars-cov- rbd-hace interaction and neutralized a sars-cov- s pseudotyped lentiviral particle ( ) . the mab- b and mab- d neutralized pseudovirus entry into host cells ectopically expressing hace ( ) . several nabs, such as b , f , and e , towards epitopes on sars-cov s manifested neutralization properties ( , ) . this article is protected by copyright. all rights reserved nucleocapsid-specific antibodies have been also demonstrated in the sera of infected patients. most studies assessing nucleocapsid antibodies have not differentiated these antibodies from other antibodies directed against sars-cov- studies that have seem to show similar kinetics to that of the general antibody response ( ) . no studies have shown the occurrence of definitive nabs directed at the n protein or the nature of the immune response triggered by such antibodies. serum igg, igm, and iga antibodies against sars-cov appeared in the patients after primary sars infection ( ) . data on the production of igg and igm is important for improved diagnosis of covid- ( ) . several studies have described the dynamics of antibody production in these patients. while it is too early to definitively summarize the characteristics of antibody dynamics, certain conclusions seem consistent across these studies. broadly, antibody titers increase and the prevalence of viral rna decrease as time progresses from the symptomatic disease onset ( , ) . elisa-based diagnostic kits often report a specificity of ~ % ( ), with some trials reporting higher percentage ( ) . while this is an impressive figure by itself, it may yield a relatively poor positive predictive value (ppv) when employed on a large scale to a disease with relatively low prevalence. elisa tests were argued to be efficient when trying to augment the sensitivity of testing of close contacts ( , ) , or deciding to allow a person to leave from quarantine. this specificity may be further reduced when testing a person recently exposed to the milder coronaviruses circulating within humans and livestock. however, to our knowledge, this question has not been directly assessed. igg and igm antibodies may appear simultaneously or sequentially, with cases of igm antibodies appearing last being described in some of the studies ( ) . conversion from seronegativity to seropositivity is likely to occur between - days after the onset of symptoms. data from some of these studies show that the patients with more severe illness were more likely to mount a high-titer and high-affinity antibody response, which was not necessarily associated with a reduction in the viral rna assayed from their blood ( ) . this is supported by the reports of recurring pcr positivity after igg seroconversion ( ) . if these studies become the prevalent findings, they may stand in a stark contrast to well established viral disease behaviors where high igg levels are thought to denote virtual immunity to the disease, allowing, at most, a mild manifestation upon re-exposure. it seems that in covid- , as our current understanding stands, antibody titers should be thought of as the disease markers and not as the definitive markers of immunity or disease resolution in the actively ill. in the antibody detection, different elisa kits, based on the recombinant sars-cov- nucleocapsid protein (rn) and recombinant spike protein (rs), show variable results. in a study of patients with confirmed covid- , % were diagnosed with rn-based igm, % with an igg, % with rs-based igm, and % with igg tests. the positive rates for rn-based and rs-based elisa detections were % for igm and % for igg. the sensitivity of the rs-based elisa for igm was higher than that of the rn-based test. here also, the antibody positivity increased as disease time progressed ( ) . another stratum of results expected from antibodies is the identification of immune and recovered persons who may be able to work in the critical locations during the times of pandemic. the ability to definitively identify specific nabs in the serum of recovered patients could also allow identifying the potential plasma donors for the development of passive immunization, and may assist in evaluating the effectiveness of various treatments in addition to assisting in determining the prognosis ( ) . most convalescent plasmas obtained from individuals who recover from covid- do not contain high levels of nabs. a recent analysis of covid- convalescent individuals evaluated plasmas collected an average of days after the onset of symptoms showing variable half-maximal pseudovirus neutralizing titers below : in % and below : , in %. only % showed titers above : , . expanded clones of rbd-specific memory b cells expressing closely related abs in different individuals were identified. the abs were directed against three distinct epitopes on rbd. rare but recurring rbdspecific antibodies with potent antiviral activity were identified in all subjects recovered ( ) . the relevance of the titers for the clinical effect are yet to be determined. a recent review analyzed the diagnostic accuracy of antibody tests for sars-cov- infection, for assessing past infections and for use in seroprevalence surveys ( ) . a total of publications reporting cohorts with , samples, of which were from cases of sars-cov- infection, were evaluated. substantial heterogeneity in sensitivities of iga, igm and igg abs, or combinations thereof, for results aggregated across different time periods postsymptom onset. pooled results for igg, igm, iga, total antibodies and igg/igm showed low sensitivity during the first week since onset of symptoms, rising in the second week and reaching their highest values in the third week. the sensitivity of antibody tests was proposed to be too low in the first week since symptom onset, to have a primary role for the diagnosis, but were suggested to have a role complementing other testing in individuals presenting later, when rt-pcr tests are negative. antibody tests are useful for detecting previous sars-cov- infection if used or more days after the onset of symptoms ( ) . several currently-available covid- antibody tests that are used in diagnostics and epidemiology, with a focus on their strengths and weaknesses are summarized in table . the lack of specific sars-cov- -targeted treatments and vaccines poses great challenges for the management of patients with severe illness. igg levels against sars-cov, drawn from affected patients, reach peak serum concentration during the convalescent phase and are reduced following recovery ( ) . while the capacities of antibodies to neutralize the virus were highly variable in the required concentration, some of them indeed showed such capability, and have been shown to provide protection against re-infection in a mouse model ( ) . use of convalescent plasma and development of nabs are attractive methods for the treatment of viral infections ( , ) . blocking mabs with high antigen specificity were proposed as potential candidates for neutralizing infections ( ) ( ) ( ) . convalescent plasma has intermittently emerged this article is protected by copyright. all rights reserved during the last few decades as a treatment for various infectious diseases ( ) ( ) ( ) , enjoying attention whenever diseases prove resistant to more conventional treatment methods. plasma-derived nabs can provide passive immune responses to viral infections and were effective in patients with severe illnesses caused by other viruses ( , ) . a meta-analysis showed that the mortality was reduced after receiving various doses of convalescent plasma in the patients with severe acute respiratory infections, with no adverse events or complications after treatment ( ) . antibodies from convalescent plasma were proposed to reduce the viremia by enhancing viral clearance, blocking infection of new cells, and contributing in the clearance of infected cells ( , , ) . during the sars epidemic, severely ill patients who deteriorated despite the treatment with methylprednisolone were given convalescent plasma at around th day of the disease onset. earlier plasma administration correlated with a better prognosis and higher rate of hospital discharge at day ( ) . convalescent plasma or immunoglobulins were effective in sars patients whose condition continued to deteriorate. some studies suggested a shorter hospital stay and lower mortality rate following convalescent plasma administration ( , , ( ) ( ) ( ) ( ) ( ) . a similar trend for the treatment timing was described in patients with lassa fever in nigeria treated with convalescent plasma ( ) . the empirical use of convalescent plasma for ebola virus disease showed some positive results ( ) ( ) ( ) . experimental and clinical data on the use of convalescent plasma products and humanized monoclonal antibodies for h n influenza infection have also shown positive outcomes, and this treatment was proposed as a mean for overcoming anti-viral drug resistance ( , , ) . in a study involving patients with severe pandemic influenza a (h n ) virus infection, administration of convalescent plasma reduced respiratory tract viral load, serum cytokine response, and mortality ( ) . a prospective cohort study during the pandemic showed reduction in the relative risk of mortality in the patients treated with convalescent plasma, demonstrating reduction of the viral loads without any adverse effects ( ) . a randomized trial of convalescent plasma failed to achieve its primary end point, a reduction of mortality; however, a subgroup multivariate analysis performed on of the patients enrolled in the trial demonstrated that h-ivig treatment was the only factor independently associated with reduced mortality ( ) . development of nabs against sars-cov- was proposed as a method for developing therapeutic agents for covid- ( , , , ) . several sars-cov- proteins (discussed above) prove attractive targets for nabs. the sars-cov- s protein is a target for developing nabs to block its binding and fusion ( ) . currently, no sars-cov- -specific nabs have been reported ( ) . however, polyclonal antibodies from recovered sars-cov- -infected patients are being used to treat the patients with severe infections. while many patients will develop an antibody response following their illness, specific characterization of these antibodies and their properties as nabs has yet to be determined ( , ) . early administration of convalescent plasma was advised in order to maximize its viral clearance effect ( ) . this article is protected by copyright. all rights reserved plasma collection is done via apheresis. in order to qualify for the donation, the donor must meet several conditions: diagnosis of prior covid- infection confirmed by pcr, donation needs to take place - days after resolution of the symptoms followed by two consecutives negative pcr results, donors need to be tested for absence of transmissible pathogens, donation should be done from male or nulliparous female donors, with no previous exposure to blood products in order to minimize the risk of transfusion associated acute lung injury (trali). plasma ( - ml) is donated according to the abo compatibility. pathogen inactivation measures need to be undertaken ( ) . it is advised to administer up to two units of plasma, possibly from two different donors ( ) . several studies that described the administration of convalescent plasma to critically ill covid- patients suggested post transfusion viral elimination and clinical improvement. a study of critically ill patients (n= ) reported clinical improvement in patients' status and laboratory indication of viral clearance for up to days post transfusion of two consecutive doses of convalescent plasma (total ml).( ) three of the patients were on mechanical ventilation and two on ecmo, and the treatment was provided between - days after hospitalization, following which improvement in fever, pao /fio ratio, and viral clearance were noted. three patients were discharged from the hospital, and two were in stable condition at the end of the follow-up period. although a clinical effect was obtained, the delay of up to three weeks in the administration, and the concurrent use of other therapies, make it difficult to assess the effect of plasma ( ) . administration of convalescent plasma in six critically ill patients was followed by discontinued viral shedding three days after infusion without reducing the mortality ( ) . a study in six covid- patients showed clinical, radiological, and laboratory improvement following administration of abo-compatible convalescent plasma indicating that this therapy is effective and specific ( ) . in a study of severe patients administration with ml of convalescent plasma, showed improved clinical, laboratory, and radiological status without severe adverse effects ( ) . in this study, the antibody titers of donor's plasma were assessed and found to be elevated in the majority of donors, along with a concurrent increase in nabs titers in the patients' sera following transfusion. treatment within two weeks of initial symptoms has improved the response ( ) . differences in the outcomes between the studies may reflect temporal variations of administration including the time lag between plasma donation and administration as well as the time from disease detection to treatment. safety evaluation of candidate antibodies must not be overlooked. although antibodies are generally protective, the antibody-dependent enhancement (ade) phenomenon of viral infections is documented for dengue virus and other viruses ( ) . in sars-cov infection, ade is mediated by the engagement of fc receptors (fcrs) expressed on various immune cells, including monocytes, macrophages and b cells ( ) . pre-existing sars-covspecific antibodies were proposed to promote viral entry into fcr-expressing cells. internalization of virus-antibody immune complexes may induce inflammation and tissue injury by activating myeloid cells via fcrs ( ) . this article is protected by copyright. all rights reserved figure presents several putative and proven nabs interactions in covid- , such as antibody targets and functions including those associated with disruption and non-disruption binding mechanisms, and those targeting the virus itself. in addition, non-neutralizing antibodies, cross reactive antibodies, and antibodies with low specificity or low titers, which are unable to act as nabs, are also generated. several large trials using convalescent plasma are being conducted ( ) . identifying and cloning mabs that target viral proteins to block the entry into host cells is being explored for preventing and treating covid- ( , ) . computational simulation of antibody-antigen complexes can improve the design of these therapies. key residues between rbd and nabs can be identified, and models are being used to assess the interaction between s protein and human ace or antibodies ( , , ( ) ( ) ( ) ( ) . several methods for improving the effectiveness of convalescent plasma or nabs are being considered. the outcomes of passive convalescent plasma therapy from recovered donors are unpredictable due to variability among the donors in both the levels and types of antibodies ( ) . appropriate selection of the donors is required for improving the quality of the collected plasma. assessment of the antibody titers needs to be performed prior to harvesting due to a marked variability in titers among the donors. titers correlate with the disease severity, timing of donation, use of steroids during acute illness ( , ) , and quality of antibodies (i.e. whether they are nabs or not) nonwithstanding. timing of plasmapheresis is a major factor as lower levels of antibodies are detected within the first two weeks following recovery ( ) . more data is required on the amount of virus neutralization by antibodies upon exposure to convalescent plasma. in vitro testing for neutralizing and/or crossneutralizing activity, and in vivo evaluation in available covid- animal models for protective efficacy, along with preclinical studies and clinical trials testing the safety and efficacy, are needed for optimizing this therapeutic option ( ) . the gender of the donor also plays a role in mounting a significant response. the degree of activation of the immune cells is higher in women than in men, which correlates with the triggering of tlr and production of proinflammatory cytokines. tlr is expressed in innate immune cells, which recognize single strand rna virus by promoting the production of viral antibodies and generation of il- and il- inflammatory cytokines. tlr is higher in women than in men and its expression may lead to better immune responses and increased resistance to viral infections ( ) . pairing hla-typing with covid- was proposed to improve the assessment of disease severity and assist in preferred donor selection ( ) . the use of hyper-immune globulin rather than whole plasma was proposed for improving efficacy and validity of the therapy. the main advantages are associated with an ability to provide the patients with controlled quantities of antibodies in lower volumes ( ) . similar techniques for concentrating this article is protected by copyright. all rights reserved antibodies are being used for the treatment and prevention of other diseases ( ) . this is similar to the concept of using hyper-immune globulin for various indications, including viral diseases in immunocompetent and immunocompromised hosts ( ) ( ) ( ) . a "cocktail antibody approach" for sars-cov- was proposed based on the studies suggesting that the combination of antibodies from diverse donors may exert a synergistic neutralization effect ( ) . a mixture of two antibodies showed a synergistic neutralization effect due to recognition of different epitopes on rbd ( ) . the use of immune adjuvants may also improve the response to the antibodies ( ) . sphingolipid-based adjuvants, when administered with antibodies, augmented the anti-viral response ( ) and improved the systemic anti-inflammatory effects of antibodies ( ) . the use of hyper-immune bovine colostrum comprised of antibodies and sphingolipids was effective in reducing systemic inflammation ( ) ( ) ( ) . mode of antibody administration may also have an impact on the effect of antibody-based therapy. oral administration of antibodies ameliorated viral-mediated chronic inflammation via promotion of regulatory t cells ( ) , and oral administration of viral antigens augmented an anti-viral immunity while reducing the inflammation ( , ) . the data on the possible harmful effects of antibody-mediated immune response in the development of pulmonary complications of sars-cov is controversial. several patients died of sars manifested strong nabs responses and pulmonary inflammation, suggesting that the nabs could be associated with the deterioration of the lung disease ( , ) . similar notions have been proposed as explaining the more severe phenotype of covid- prevalent in china. this may be related to the higher degree of exposure to milder coronaviruses and a "priming" of the immune system by pre-existing antibodies, leading to immune dysfunction and over-function ( ) . this notion is supported by the mild disease manifestations in the patients with agammaglobulinemia ( ) . previous exposure to coronaviruses may also explain a relatively high prevalence of spike protein-reactive cd cells in the healthy donors in a study ( ) . a major obstacle for implementing immune-based therapies for the viruses, including the administration of mabs, is associated with the development of viral resistance due to the immune evasion mechanisms, which the virus generates in response to the immune-pressure imposed on it by the immunomodulatory agents ( ) . prolonged exposure to the anti-viral drugs is associated with drug resistance, leading to persistent viremia or severe disease. in cases where anti-viral treatment is highly effective leading to viral elimination, resistance is less likely to occur. however, immunotherapy, including the administration of antibodies, are associated with selective pressure that may result in rapid viral and host adaptations leading to resistance to the therapy ( ) . both host and viral factors are associated with the development of resistance. viral-related tools include mechanisms of viral replication, genomic inference, and high rates of viral mutations ( , ) , ( ) . an immune adaptation process towards antibody-induced pressure on the virus or on anti-viral humoral and cellular responses may limit the efficacy and longevity of these therapies. combination of several potent nabs could improve the sensitivity to neutralization ( ) . methods for overcoming resistance by implementing host-tailored variability are being developed based on the data generated from the use of these methods for improving the effects of other immunomodulatory drugs ( ) ( ) ( ) ( ) . these include implementing artificial intelligence methods for overcoming host compensatory responses in sepsis and its sequela ( ) , and for improving the effects of adjuvants ( ) . algorithmcontrolled treatment regimens are now being used in several clinical trials for overcoming drug resistance (nct ; nct ). the lack of accurate diagnostic and effective therapeutic methods for the sars-cov- -infected patients led to the need of developing humoral-based approaches. while this approach holds promise, more data is needed for optimizing the antibody-based diagnosis, and for improving the implementation of convalescent plasma and other antibody-based therapies. the potential development of effective vaccines will benefit from the results achieved from these diagnostic and therapeutic attempts. immunotherapeutic methods are expected to require targeting the cellular arm of the immune system either in addition or as part of the design of antibody-based approaches, mainly for alleviating the immune-mediated target organ damage in covid- . this article is protected by copyright. all rights reserved sars-cov- : severe acute respiratory syndrome coronavirus ; s protein: spike protein; s and s : s protein subunits; rbd: receptor binding domain; abs: antibodies; nabs: neutralizing antibodies; and ace : angiotensin-converting enzyme . how to reduce the likelihood of coronavirus- (cov- or sars-cov infection and lung 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causing an outbreak of pneumonia (covid- ) this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved accepted article key: cord- -wgt kg f authors: diego-martin, borja; gonzález, beatriz; vazquez-vilar, marta; selma, sara; mateos-fernández, rubén; gianoglio, silvia; fernández-del-carmen, asun; orzáez, diego title: pilot production of sars-cov- related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: wgt kg f the current covid- crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. a critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. on the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. in this work we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. for this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against sars-cov- in nicotiana benthamiana. for the design of the antibodies we took advantage, among other data sources, of the dna sequence information made rapidly available by other groups in preprint publications. mabs were all engineered as single-chain fragments fused to a human gamma fc and transiently expressed using a viral vector. in parallel, we also produced the recombinant sars-cov- n protein and its receptor binding domain (rbd) in planta and used them to test the binding specificity of the recombinant mabs. finally, for two of the antibodies we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. our results indicate that gram amounts of anti-sars-cov- antibodies could be easily produced in little more than weeks in repurposed greenhouses with little infrastructure requirements using n. benthamiana as production platform. similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses. the current pandemic is evidencing several weaknesses in our ability to respond to a global crisis, one of which is the insufficient and heavily centralized distribution of the world manufacturing capacity of bioproducts such as antibodies, vaccines and other biological reagents, specially proteins. since it is economically impracticable to ensure readiness by maintaining inactive infrastructures during large periods of normality, the development of dualuse systems has been proposed, which would serve regular production needs in normal times but could be rapidly repurposed to strategic manufacturing requirements in times of crisis. ideally, such adaptable infrastructures should be widespread to serve local demand in case of emergency. recombinant protein production in plants is a technologically mature bioengineering discipline, with most current plant-based bioproduction platforms making use of non-food crops, mainly the nicotiana species tabacum and n. benthamiana as biomanufacturing chassis (moon et al., ; capell et al., ) . n. benthamiana is most frequently used in association with agrobacterium-mediated transient expression, also known as agroinfiltration, a technology that dramatically reduces the time required for product development. briefly, agroinfiltration consists in the massive delivery of an agrobacterium suspension culture to the intercellular space of plant leaves, either by pressure, using a syringe (small scale), or applying vacuum to plants whose aerial parts have been submerged in a diluted agrobacterium culture (large scale). agrobacterium transfers its t-dna to the cell nucleus, therefore massively reprogramming the plant cell machinery towards the synthesis of the t-dna-encoded protein(s)-of-interest. transient expression of the transgene is often assisted by self-replicating deconstructed virus vectors that amplify the transgene dose, thus boosting protein production by several orders of magnitude (gleba et al., ) . other systems, such as the peaq system, rely on viral genetic elements for boosting expression without recurring to viral replication (sainsbury et al., ). transient expression in n. benthamiana has become the standard in plant-based recombinant protein production due to a unique combination of advantages, with speed and high yield as the most obvious ones. maximum production levels in the g/kg fresh weight (fw) range for certain highly stable proteins such as antibodies have been reported (marillonnet et al., ) . regarding speed, the in-planta incubation times required to obtain maximum yield of recombinant protein are no more than two weeks. an important, often insufficiently highlighted feature of n. benthamiana transient expression is its relatively small infrastructure requirements, partially overlapping with those employed in more conventional, medium/high-tech indoors agriculture, such as hydroponics, vertical farming, etc. (buyel, ) . in this context, when confronted with the covid- crisis, we decided to exercise a partial reorientation of the activities in our academic lab, which is equipped with a multipurpose glass greenhouse facility, towards the production of sars-cov- antigens and antibodies against the virus. here we describe the recombinant production, purification and analysis of six anti-sars-cov- monoclonal antibodies at laboratory scale, plus a pilot upscaling of two of those six antibodies. next to production scale, a critical parameter to assess was the response time. the process described here started in mid-april with the selection of literature-available antibody variable sequences and finalized nine weeks later with approximately . g of anti-sars-cov- antibody (ab) produced in modular badges of n. benthamiana plants and formulated as one litre of ab-enriched plant apoplastic fluid. based on this experience, we estimate that the same process can be reduced up to - weeks with small pre-adaptations, a remarkably short reaction time for a de novo antibody production system. absolutely key for this fast reaction is the immediate availability of scientific data including antibody sequences in pre-print repositories. this is in our opinion one of the most positive lessons that can be extracted from the covid- crisis. we discuss here the possible applications of the fast plant-produced antigens and antibodies in diagnostics and therapy and propose the repurpose of high-tech agricultural facilities as an alternative for decentralized biomanufacturing in times of crisis. both, nicotiana benthamiana wild type plants and , -xylosyltransferase/alpha , fucosyltransferase (Δxt/ft) rnai knock down lines (strasser et al., ) were grown in the greenhouse. growing conditions were °c (light)/ °c (darkness) with a -h-light/ -hdark photoperiod. all sequences were cloned and assembled using the goldenbraid (gb) assembly system https://gbcloning.upv.es) . antibody sequences were obtained from literature (see table ). all antibodies were cloned as single chain antibodies fused to the human igg fc domain. those antibodies derived from synthetic or camelid single domain vhh libraries (sybody , sybody and nanobody ) were designed as direct fusions. cr , cr and cr human monoclonal antibodies were redesigned as single chain variable fragment (scfv) by connecting the variable light (vl) and heavy (vh) chains with a ggggsggggsggggssgggs peptide linker. antibody sequences were codon optimised for n benthamiana with the idt optimization tool at http://eu.idtdna.com/codonopt . the sars-cov- antigen sequences used (n protein, yp_ . ; and s-protein rbd domain, yp_ . , aa - ) derive from the wuhan strain nc_ . four different versions of rbd were designed corresponding to (i) the native sequence with a cterminal xhis-tag or (ii) an n-terminal xhis-tag and a c-terminal kdel sequence for er retention, and (iii and iv) their corresponding n. benthamiana codon optimised counterparts, using the same tool as above. dna sequences were domesticated as level phytobricks for gb cloning and ordered for synthesis as double-stranded dna fragments (gblocks, integrated dna technologies). gblocks were first cloned into the domestication vector pupd (vazquez-vilar et al., ) in a bsmbi golden gate restriction/ligation reaction ( °c - min, x ( °c - min / °c - min), °c - min, °c - min). the ligation product was transformed into e. coli top electrocompetent cells and positive clones were verified by restriction digestion analysis and sequencing. pupd level phytobricks were then cloned into the expression vectors pgreen sp-higg (antibody sequences), pcambiav (rbd sequences) or pcambiav (n sequences). pgreen sp-higg is a pgreen vector based adaptation of the the magnicon® ' provector pich (icon genetics) that is designed for bsai cloning of gb (b -b ) standard parts as in-frame fusions with the tobacco ( - )-beta-glucanase signal peptide and the human igg fc domain. similarly, pcambiav and pcambiav are pcambia based adaptations of the magnicon® ' provector pich that are designed for bsai cloning of gb standard parts as in-frame fusions with tobacco ( - )-beta-glucanase signal peptide (pcambiav , for expression of secreted proteins) or without any subcellular localization signal (pcambiav , for expression of cytoplasmic proteins). assembly reactions were performed as above, and the ligation reactions were transformed into e. coli top electrocompetent cells. positive clones were verified by restriction digestion analysis. all level parts generated in this work are listed in supplementary table . for transient expression in n. benthamiana, the plasmids were transformed into agrobacterium tumefaciens strain gv c c by electroporation. the same strain but carrying the psoup helper plasmid was employed to allow the replication of the pgreen vectors which encode the antibodies. overnight grown exponential cultures were collected by centrifugation and the bacterial pellets were resuspended in agroinfiltration solution ( mm mes, mm mgcl , µm acetosyringone, ph . ) and incubated for h at rt in a horizontal rolling mixer. for small scale agroinfiltration, culture optical density at nm was adjusted to . with agroinfiltration solution and the bacterial suspensions harbouring the ' antibody or antigen modules, the integrase (pich ), and the ' module (pich ) were mixed in equal volumes. control samples were agroinfiltrated with pich _dsred and integrase module. agroinfiltration of to -week-old n. benthamiana plants was carried out through the abaxial leaf surface using a ml needle-free syringe (becton dickinson s.a.). for pilot scale production, the bacterial suspensions were prepared as above except that a lower od was used ( . for sybody agroinfiltration and . for nanobody agroinfiltration). additionally, for sybody agroinfiltration, a bacterial suspension of pich _dsred, a magnicon® ´module encoding the fluorescent protein dsred, was added to the final agrobacterium infiltration solution in a ratio : : . : . (pich :pich :pgreensp-sybody -higg :pich _dsred). delivery of agrobacterium to the plant cells was carried out by vacuum infiltration in a vacuum degassing chamber (model dp , applied vacuum engineering) provided with a l infiltration tank. the aerial part of whole plants (seven plants at a time) was immersed into the agrobacterium infiltration solution; vacuum was applied for min at a vacuum pressure of . bar and then slowly released. days post-vacuum agroinfiltration leaves were excised and then infiltrated with mm phosphate buffer ( . mm nah po , . mm na hpo . h o, ph ), without (sybody ) or with (nanobody ) . mm pmsf (sigma-aldrich, # ), following the same procedure as the vacuum agroinfiltration. after eliminating the buffer excess with tissue paper, the leaves were introduced into mesh zipped bags and then centrifuged using a portable cloth dryer orbegozo sc . thus, the apoplastic fluid was obtained from the drain tube. the apoplastic fluid was centrifuged ( min, x g, at °c) to remove any cell debris and agrobacterium, the supernatant was collected and then fractions of ml were concentrated times using kda amicon ultra- k centrifugal filters (millipore) after centrifugation ( min, x g, at °c). protein crude extracts were obtained by homogenizing ground frozen leaf tissue with cold pbs buffer ( mm nah po , mm na hpo . h o, mm nacl, ph . ) in a : (w/v) and were centrifuged at rpm for min at °c. for antibody purification, g of ground agroinfiltrated tissue were extracted in ml of cold mm phosphate buffer. samples were centrifuged at x g for min and the supernatant was transferred to a clean tube and further clarified by filtration through a . µm membrane filter. the recombinant antibodies were purified by affinity chromatography with protein a agarose resin (abt technology) following a gravity-flow procedure according to the manufacturer's instructions. mm citrate buffer ph was used for elution and m tris-hcl ph was used for neutralization of the eluted sample ( . µl for each µl elution fraction). purified antibodies were quantified using the bio-rad protein assay following the manufacturer's instructions and using bsa for standard curve preparation. the n. benthamiana leaves infiltrated with the different sars-cov- proteins were collected (rbd) or - (n protein) days post infiltration (dpi). leaves were frozen in liquid nitrogen and stored at - °c until used. protein extraction was performed using - g of ground frozen tissue in volumes of coldextraction buffer. three different buffers were tested as a first approach, in order to optimize the purification yields. buffer a: pbs buffer with mm imidazole, ph . buffer b: buffer a supplemented with % triton x- , and buffer c: buffer b supplemented with % glycerol, % sucrose and . % -β-mercaptoethanol. samples were vigorously vortexed and centrifuged at x g for min at °c. the supernatant was carefully transferred to a clean tube and filtered through a . µm syringe filter. protein purification was carried out by ni-nta affinity chromatography as described in (fernandez-del-carmen et al., ) . purified proteins were quantified using the bio-rad protein assay following the manufacturer's instructions and using bsa for standard curve preparation. proteins were separated by sds-page electrophoresis on nupage % bis-tris gels (invitrogen) using mes-sds running buffer ( mm mes, mm tris-base, . mm sds, mm edta, ph . ) under reducing conditions. gels were visualized by coomassie blue staining. for western blot analysis, proteins were transferred to pvdf membranes (amersham hybond™-p, ge healthcare) by semi-wet blotting (xcell ii™ blot module, invitrogen, life technologies) following the manufacturer's instructions. blots were blocked with % ecl prime blocking agent (ge healthcare) in pbs-t (pbs buffer supplemented with . % (v/v) tween- ). for anti-sars-cov- antibody detection, the blots were incubated with : hrp-conjugated rabbit anti-human igg (sigma-aldrich, #a ). for sars-cov- antigen detection the blots were incubated with : anti-his mouse monoclonal primary antibody (qiagen, # ) and then incubated with : peroxidase labelled anti-mouse igg secondary antibody (ge healthcare). blots were developed with ecl prime western blotting detection reagent (ge healthcare) and visualised using a fujifilm las- imager. the overnight coating of costar well eia/ria plates (corning) was carried out at °c with µl of µg/ml rbd (raybiotech, # - ) or bsa (used as control) in coating buffer ( mm na co , mm nahco , ph . ). after washes with µl of pbs, the plate was blocked with µl of a % (w/v) ecl advance blocking reagent (ge healthcare) solution in pbs-t (pbs supplemented with . % (v/v) tween- ) for h at rt. the plate was washed times with pbs, and then, starting at µg of the purified antibody per well ( µl), : serial dilutions in blocking solution were incubated for h min at rt. after washing steps with pbs-t (pbs buffer supplemented with . % tween- ), : hrp-labelled rabbit anti-human igg (sigma-aldrich, #a ) in blocking solution was added. after h, the plate was washed with pbs and the substrate o-phenylenediamine dihydrochloride sigmafast™ opd tablet (sigma-aldrich, #p ) was added (following manufacturer's instructions). reactions were stopped with µl m hcl per well and absorbance was measured at nm. the endpoint titer was determined as the last concentration of each purified antibody showing an absorbance value higher than the value defined as cutoff (mean blank + sd). blank is defined as the values from each elisa test against bsa (zrein et al., ; armbruster and pry, ) . the sandwich elisas were performed as described in the antigen elisa section with a few changes. the plates were coated with µl of µg/ml murine anti-his mab (qiagen, # ), and after blocking, the plates were incubated with µl of the crude extracts of the (rbd/n) antigen expressing leaves serially diluted ( : ) in bsa . %. wt crude extracts were used as negative control. the crude extracts were prepared by adding a volume of pbs buffer corresponding to times the mass of the ground tissue in liquid nitrogen. then the mix was centrifuged ( rpm, °c, min) and the supernatant was subjected to sonication before use. the antigens were sandwiched with µg of the corresponding purified antibody (or µl of the apoplastic fluid in % blocking reagent) per well ( h min incubation, rt). the same procedure as in the antigen elisa was followed for the incubation with the conjugated secondary antibody, colorimetric reaction and measurement. six different antibody sequences were selected for recombinant production in n. benthamiana, following a plant deconstructed viral strategy based on magnifection technology, as described earlier (marillonnet et al., ) (see table ). four of those were directed against the receptor binding domain (rbd) of the sars-cov- spike (s) protein, whereas the remaining two were directed against the n protein. all six antibodies were engineered as single polypeptide chains fused to the human cɣ -cɣ constant immunoglobulin domains. three of them, those derived from single chain camelid or synthetic vhh antibody libraries, were produced as direct fusions. the other three, derived from full-size human monoclonal antibodies, were redesigned as scfvs, using a linker peptide that connects vh and vl regions (see fig a) . the nucleotide sequences of the different variable regions were all obtained from the literature, then chemically synthesized with appropriate extensions and cloned into a destination magnifection-adapted vector using a type iis restriction enzyme strategy. the cloning cassette was flanked by a β-endoglucanase signal peptide for apoplastic localization in n-terminal and the human cɣ -cɣ domains of the human igg in the c-terminal side. the resulting vectors were transferred to agrobacterium cultures and agroinfiltrated in n. benthamiana leaves in combination with a ´ magnicon® module, containing the rna polymerase and movement protein, and with an integrase module (fig b) . for antibody production we used wild-type and rnai Δxt/ft glycoengineered n. benthamiana plant lines, the latter lacking plantspecific xylose and fucose glycosylation (strasser et al., ) . infiltrated leaves were examined daily, and only minimal damage was observed in the agroinfiltrated tissues during the incubation period. after seven days, leaf samples were collected, ground, and crude extracts were analyzed in sds-page. as can be observed in fig a-b (upper panel) , all samples produced coomassie-detectable bands of the expected antibody size. scfv-igg - kda antibodies migrated slightly above the - kda rubisco large subunit, partially masking its detection. vhh-igg antibodies migrated at the expected - kda size. the identity of the coomassie bands was confirmed by western blot using an anti-human igg antibody for detection (fig a-b , lower panel). as shown in fig a-b , under reducing conditions lower molecular weight (mw) bands were also detected, probably as a result of partial proteolytic degradation. small-scale affinity purification was carried out for all six antibodies produced in Δxt/ft plants using protein a affinity chromatography (fig c-d) . the resulting purified antibodies were used to estimate the yield of the final product, which ranged between . µg/g fw (cr antibody) to . µg/g fw (nanobody antibody) (see table ). the in-planta production of sars-cov- rbd and n protein antigens was also assayed in parallel using a similar strategy as described for antibody production. for this purpose, two versions of the expression vector were designed for rbd, one with the native viral sequence and the other with a n. benthamiana codon-optimized sequence. for the n protein, only the n. benthamiana codon-optimized sequence was employed. for rbd, native and codon optimized versions were targeted to the apoplast with the tobacco glucan endo- , -beta-glucosidase signal peptide and versions containing a kdel peptide for er retention were also generated. all nucleotide sequences were chemically synthesized with a small nucleotide extension coding for a histidine tag for detection ( fig a) . as described for antibody production, magnicon®-derived ´ vector modules encoding rbd and n proteins were agroinfiltrated in combination with an integrase module and a ´-module lacking any additional subcellular localization signal. shorter incubation times were decided in antigen production as compared to antibodies because antigen constructs produced different degrees of necrotic lesions in the leaves, ranging from mild symptoms in n protein to severe necrosis after four days in native rbd. for those constructs producing more severe lesions, incubation time was reduced to five days, and for the rest the incubation period was extended to seven days. rbd was extracted and purified using small-scale affinity-chromatography with niquel columns and the resulting coomassie and a western blot analysis are shown in fig b. rbd can be detected as a major estimated kda band, with the presence of higher mw bands that suggest multimerization. er retention did not improve expression levels of rbd for the native version, nor for the n. benthamiana optimized one (data not shown). addition of % triton x- to the standard extraction buffer (see materials and methods) did not improve the yield, which was estimated as - µg/g fw (table ) . n protein was extracted from agroinfiltrated leaves and affinity purified following the same procedure described for rbd. a major kda band was detected both on the crude extract and upon purification (fig b) . small-scale affinity-chromatography with niquel columns gave an estimated yield of µg/g fw for n protein ( table ) . binding activities of affinity purified anti-rbd antibodies were analysed by antigen elisa as shown in fig a. as expected, all assayed antibodies were active in binding their respective antigen. endpoint dilution titers were calculated for anti-rbd antibodies using a commercial antigen. sybodies and and nanobody showed high dilution titers, ( . µm, . µm and . µm, respectively), but the performance of cr was significantly lower ( . µm). in a parallel experiment, we tested the ability of plant-made antibodies to selectively detect our own plant-made antigens, including here also the n protein, using a sandwich elisa approach. for this analysis, elisa plates were coated with a murine anti-his mab, incubated with serial dilutions of crude plant extracts from antigen-producing plants and sandwiched with purified plant-made antibodies. as shown in fig b- c, all antigen-producing plant extracts gave sandwich-elisa signals significantly above the background when assayed using their cognate antibodies, thus evidencing the capacity of both, antibodies and antigens, to function as potent diagnostic tools. background signals in this experiment are likely due to cross-reaction of the anti-human igg secondary antibody with the murine anti-his mab, and could be easily reduced for more potent diagnostic applications by employing recombinant antibody formats other than igg. in the design of a pilot upscaling experiment, we favoured modularity and tried to maximize the affordability and adaptability of the process by reducing the requirements for highly specialized lab equipment. we carried out a final agroinfiltration for recombinant antibody production using a total of plants, equivalent to approximately . kilograms of fresh plant material. the plants were divided in two batches of plants each, and used to produce sybody and nanobody respectively, as these antibodies showed the most promising binding activities and yields. to facilitate the upscaling of the agroinfiltration process, plant seedlings were transplanted in growth modules, each module comprising seven pots kept together in a double layer of disposable plastic-board hexagons as shown in fig a. each production batch consisted in eight hexagonal modules. when plants were six weeks old, they were agroinfiltrated by submerging each hexagon upside down into a cm diameter cylindrical tank filled with l of an agrobacterium suspension, set inside a cylindrical vacuum degas chamber (fig a) . in this way, seven plants at a time were vacuumagroinfiltrated by slowly releasing vacuum while leaves remained submerged in the solution. next, plants were rinsed, brought back to the growth chamber and incubated for days before harvest. two different concentrations of the agrobacterium suspension were used in this experiment. one of them (sybody ) consisted in an od . final mix containing plasmids pich , pich , pgreensybody -igg , and pich _dsred at : : . : . ratio, where pich _dsred is a magnicon® ´module encoding dsred. the fluorescent marker was added to the infiltration mix to monitor the extension of the viral infection foci. as described elsewhere (julve et al., ; julve parreño et al., ) superinfection exclusion among virial clones yields mosaic-like expression patterns of individual clones, therefore the tiles produced by red fluorescent proteins were used as an indication of the extension and distribution of the unlabelled foci producing the recombinant antibody. in parallel, nanobody upscaled production was undertaken by agroinfiltration of an od . agrobacterium culture mix containing pich , pich and pgreennanobody -igg at : : ratio. after days, dsred tiles in sybody experiment, clearly visible with the naked eye, finalized their expansion in most agroinfiltrated leaves, an indicator that the expression tiles had covered the whole leaf surface (fig b) . at this stage, leaves were harvested and submitted to an apoplastic fluid recovery assay, where > . kg batches of detached leaves were vacuum infiltrated in mm phosphate buffer using the same vacuum device as described above. once rinsed to remove the excess of buffer, leaves were packed in mesh zipped bags, spinned down in a spin portable cloth dryer, and the intercellular apoplastic fluid was recovered from the drain tube. with this simple procedure, between and millilitres of apoplastic fluid (sybody and nanobody , respectively) was recovered from . kg of detached leaves. a fraction of the apoplastic fluid of both antibodies was concentrated times in kda centricons, and the rest was kept refrigerated for further analysis. fig c-d show the coomassie-staining and western blot analysis of crude extracts as well as apoplastic fluid preparations, and their corresponding purifications. crude extracts in this pilot experiment showed a vhh-igg band similar in intensity to that obtained in small scale experiments (data not shown). interestingly, apoplastic fluid consisted in a very simplified mix of proteins, with the recombinant antibody being among the most predominant ones. as shown, the different optical density of the agrobacterium culture, together with the presence of a competing dsred clone clearly influenced the accumulation levels, with the yields of nanobody clearly outperforming those of its sybody counterpart. unfortunately, the antibodies seemed partially degraded as indicated by the presence of two bands smaller than the expected vhh-cɣ -cɣ size, which could be compatible with degradation fragments. degradation was only partially solved with the addition of the protease inhibitor pmsf into the recovered phosphate buffer, as shown with nanobody production (fig d) . despite degradation, in a densitometric analysis we estimate that the recovered apoplastic fluid contains . g per liter of intact mab full-size. finally, we performed sandwich elisa tests of sybody and nanobody ( fig e and fig f, respectively) using the total and concentrated apoplastic fluid as detection reagent against serial dilutions of crude plant extracts from rbd-producing plants, showing that this simple antibody preparation can be directly employed in detection procedures without the need of additional purification steps. several n. benthamiana-dedicated bioproduction facilities are functioning worldwide, as those from leaf expression systems in uk (dobon, ) , icon genetics (giritch et al., ) and fraunhofer in germany (wirz et al., ) or kentucky bioprocessing in us (olinger et al., ) , among others. notably, medicago recently announced the building a new sqm facility with capacity for around - million of planned doses of flu vaccine per year. such facilities usually involve separated modules for upstream processing, namely a wet-lab module for preparation of the bacterial inoculum, a regular plant growth chamber, and agroinfiltration room, and a post-infiltration growth chamber. in addition, downstream processing facilities are often situated next or to the production ones to minimize the handling time of fresh tissues. whereas installed capacity of plant-dedicated biofactories is in continue growth, they are clearly insufficient to respond to global or even regional demands in times of crisis. we reasoned that, at least for upstream processes, the infrastructures required for medium scale n. benthamiana-based production are not radically different to those employed in high-tech agriculture practices as hydroponics, aeroponics or vertical farming, and thus high-tech agriculture facilities could be easily repurposed as biomanufacturing facilities in a matter of days or weeks (mcdonald and holtz, ) . as an exercise to practically test the repurposing requirements, we describe here the partial adaptation of our research laboratory and greenhouse facilities to the production of sars-cov- -related antigens and antibodies using n. benthamiana agroinfiltration as manufacturing platform. in figure we show a chronogram of the activities undertaken by our team towards the production of sars-cov- antigens and antibodies, from the initial selection of the nucleotide sequences of the genes-of-interest to the production of one litre of plant apoplastic fluid of recombinant sybody and nanobody . in our hands, the whole process took a total of nine weeks with non-exclusive personnel dedication and partially restricted access to our facilities. the process can be divided in three periods: the first step (design), taking approximately ten days, was dedicated to construct design and gene synthesis. it was pivotal in this step to have open access to viral and antibody sequences deposited in pre-print repositories. particularly remarkable was the openness of academic labs that immediately released primary sequence information of partially characterized anti-sars-cov- monoclonal antibodies, an exercise that should serve as an example in the future. due to our limited testing capacity, the number of parallel designs per product was maintained relatively low, and several design decisions (e.g. codon optimization, purification tags) were taken based in a best-guess approach. ideally, proper crisis preparedness should involve a centralized automated equipment such as a biofoundry (hillson et al., ) , with which the design space could be extended dramatically without causing delay. the second phase (build) was dedicated to cloning and construct building and lasted less than three weeks. our lab counts with adapted plasmids and cloning procedures from previous projects (sarrion-perdigones et al., ; vazquez-vilar et al., ) , therefore no significant time lag occurred in this step. importantly, this period also involved seeding a new plant batch at the scale required for pilot production in week seven ( plants distributed in hexagonal modules in this case). in a third phase (test), starting on week , all constructs were infiltrated at a small scale (three replicate leaves each), shortly incubated ( or days) and then tested functionally in parallel analyses. this small-scale assay took two additional weeks, summing a total of approximately days for the complete process. the synthetic biology-inspired design-build-test (dbt) process described above is conceived as an iterative one, so that new dbt cycles can be run fuelled by the conclusion of previous cycles to generate new optimized versions of the product. based on this experience, we estimate that the whole dbt cycle could be shortened to days or less by optimising the pipeline (e.g. introducing centralized, automatized design and build phases), and by improving preparation and anticipation in the facilities (fig ) . for instance, note that moving from step to step without delay requires a small batch of plants be always maintained in the facility, as it was in our case to supply our research requirements. this only involves transplanting - seedlings every three weeks, and then disposing of them every other three weeks once they start flowering. if a continuous plant supply is not maintained, a minimum of three extra weeks needs to be considered to have plants ready for the first test iteration. whereas the first version of products shown here lack iterative optimization, it would serve eventually to respond to the most urgent demands. in our case, as the results of the first dbt process arose, the best performing version (v . ) of two of the products were taken to production phase. in the exercise shown here, the upscaling was relatively small ( plants, approximately . kg fw). post-agroinfiltration incubation time was extended to days to maximize yields. in the meantime, optimization of the purification/extraction methods were undertaken at small scale, so that the new knowledge acquired could be applied in the batch purification of the pilot experiment. in a crisis-scenario, and given the modularity of the proposed scheme, several medium-size production modules can be replicated in a farming facility, and reproduced in several farms, allowing easy scalability. successive iterations with small scale agroinfiltration could be an effective way to maximize yields and reduce development times by comparing different small-scale strategies. it should be mentioned that the basic apoplast-based downstream processing proposed here could only be used, with the necessary adaptations, in a limited number of crisis-related applications, mainly related with detection and diagnosis. other uses, certainly therapeutic ones, would involve additional regulatory considerations including gmp downstream facilities, which are beyond the scope of this exercise. as a result of this experience, several improvements can be envisioned. we employed the magnicon® vector system with few adaptations for all the attempted proteins. although magnicon® produces maximum yields for many products, some proteins, particularly viral antigens may express better with other (e.g. non-viral) systems. in our experiments, antigens showed rather low expression levels despite optimization attempts using codon optimization and different localization signals. in adapting to an emergency, it would be advisable to perform initial expression tests using different production platforms also involving nonreplicative methods (sainsbury and lomonossoff, ) or dna viruses (yamamoto et al., ; zhang and mason, ) , and to incorporate them to the initial optimization test. as mentioned, this could be done in a centralized manner, later distributing expression clones to several repurposed production facilities. in contrast to antigens, recombinant single-chain antibodies showed in general higher and more uniform expression levels, as could be expected from their more similar structure. we chose to adapt full human iggs to a scfv-igg format to facilitate cloning and expression procedures, since it has been earlier described in plants that single chain formats reproduce the binding activities of the original full-size antibodies from which they derive. this format also facilitates comparisons with vhh antibodies, also produced as igg fusions. the plant-made sars-cov- products described here have several potential applications in the diagnosis area. both rbd and n proteins can be used as reagents for serological assays (amanat et al., ; liu et al., ) , although further yield optimizations should be required. for those assays where antigen glycosylation is an important factor, glycoengineered plants (strasser et al., ) can provide a competitive alternative to mammalian cells cultures (o'flaherty et al., ) . regarding antibodies, they can serve as internal references for the quantification of serological responses. with small modifications, the same antibodies can be adapted for sandwich elisa and employed in the detection and quantification of viral particles, a better proxy for infectiveness than rna. we also show here that apoplastic fluid is an inexpensive antibody preparation suitable for certain applications that require low-cost preparations, e.g. the concentration of the virus from environmental samples. as shown here, the protein complexity in the apoplast is greatly reduced, therefore the apoplast could be regarded as a plant-equivalent of hybridoma supernatant or ascited fluid, although at much lower cost. unfortunately, apoplastic preparations are prone to partial antibody degradation, probably due to endogenous proteases, however this can be minimized using extraction buffers with appropriate protease inhibitors, as it was shown for nanobody . the current pandemic crisis has evidenced the power of new antibody selection procedures, either based on single-cell selection from human peripheral blood mononuclear cells, in the case of full-size antibodies, or based on ultra-high throughput selection of synthetic libraries (sybodies) in the case of camelid-derived nanobodies (zimmermann et al., ; walter et al., ) . large collections of anti-sars-cov- , potentially neutralizing antibody sequences were made available to the scientific community in a question of weeks rather than months. it does not go unnoticed that the combination of rapid antibody selection procedures with fast, modular and scalable plant expression also has implications in the therapeutic arena as an ideal system for passive immunization. intravenous polyclonal immunoglobulins (ivig) from recovered patients have been shown a very effective covid- treatment in several studies (montelongo-jauregui et al., , and references herein) however the limited availability of patient sera hampers its application in practice. interestingly, we showed in a recent work that large recombinant polyclonal antibody cocktails (pluribodies), mimicking a mammalian immune response can be produced in n. benthamiana with high batch-to-batch reproducibility (julve parreño et al., ) . passive immunization with recombinant antibody cocktails resembles a natural response more than a monoclonal therapy, requires shorter developmental times and is probably more robust against the development of resistances. in conclusion, based on the results of the exercise described here, we propose the repurposing of indoors farms into plant-based biomanufacturing facilities as a viable option to respond to local and global shortages of bioproducts such as diagnostics and therapeutic reagents in times of crisis. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. all authors designed and performed the experiments, and analyzed the data. d.o. wrote this manuscript. all authors revised and edited the written manuscript. this work was supported by h eu projects newcotiana and pharmafactory. s.s. is recipient of a fpi fellowship bio - -r from the spanish zimmermann, i., egloff, p., hutter, c. a., arnold, f. m., stohler, p., bocquet, n., et al. ( the upper timeline represents the actual timespan of the experiments. note that the time points represent approximately the days required to produce and initially characterize the designated products; notwithstanding, some of the results shown in previous figures correspond to extended analysis obtained at a later stage, during the preparation of this manuscript. the lower timeline describes the estimated minimal timespan that would result by introducing some of the optimizations described in the text. a serological assay to detect sars-cov- seroconversion in humans limit of blank, limit of detection and limit of quantitation plant molecular farming -integration and exploitation of side streams to potential applications of plant biotechnology against sars-cov- transient gene expression seeds plant-based bioproduction systems: leaf expression systems' hypertrans technology promises low costs and high yields recombinant jacalin-like plant lectins are produced at high levels in nicotiana benthamiana and retain agglutination activity and sugar specificity rapid high-yield expression of full-size igg antibodies in plants coinfected with noncompeting viral vectors engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies building a global alliance of biofoundries a coat-independent superinfection exclusion rapidly imposed in nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein a synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov- systemic agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants from farm to finger prick-a perspective on how plants can help in the fight against covid- convalescent serum therapy for covid- : a th century remedy for a st century disease development of systems for the production of plant-derived biopharmaceuticals. plants mammalian cell culture for production of recombinant proteins: a review of the critical steps in their biomanufacturing delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques extremely high-level and rapid transient protein production in plants without the use of viral replication peaq: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants goldenbraid: an iterative cloning system for standardized assembly of reusable genetic modules goldenbraid . : a comprehensive dna assembly framework for plant synthetic biology generation of glyco-engineered nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like n-glycan structure: xylt and fuct down-regulation in n. benthamiana potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus gb . : a platform for plant bio-design that connects functional dna elements with associated biological data sybodies targeting the sars-cov- receptor-binding domain automated production of plant-based vaccines and pharmaceuticals structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies improvement of the transient expression system for production of recombinant proteins in plants bean yellow dwarf virus replicons for high-level transgene expression in transgenic plants and cell cultures the authors want to thank to the staff of the ibmcp and the polytechnic university of valencia who help us to access safely to our laboratory and greenhouses during the covid- lockdown in spain, and specially eugenio grau for his readiness to help us with sanger sequencing during that period. we are grateful to prof. steinkellner and strasser for providing glycoengineered plant lines and to prof. gleba for sharing magnifection plasmids. ministry of science and competitiveness and r.m. is recipient of a gva fellowship (acif/ / ). key: cord- - xy s authors: hu, dan; zhu, zhongyu; li, shun; deng, yongqiang; wu, yanling; zhang, nana; puri, vinita; wang, chunyu; zou, peng; lei, cheng; tian, xiaolong; wang, yulu; zhao, qi; li, wei; prabakaran, ponraj; feng, yang; cardosa, jane; qin, chengfeng; zhou, xiaohui; dimitrov, dimiter s.; ying, tianlei title: a broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain iii date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: xy s dengue is the most widespread vector-borne viral disease caused by dengue virus (denv) for which there are no safe, effective drugs approved for clinical use. here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the denv envelop protein domain iii (diii) combined with depletion by an entry defective diii mutant, we identified a cross-reactive human monoclonal antibody (mab), m . , which bound with high affinity to denv diii from all four denv serotypes. immunogenetic analysis indicated that m . is a germline-like mab with very few somatic mutations from the closest vh and vλ germline genes. importantly, we demonstrated that it potently neutralized denv both in vitro and in the mouse models of denv infection without detectable antibody-dependent enhancement (ade) effect. the epitope of m . was mapped to the highly conserved regions on diii, which may guide the design of effective dengue vaccine immunogens. furthermore, as the first germline-like mab derived from a naïve antibody library that could neutralize all four denv serotypes, the m . can be a tool for exploring mechanisms of denv infection, and is a promising therapeutic candidate. a a a a a dengue virus (denv) causes the most prevalent mosquito-borne viral disease. over . billion people are at risk for infection in over countries, - million are infected with symptoms, and up to , die from dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) each year [ , ] . no specific antiviral drug has been available against denv infection; the only approved vaccine, dengvaxia, has caused considerable controversy regarding its safety and potential benefits [ ] [ ] [ ] [ ] . for decades, anti-denv vaccine and biological drugs development has been hampered by the high sequence divergence ( - %) among the four denv serotypes [ , ] . such divergence leads to the fact that one antibody may not be sufficient to neutralize all denv infection. instead, the induced humoral immune response to one denv infection can enhance the infection and disease processes brought by a subsequent infection with another denv serotype [ ] [ ] [ ] . these findings suggest that the development of new and broadly neutralization antibodies against all the serotypes of denv could be promising candidate anti-denv agents, and may also guide the design of effective and safe vaccine immunogens. the denv envelope glycoprotein (e protein), which mediates virus entry into cells, is the major neutralizing target of antibodies [ ] [ ] [ ] [ ] [ ] . e protein is a type ii fusion protein and consists of three domains: di, dii, and diii of which diii has been proposed to contain a receptor binding domain [ ] [ ] [ ] [ ] . recent studies revealed that cross-reactive conserved epitopes exist on dii as well as diii of the denv e protein [ , [ ] [ ] [ ] . during the naturally-occurring primary denv infection, a large fraction of the antibody repertoire consists of dii-specific antibodies which are, unfortunately, typically poor in neutralization and may increase the likelihood of severe disease upon subsequent infection through a mechanism known as antibody-dependent enhancement (ade) [ ] [ ] [ ] . in contrast, antibodies targeting diii have proven to be the most potent neutralizing antibodies, but very few could be elicited in naturally infected individuals [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite this, previous studies indicated that anti-denv diii serotype-specific and cross-reactive antibodies could be elicited using denv diii as vaccine immunogen [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in infected humans [ ] [ ] [ ] [ ] . it has also been demonstrated that the lysine at position on diii is the critical residue in the cross-reactive epitope [ ] . therefore, the conserved epitope on diii represents an attractive target for the development of broadly neutralizing denv antibodies. here, we report the isolation of a potent denv diii-specific human monoclonal antibody (mab), designated as m . , from a large naïve antibody library constructed by the blood of healthy adult donors. a competitive sorting strategy using a diii mutant as competitor was applied to identify antibodies precisely targeting the conserved neutralizing epitope. to our knowledge, m . is the first human mab isolated from a naïve antibody library which could neutralize all the four serotypes denv viruses. importantly, both heavy and light chain genes of m . are very close to their putative germline predecessors. its fully human origin, the germline-like nature, combined with high-affinity and broad neutralizing activity toward all denv serotypes, suggest that m . is a promising candidate antiviral agent and may also provide a unique template for designing effective dengue vaccine immunogens. we previously prepared some large naïve antibody libraries using peripheral blood b lymphocytes of non-immunized healthy donors and used them for panning/screening against viral and cancer targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we used a competitive library sorting strategy to isolate broadly neutralizing antibodies against denv - ( fig a and b) . the yeast-displayed naïve single chain antibody fragment (scfv) library was used to screen against the biotinylated denv diii, and, importantly, ten times concentration unbiotinylated diii k e mutant was used as the competitor. the yeast cells were selected to present the antibody-expressing cells that could bind well to the wild-type diii instead of the diii mutant, resulting in the isolation of antibodies that can target the cross-reactive neutralizing epitopes covering the residue lysine [ ] . potent enrichment was achieved after four rounds of sorting, and a panel of antibodies were identified ( fig b) . two antibodies, designated as m and m , bound potently to denv diiis. their scfv gene were fused with human igg fc for protein expression, and surface plasmon resonance (spr) experiments were used to evaluate the antigens binding. the equilibrium dissociation constant (k d ) of m for the denv - diiis were . nm, . nm, . nm and . nm, respectively. the mab m displayed a broader binding profile compared with that of m , with the k d of . nm, . nm, . nm and nm to denv - , respectively (table , s and s figs). to further improve the affinity of m and m with the four denv serotypes, we constructed a mutant library using the error-prone pcr technologies. following three cycles of mutagenesis and selection, two clones were identified from the enriched pool of yeast sorting, designated as m . and m . . biacore analysis showed that the cross-reactive binding activities of m . and m . to all diiis were preserved after the affinity maturation process. the k d of m . for the denv - diiis were . nm, pm, . pm and nm, respectively (s fig). although the binding to denv - diiis was improved, the m . had only slightly increased binding affinity to denv diii compared to its parental mab m . notably, the m . exhibited high affinity to all the denv diiis. the k d of m . for the denv - were . nm, . nm, . nm, and . nm respectively, which demonstrated that m . could bind to all the four serotype denv viruses with high avidity (table , fig ) . we also assessed the binding specificity of m . by elisa, and the results showed that m . had weak cross-reactivity with zika virus (zikv) diii and no binding with other irrelevant antigens (s fig). next, we assessed the neutralization capacity of m . and m . against the four denv serotypes using a denv luciferase reporter viral particle (rvp) neutralization assay. we used denv rvps against the four dengue serotypes that are common strains in denv research: denv- (westpac ), denv- (s ), denv- (ch ), and denv- (tvp ). the luminescent reporter expression was proportional to the number of rvps added to bhk dc-sign cells, confirming the linear correlation between the extent of rvp infection and reporter gene expression. in consistent with the biacore binding results, both m . and m . could neutralize all the four serotype denv, and m . displayed better neutralization than m . , with the % neutralization titers (ic to further evaluate the neutralization breadth of m . igg against the four denv serotypes, a standard plaque reduction neutralization assay (prnt) on bhk- cells was performed using denv - live viruses, including denv- (genbank fj ), denv- gz / (s fig, isolated from a denv- infected patient in guangzhou, china), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ). an irrelevant human mab g was used as the negative control [ ] , and a g , a broadly neutralizing mab against all the four denv serotypes, was used as the positive control [ , ] . as shown in fig , m . igg could neutralize all the four denv serotypes. the % neutralization titers (ic ) of m . against denv - was . , . , . , and . μg/ml respectively ( table ). we next used a well-established ade assay to detect the in vitro ade effect of m . igg. a mutated form of m . igg was also generated containing the leucine to alanine substitutions at positions and (m . igg-lala), which lacked binding to fcγ receptors. the ade effects of denv- or denv- by m . igg, m . igg-lala, as well as a g were measured. interestingly, neither m . igg nor m . igg-lala presented any ade effect against different serotypes of denv ( fig f, s fig) . in contrast, potent ade effects were observed for the dii-specific mab a g . these results showed that m . igg is a denv diii-specific mab without detectable ade effect. we further analyzed the sequences of mabs using the imgt tool to identify their closest vh and vλ germline genes. the results indicated that m . and m . originated from different b-cell lineages ( table ). the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . in contrast, the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . interestingly, we found that the encoding genes of both m . and m . closely resembled their corresponding germline gene segments. notably, m . vh and vλ gene shared . % and . % sequence identities with the ighv - � and iglv - � germlines respectively (fig a and b ). these results indicated that the mab m . is a germline-like antibody, which, in general, could show better drug properties and lower immunogenicity compared to somatically hypermutated antibodies [ ] . to further investigate the immunogenetic characteristics of m . -like antibodies, we analyzed in detail the ighv - recombination frequencies with specific ighd and ighj genes families from naïve immunoglobulin m (igm) repertoires of health adult donors and neonatal igm repertoires of newborn babies, using next-generation sequencing data previously generated from our antibodyome studies [ ] . by querying the m . sequence from the igm repertoires, sequences were found to display m . -like v(d)j recombination from the genes ighv- - , ighd , and ighj out of a total of , , sequences from healthy adult igm repertoires. in igm repertoires of newborn babies, a similar recombination frequency was also observed, in which sequences with m . -like v(d)j recombination were found from , , sequences. our analysis showed that ighv - is one of the most frequently used ighv genes, and identified that many of those sequences sharing a significant degree of resemblance to m . ( fig c) . in brief, analysis of these data showed the potential of eliciting robust immune responses with the m . -like germline antibodies by vaccination. to determine whether m . can protect denv infections in vivo, we firstly used a lethal denv - infection suckling mouse model. the mice were challenged with denv - at pfu/mouse via intracranial injection. four hours later, the mice were treated intracranial with a single dose ( μg) of m . igg, m . igg-lala mutant and g (unrelated antibody control). these animals were monitored for morbidity and mortality daily. as shown in fig , all the mice in control groups died from denv infection, and most of them died within the first two weeks of viral challenge. interestingly, there was no significant difference in therapeutic efficacy against denv - infection between m . and the lala-mutated m . . the m . igg protected % denv- , denv- , denv- and % denv- infection whereas (c) germline-rooted circular phylogenetic tree of m . -like antibody sequences found in igm libraries derived from healthy human adults and neonates. the m . and a sequence showed highest similarity to m . were shown in red. sequence id started with cb represents sequence derived from the neonates, and that started with hh represents sequence derived from the healthy adults. the phylogenetic tree was constructed by the neighbor-joining method. https://doi.org/ . /journal.ppat. .g lala-mutated m . protected % denv- , denv- and % denv- , denv- respectively. therefore, m . has no detectable ade as confirmed in both in vitro and in vivo experiments. we also used the ag (types-i and -ii ifn receptor deficient) mice to test the therapeutic effect of m . against denv- (s fig). the results showed that all the mice in the control antibody treatment group died while the survival rate of mice can reach % in m . treatment group, indicating that the antibody can also protect the lethal infection of denv- in ag mice. taken together, these results indicated that m . can protect denv - infections in vivo. to map the epitope of the germline-like mab m . and identify in greater detail the structural basis of denv neutralization, we employed multiple approaches (fig ) . sequence alignment of different denv genotypes and mapping of the conserved amino acid residues of denv diii showed that four serotypes denv diiis amino acid residues were different from one another between amino acids - ( fig a) . subsequently, serotype derived diii consensus gene was randomly mutated to construct a yeast-displayed mutant library. two rounds of sorting of those yeast cells showing expression on surface but lacking the binding to m . was performed. a total of binding escape mutants were aligned with the serotype consensus protein sequence. mutation frequency at each position was plotted against the residue position number. similarly, unique diii sequences derived from naturally isolated serotype dengue viruses from genebank were also aligned with the consensus sequence (fig b and c) . the superimposed profiles of the two set of sequences showed that many of the escaped mutations located in the well-conserved area, indicating the broad crossreactivity of m . to naturally isolated dengue viruses. besides, the epitope mapping shows that the m . epitope is at close to or partially overlaps the dimerization interface between domains ii and iii. these results may explain why m . is a potent cross-reactivity antibody to all the four denv serotypes. furthermore, computational docking of denv diii-m . antibody complex was performed using zdock method. we selected the three top scored docked complexes that contained the key residues identified from an experimental epitope mapping approach. one of the top scored docked models exhibited minimum clashes with appropriate protein interface parameters and was used to demonstrate the lactation the potential epitopes and their interactions with m . antibody, which might shed light on the molecular mechanisms of broadly cross-reactive neutralization. fig d showed the docking model of the diii-m . antibody complex in which these epitopes are highlighted in green surfaces. the docking model revealed a different orientation of antibody binding as compared to the diii complex structure with fab a d- that was previously determined [ ] . the epitope comprised of three structurally proximal regions, residues - in green, , and in dark green, and - at the c-terminal in lime. one of the key residues, k , contacts the cdr-l of m . which has a germline mutation. in env-diii-fab- a d- complex structure, the residue k contacts the cdr-h . the hydrophobic residues, ile and trp, of cdr-h contact the center part of the epitope, and other loops h , h , l and l also involve in the binding. the surface area of the interface between diii and m . antibody in the model complex is Å , a typical of antibody-antigen interactions. there are six hydrogen bonds likely to form and no salt bridges at the interface. in brief, the binding regions of the m . may be close to or partially overlaps the dimerization interface between domains ii and iii, which might indicate the broad crossreactivity of m . to the four serotypes of denv. dengue is a disease with a complex immune response orchestrated by host cells partially due to the presence of four serotypes of denv. importantly, after a primary denv infection, one can be protected against or aggravate of a secondary infection with a different serotype, which bring many difficulties to develop an effective vaccine. thus, it is very urgent to develop an effective and cross-reactive antiviral therapy against denv infection. monoclonal antibodies (mabs) are of growing importance for protective and pathogenic immune responses to viruses. at present, there are many therapeutic antibodies to treat viral infections under development, such as antibodies for hiv- , sars-cov, mers-cov, nipah and hendra viruses, and h n influenza virus [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . fortunately, screening antibodies from the large naïve libraries has enabled the rapid development of high-affinity human mabs, especially for the rapid response to the outbreak of emerging viruses and diseases. we recently successfully identified two human germline-like mabs against mers-cov and h n influenza virus from the naïve library, named m and m , respectively [ , ] . they both can naturally exist with very low level of somatic hypermutation in the naïve library with which they have potent binding activity against the envelop proteins of mers-cov and h n influenza virus. most importantly, m and m all showed highly therapeutic effective in the animal models. therefore, the naïve library screening can be quickly used to isolate germline-like antibodies that effectively bind to complex protein targets like those in denv viruses. how to increase the neutralization breadth is a key issue in developing anti-denv antibodies. previous studies revealed two classes of broadly neutralizing antibodies to flaviviruses, including antibodies targeting the conserved epitopes in dii or diii [ ] [ ] [ ] ]. while the conserved fusion loop epitope (fle) in dii is the immunodominant epitope in e protein, unfortunately, this epitope frequently induced poorly neutralizing and strongly infectionenhancing antibodies via ade [ ] [ ] [ ] . therefore, diii represents the ideal target for neutralizing antibodies. in this study, we applied a highly efficient yeast-display-based sorting strategy by using the highly diverse denv diiis as antigen and the competitive sorting technique. by applying this method, we quickly and efficiently identified two human germline-like broadspectrum anti-denv mabs (m and m ) from the naïve scfv yeast library using the diii antigen that make them as promising candidate therapeutics as well as the template for vaccine development. another class of highly efficient broadly neutralizing antibodies that target the envelope dimer epitopes (ede) from the secondary acute denv infection plasmablasts has been identified by dejnirattisai et al. [ ] . these antibodies may especially get through with high somatic mutations from the secondary virus infection. compared with the highly somatically mutated antibodies, germline-like antibodies typically have better safety and drug-related property [ ] . importantly, the hendra and nipah antibody m . is a near germline antibody and exhibited a very good drugability, which was from a similar library that was also used to isolate our m and m -like antibodies. m . was successful as a candidate therapeutic mab in animal models and was also completed the phase i clinical trial (actrn ) without side effects [ ] . to further improve the affinity of m with the four serotypes denv diiis, we subjected m to affinity maturation process, and named it as m . . subsequently, we analyzed m . sequence using the imgt tool to identify its closest vh and vλ germline genes. interestingly, we found that m . is still a germline-like antibody although it went through the mutation process in vitro, with over % identities of its vh and vλ genes to the ighv - � and iglv - � germline respectively. in order to evaluate the neutralization effect of the m . igg, we used a standard plaque reduction neutralization with bhk cells to measure denv infection and neutralization. the m . igg showed broadly neutralization towards the four serotypes denv as well as a recent denv isolate from clinical samples. more importantly, m . did not present any ade effects in different serotypes of denv. the in vivo study results demonstrated the therapeutic potential of m . against severe denv - infections. in brief, the m . could neutralize the four serotypes denv in vitro and protect the denv infection mouse model in vivo without detectable ade effects. we therefore expect that m . has a likeness drugability of m . and could be developed as a candidate therapeutic in the future. we have also localized the m . epitope by using a combination of computational structural modeling, display-based antigen mutagenesis, and sequence-based analysis of mutants. the epitope appears to overlap with the epitope previously explored as targets for cross-reactive murine mabs and close to or partially overlaps the dimerization interface between domains ii and iii. this further indicates that this epitope could be an important component of vaccine immunogens intended to elicit cross-reactive neutralizing antibodies. in progress are our experiments to crystallize the complex of m . with denv diii that would allow precise determination of the m . epitope. the major result of this study is the identification of a germline-like human mab, m . , from a naïve yeast antibody library which binds with high (picomolar) affinity to diiis from all serotypes and neutralizes the four denv serotypes. there are two major implications from this finding: ) m . is a potential candidate therapeutic which could be further developed in preclinical and clinical settings. ) the epitope of the germline-like mab m . could guide the design of effective candidate vaccine immunogens capable of eliciting m . and/or m . -like antibodies. bhk cells were cultivated in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) (biowest). mosquito cells c / were cultured in rpmi- medium supplemented with % fbs. all cells were maintained in a humidified atmosphere of % co at ˚c incubator, except for c / cells, which were cultivated at ˚c. denv- (genbank fj ), denv- gz / (isolated from denv- infected patient in guangzhou), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ) were propagated in c / cells by using rpmi medium and the titers were measured by standard plaque forming assay in bhk cells. denv diii genes from all serotypes were synthesized by genescript, inc (nanjing, china), fused with igg fc and a c-terminal avi-tag, and cloned into psectag expression vector. the diii. (serotype ) k e mutant was generated through overlapping pcr. for the conversion of igg from scfv, the heavy and light chains of scfv were amplified and recloned into the ptt-igg vector. the plasmids were transfected into expi cells (thermo fisher) for transient expression, and purified using protein g column (ge healthcare, piscataway, nj) according to the manufacturer's instructions. the purified protein was biotinylated by mixing with biotinylation reagents in pbs for min on ice, according to the manufacturer's instructions (pierce). a large yeast-displayed scfv library was used for antibody screening, and the screening protocols were essentially carried out as described previously [ ] . briefly, μg of binotinylated diii. -fc and cells of the initial naïve library were mixed and washed by pbsa, and incubated with μl streptavidin conjugated microbeads (miltenyi biotec, auburn, ca) before loading onto the automacs system (miltenyi biotec) for sorting. after three rounds of sorting, the downsized library was further sorted against binotinylated diii. -fc ( μg/ml) but also using unbiotinylated k e mutant ( μg/ml) as the competitor. to generate the m scfv and m scfv mutant libraries, random mutagenesis of the scfv genes were performed through error-prone pcr by using a genemorph ii kit (stratagene) following the manufacturer's instructions with minor modifications. to further diversify the mutation profile, um of each of the two nucleotide analogues ( -oxo-deoxyguanosine triphosphate and '-deoxy-p-nucleoside- '-triphosphate) was mixed in the pcr reaction mixture. for the second and third cycle library constructions, an extra step of dna shuffling pcr was inserted into the regular pcr cycles to combine the beneficial mutations obtained from previous maturation process. dna shuffling pcr step was performed as following: cycles of denature at ˚c for seconds followed by annealing/extension at ˚c for second on the bio-rad mycycler. binding affinities of m scfv, m scfv, m . scfv, and m . scfv to the denv diiis were analyzed by surface plasmon resonance technology using a biacore x instrument (ge healthcare). the antibodies were covalently immobilized onto a sensor chip (cm ) using carbodiimide coupling chemistry. a control reference surface was prepared for nonspecific binding and refractive index changes. for analysis of the kinetics of interactions, varying concentrations of antigens were injected at flow rate of μl/min using running buffer containing mm hepes, mm nacl, mm edta, and . % surfactant p- (ph . ). the association and dissociation phase data were fitted simultaneously to a : langumir global model by using the nonlinear data analysis program biaevaluation . . all the experiments were done at ˚c. neutralizing activity of mabs was measured using a standard plaque reduction neutralization with bhk cells as previously described [ ] . briefly, -fold serial dilutions of mabs were added to approximately pfu of a variety of dengue virus strains and incubated for h at ˚c. then, the mixture was added to bhk cell monolayers in a -well plate in duplicate and incubated for h at ˚c. the mixture was removed, and ml of . % (w/v) lmp agarose (promega) in dmem plus % (v/v) fbs was layered onto the infected cells. after further incubation at ˚c for days, the wells were stained with % (w/v) crystal violet dissolved in % (v/v) formaldehyde to visualize the plaques. prnt values were determined using non-linear regression analysis. prnt data were calculated by doing a non-linear regression analysis using sigmaplot (version . , systat software, inc., ca) as previously described [ ] . denv rvps from all four serotypes were pre-incubated with an equal volume of serially diluted antibodies ( μg/ml to . μg/ml pre-dilution or . μg/ml to . μg/ml predilution, as measured based on the dilution of antibody prior to combining with rvps) in dmem infection media for h at room temperature and transferred to wells of a -well plate. an equal volume of denv rvps were added to each well followed by slow agitation for h at room temperature. bhk dc-sign cells were added to each well at a density of , cells per well followed by incubation at ˚c in % co for h. cells were subsequently fixed in lysed and analyzed for luminescent reporter expression using the wallac victor. the percent infection for each concentration of mab or serum was calculated, and the raw data was expressed as percent infection versus log of the mab concentration or the reciprocal serum dilution. the data were fit to a sigmoidal dose-response curve using prism (graphpad software, la jolla, ca) to determine the titer of antibody that achieved a % reduction in infection. maximum infection was determined in the absence of antibodies. the in vitro ade assay was performed using k cells [ ] . briefly, serial -fold dilutions of antibodies under concentrations ranging from to . μg/ml were mixed with denv- or denv- , and incubated for h at ˚c. mixtures were then added to × k cells at multiplicity of infection of . ~ . for h in -well plates. the cells were subsequently washed times with serum free rpmi- medium. after collecting cells by centrifugation, the cell pellets were re-suspended with rpmi- medium containing % fbs and added to -well plates, then incubated for days at ˚c with % co . the titer of viruses in the supernatant was then measured using a plaque assay. the ade effect was calculated as different viral yields in the supernatant after infection in the presence of the added antibodies. the epitope mapping of m . was performed using previously described protocols [ ] . briefly, random mutagenesis of the denv diii. gene was performed using a genemorph ii kit (stratagene). as described above, the yeast-displayed mutant library was mixed with biotinylated m . scfv-fc, washed, and stained by mouse anti-c-myc antibody (roche), alexa- conjugated goat-anti-mouse antibody (invitrogen), and pe-conjugated streptavidin (invitrogen). after two rounds of sorting on a facsaria ii cell sorter (bd biocsiences, san jose, ca), the sorted cells were amplified and their plasmids were prepared and sequenced. homology modeling of variable regions of heavy (v h ) and light (v l ) chains for m . scfv antibody was carried out using the swiss-model workspace [ ] by selecting the closest template structures (pdb codes: qos for heavy chain and dd for light chain), whose sequence similarities were % and % respectively. the v h -v l orientation of m . scfv structure was assigned similar with one of the templates (pdb code: dd ) that showed minimum steric clash for creating the final m . scfv model. the crystal structure of denv diii serotype (pdb code: r ) was used for docking with the modeled scfv antibody m . . docking of scfv m . to the dengue env-iii was performed by zdock server (http://zdock.bu.edu) that uses a fast fourier transform (fft)-based rigid-body protein docking algorithm with scoring functions combining pairwise shape complementarity, desolvation and electrostatic energies. based on the escape mutants that led to the loss of epitopes and available crystal structure of denv diii, we selected a list of residues as biological constrains, , , , , , and , on the surface of env-diii as potential contacting residues for docking constraints. similarly, one or two residues from each of cdr-h , h and l loops were chosen at the docking interface. cdr-h and h loops had dominant hydrophobic residues whereas cdr-l had a germline mutation, and they all had high antigen-contacting propensities [ ] . results from the top zdock predictions were filtered using the userdefined residues and a angstrom distance cutoff. three predicted complexes were only kept as all residues selected come together at the interface and were further examined by pdbepisa (protein interfaces, surfaces and assemblies). pymol was used for the analysis of docked model and graphical illustration [ ] . the suckling mice were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the animal biosafety level (bsl- ) laboratory (shanghai, china). one day mice were used for all experiments. all mice were intracerebrally injected with pfu of denv - . at h post infection, mice were passively transferred a single dose of μg antibody m . igg, m . igg lala mutant or g igg as the negative control via intracerebrally injection. survival rates and disease sings were monitored daily. the ag mice (type i and type ii interferon receptor-deficient) were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the bsl- laboratory (shanghai, china). groups of mixed-sex -to -week-old mice were used for all experiments. all mice were intraperitoneally injected with x pfu of denv- in a volume of μl. at h post infection, mice were passively transferred a single dose of μg antibody m . igg-lala, or g antibody as the control via i.p. injection. survival rates, weight loss, and disease sings were monitored daily. specific-pathogen-free ag mice ( - weeks old) and suckling mice were used for all experiments. all experimental protocols were reviewed and approved by the institutional committee of the who dengue classification and case definitions: time for a reassessment global spread and persistence of dengue the risks behind dengvaxia recommendation dengvaxia: age as surrogate for serostatus. the lancet infectious diseases : dengvaxia controversy: impact on vaccine hesitancy ade and dengue vaccination cross-reacting antibodies enhance dengue virus infection in humans the growth and potential of human antiviral monoclonal antibody 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effect a bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein therapeutic antibodies, vaccines and antibodyomes broad and potent hiv- neutralization by a human antibody that binds the gp -gp interface broad and potent neutralization of hiv- by a gp -specific human antibody structural basis for broad and potent neutralization of hiv- by antibody vrc human infection with a novel avian-origin influenza a (h n ) virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies nonneutralizing antibodies induced by the hiv- gp nhr domain gain neutralizing activity in the presence of the hiv fusion inhibitor enfuvirtide: a potential therapeutic vaccine strategy a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus swiss-model: modelling protein tertiary and quaternary structure using evolutionary information antibody-antigen interactions: contact analysis and binding site topography simplifying and enhancing the use of pymol with horizontal scripts we thank professor dane wittrup from mit for providing the yeast display vector and yeast strain. key: cord- -slv r u authors: vakharia, kunal title: the right to know: ethical implications of antibody testing for healthcare workers and overlooked societal implications date: - - journal: j med ethics doi: . /medethics- - sha: doc_id: cord_uid: slv r u after the initial surge in cases of coronavirus (covid- ), the outbreak has been managed differently in different countries. in the usa, it has been managed in many different ways between states, cities and even counties. this disparity is slowly becoming more and more pronounced with the advent of antibody testing. although many argue over the potential merits of antibody testing as an immunity passport to allow the economy to restart, there are other implications that stand at the heart of the bioethical debate that are often overlooked. particularly with covid- , there are many uncertainties and the discourse alone of antibodies presumes misinformation that may outweigh the epidemiological benefits of antibody testing. although this paper does not seek to eliminate antibody testing, it does highlight the need for appropriate counselling both on a personal level with each patient but on a more global level. this moral standard of appropriate education is key to allowing the continued autonomy needed during this pandemic. the discussion that has continued since the initial severe acute respiratory syndrome virus and avian influenza epidemics has focused on the potential for immunity among the general population and the moral obligation to treat that is often faced by healthcare professionals and institutions. the current pandemic continues to spread rapidly and there is an ever-growing unease about jump-starting the country, the world and the economy again. although these are important factors to take into consideration, there are some unique challenges faced with the current coronavirus (covid- ) that transfigure this conversation into one about responsibility. with the superfluity of different antibody tests coming into the market with different specificities and sensitivities, the word 'antibody' engenders a certain sense of relief or comfort that may not be practical addressing the general population. although our understanding of vaccinations and immunity to disease is predicated on the idea of igm antibodies being the first to develop to fight off an infection followed by igg antibodies remaining longer term, coronavirus may not be something that can be as easily pre-empted as other illnesses that have vaccines and that generate a similar response. with a virus that potentially generates a significant proinflammatory state in a multiorgan system fashion, it is hard to understand the potential for immunity, the risk with repeat infection, the potential for viral mutations and the long-term impact of this situation. in addition to the challenges of immunity, the idea of antibodies creates a challenge for understanding the true epidemiological nature of the spread of covid- . although the majority of public health professionals and institutions understand that antibody testing is not a free pass to restarting the economy, the fact that this is primarily an epidemiological study is misunderstood by a majority of the public and can easily be misconstrued to be comparable with immunity. gathering epidemiological data about the spread of covid- and its potential impacts on different demographics and populations is important. this information can be used to work to better solutions for segregated minorities and for targeted and widely infected populations. the need to understand the pattern of spread is important to allow for better long-term planning on how to manage this pandemic, as well as how to manage potential disease in the future. when looking from the lens of a bioethicist, the potential beneficence to underserved populations as well as the entire global community is immense and unparalleled. the argument is not whether this potential benefit from a public health outreach, containment and understanding is critical for better preventing disease spread, but simply if this potential benefit outweighs all the other potential risks. even in the past couple of weeks, large cities, primarily new york city, have seen a large efflux of the wealthy out of the densely populated core. a recent surge in articles has highlighted the inequity and lack of justice surrounding the antibody testing, movement and spread of the disease among the wealthy and the more indigent populations in larger cities. this inherently widens the wealth gap that already exists in the usa, but even more importantly helps determine where resources should be allocated, how hospitals should or should not proceed with cases, and where the potential long-term impacts will need more care in the future. all these details are not necessarily immediately realised, but form a broader public health narrative that can be used to curb such vast discrepancies in the future. in addition to discrepancies seen between disparate populations, healthcare workers face a distinct current controversy challenge. although the idea of antibody testing is great from an epidemiological perspective, it sadly offers limited benefit from a medical standpoint. the challenge arises, particularly in the medical community and exaggerated at the front lines, when the discourse surrounding antibody testing remains around the idea of herd immunity and using antibody testing to determine where people work and the extrapolated idea of immunity passports. although slightly dystopian in its view, the idea of immunity passports is being felt at micromanagement levels as well. workers who have had only a minor illness when testing positive for covid- or those whose antibody tests return positive and who may have been asymptomatic carriers at one point are designated 'survivors'. the notion of survivorship, like with patients with cancer, is not held in the same regard. when antibody testing is mixed with survivorship, it offers a shield of protection that blinds many to understanding this disease in its entirety. people are suddenly confronted with a moral obligation towards volunteerism in place of the current voluntarism. as noted by the hastings center, there are many who are falling on either side of the covid- antibody debate. the potential implications of antibodies and whether they confer immunity are being hotly contested and draw similarities to the great vaccine debate and whether the risks of vaccination outweigh the benefits. as a society, the moral obligation to work towards an idea of herd immunity is natural, but fraught with so many uncertainties, it is hard to justify parallel discourse. with the growing literature and data suggesting the possibility of mutations, unequal impacts on different people and the potential repercussions of the spike protein for those with igg immunity already, can society in good faith adopt a moral prerogative to put antibody-positive people in the front line? or does the uncertainty and possible long-term health consequences mean that these people should be a protected population? ethically, the central tenets of most of our discussions revolve around the ideas of non-maleficence, beneficence, justice and autonomy. although autonomy is regarded as important but sometimes justifiably sacrificed for the greater good, does autonomy and the right to know supersede the potential lack of control and lack of knowledge that antibody testing may confer? although the covid- pandemic is unique, the usa has faced a similar conundrum before in the s. when the hiv epidemic was in full throttle and our understanding limited, mandatory testing was debated, but there were no effective or consistent ways to enforce the difficult logistical and legal problems that arose. similarly, there are many challenges and potential for stigmatisation that arise with covid- testing as well. healthcare workers form a group of individuals who recognise the potential of covid- , the impact it has had, and are still willing to go to work and continue to face the challenge. although morally understandable, prudence dictates that healthcare personnel at high risk of exposure, regardless of their antibody status, take full precautions with every encounter. unlike the hiv epidemic, covid- is aerosolised and spreads not by knowingly taking sexual or intravenous risk, but even by unknowingly being exposed. if antibody testing had no impact on the daily workings of a hospital department, did not change an individual's exposure risk and had no effect on the precautions everyone must take, then why take the test? is it a risk to the patient simply to take a blood test and confer next to no benefit from this information? the epidemiological studies that could arise from testing exposed healthcare personnel are enormous. the data may show the rate of conversion. it may highlight safe practices and help change the way we handle certain situations in the hospital. but tracing is incredibly hard with the number of contacts, the number of transitions of care and the potential for every exposed surface being considered contaminated. the biggest challenge faced by antibody testing may in fact be psychological. there is no compendium of information that helps sift through the vast amount of data being collected on covid- or even the number of research papers being written on the subject. scientific sharing is crucial at this time more than ever before, but because of the lag in understanding, can we justify the potential cost to those people who are antibody-tested? when the hiv epidemic was becoming more prevalent, the idea of giving antibody testing information to patients was focused on the idea that sharing information would lead to motivating a change in habits. bad needle practices and recognition of sexual contacts were a feasible and understandable benefit to the potential psychological impact an antibody test would have on an individual. in the current era, a negative test can be deflating. but how is this even possible? how can a negative test be deflating to a healthcare worker? it seems counterintuitive. most people would be ecstatic that they did not test positive for antibodies to a potentially lethal virus. the rationally irrational response is expected. the higher your risk exposure, the higher your contacts and the more isolated one feels, the more likely it seems that healthcare personnel think they have had covid- or have antibodies. although anecdotal, many of these personnel are disappointed when they test negative and continue to protect themselves as they had before. but knowing that they have not contracted the disease and that the worst may yet still be coming is a feeling that, although pervasive in the current climate, can weigh on anyone who has to face it daily. in addition, those who do test positive seem fearless. they are the ones willing to go into any room and help out even without an n mask on. these cavalier feelings, particularly among younger physicians who feel that they will likely fight off covid- better, are simply unsupported by the literature. but cavalier actions can lead to a slippery slope of consequences. recognising these psychological tolls, is there any role to testing people to understand the epidemiological risks and not telling people their antibody status? this flies in the face of autonomy and mirrors the earlier paternalistic sentiments of medicine in the early s, but also allows for more control of information and limits the spread of false predispositions. this may not be the perfect answer, but these are the two ends of the spectrum. somewhere in between will likely be the safest ethical and social decision. in these unusual circumstances where every action is magnified by a certain magnitude by the potential for public health benefits, it is important to increase testing. the centers for disease control has continued to expound the need to increase our testing capabilities and suggests that our current testing is not nearly where it needs to be. there is built-up momentum for antibody testing and this will likely have beneficial repercussions regarding opening up businesses, potentially helping allocate healthcare resources, and focus our research on the best treatment modalities, but this is not being realised at this time with current controversy the current knowledge of the virus and with the current rate of testing. within a week, the tests that have been done may be obsolete as the world begins to see reopening as a possibility. there are really no ethical justifications not to notify patients about their antibody status if they are tested. the focus on changing behaviours seen by prior epidemics has transformed during this pandemic. it is a moral imperative, more than the benefit to the community and public health, that those who are tested undergo appropriate counselling. because of the inundation of covid- stories, research and data, it is important to temper patients' expectations, guide them to understanding the ephemeral nature of testing and recognise the potential longterm consequences of this testing. some might hear all of this and say that testing allows for us to be informed and more information is more power, but implicitly in this statement is that medicine and healthcare professionals are morally obligated not only to serve their patients and their community, but to make sure that they fully understand the ramifications of any test result. with limited knowledge about the significance of covid- antibody testing at this time, it is hard to use this to stratify work in a healthcare setting or to use it for any purpose beyond epidemiological studies on the spread of the disease. the promise and peril of antibody testing for covid- antibody testing for covid- the potential danger of suboptimal antibody responses in covid- emergence of drift variants that may affect covid- vaccine development and antibody treatment hiv antibody screening in a general hospital population the richest neighborhoods emptied out most as coronavirus hit new york city show me your passport: ethical concerns about covid- antibody testing as key to reopening public life mandatory screening for hiv antibody hiv antibody screening. an ethical framework for evaluating proposed programs a short review on antibody therapy for covid- contributors this is the sole work of kv and is a product of his own viewpoint.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared. patient consent for publication not required.provenance and peer review not commissioned; internally peer reviewed.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. kunal vakharia http:// orcid. org/ - - - references key: cord- -ro x qa authors: ingram, george a.; al-yaman, fadwa title: a comparative assessment of four serological methods used in the detection and measurement of anti-parasite antibodies in the serum of the amphibian, bufo viridis date: - - journal: international journal for parasitology doi: . / - ( ) - sha: doc_id: cord_uid: ro x qa abstract antibodies against crithidia fasciculata choanomastigotes were detected in green toad (bufo viridis) sera by direct agglutination, indirect haemagglutination (iha), complement-fixation test (cft) and enzyme-linked immunosorbent assay (elisa), correlation coefficients (r) were calculated for comparisons between each of the techniques and regression formulae derived in order to convert antibody levels as determined by one immunological method to that of another. the highest mean titre obtained by elisa was approximately . – . times greater than those obtained by the other techniques whilst cft gave the lowest values. iha and elisa titres were affected by different preparations of the crithidial antigen extracts. highly significant r values were determined for control sera when iha was compared to elisa (r > . ), and to both cft and elisa with immune animals (r > . ). elisa would seem most applicable for screening other lower vertebrates for anti-parasite antibodies especially in areas of human disease prevalence. amphibians are frequently parasitized by various protozoans present in body fluids and tissues and in some cases the parasites cause serious and debilitating diseases (abrams, ; roudabush & coatney, ) . trypanosomatid flagellates, in particular trypanosomes, have been reported in and isolated from the blood of frogs, toads and newts (bardsley & harmsen, ; woo & bogart ) . throughout the investigations into trypanosomatid infection in humans and mammals, sera have been examined for both anti-parasite antibodies and parasite antigens using various immunodiagnostic techniques (strickland & hunter, ; tiru & hennessen, ) . however there is a dearth of information pertaining to the use of serodiagnostic methods to detect kinetoplastid infections and resultant antibody production in poikilothermic vertebrates with only reptiles having been studied (dollahon, hager & hua, ; ingram & molyneux, a , b. a . in this paper we present the results of acomparative assessment of four serological tests (direct agglutination, indirect haemagglutination, complement-fixation test and elisa) used to detect and to determine the levels of antibodies in the sera of green toads (b. viridis), used as experimental models. these amphibians had been injected with the choanomastigotes of c. fusciculutu, a trypanosomatid flagellate. the parasites were chosen because of their ease of culture under laboratory conditions and more importantly because of their presence in the blood and alimentary canal of ranid anurans under natural conditions (smyth & smyth, ) . to the authors' knowledge, this is the first report of the use of an immunoenzyme method to detect antibodies in amphibians. furthermore, there are no previous data concerning anti-parasite antibody detection in amphibian serum. materials and methods injecrion und serrr. before injection of the parasites. a small sample of blood was taken by cardiac puncture from each of the toads and inspected for any current infection (with naturally-occurring trypanosomatid flagellates) by smear, wet mount preparation and slope culture as described elsewhere (ingram & molyneux, a. b ). in addition. blood smears were also examined by an immunoenzyme method reported previously by ingram and molyneux ( y b, c) but using a rabbit anti-c., firscic~tkctcr serum/swine anti-rabbit immunoglobulins antiserum/ peroxidase-rabbit antiperoxidase/amino-ethylcarbazole substrate system. furthermore, the gut contents and pbs extracts of randomly selected insects and worms respectively were also examined microscopically and by culture for the presence of trypanosomatids. the choanomastigotes in culture overlay were centrifuged at x g for min. the overlay was removed and the parasites were washed three times in phosphate buffered saline-pbs. ph . (i mm-naci; . mm-na?hpo, and i mm-nah,po,. h,o in de-ionized water). the number of parasites was counted and the suspension adjusted with pbs to give the required dose for injection purposes.toads were given a single intraperitoneal (ip) injection of x io" choanomastigotes in pbs. control animals consisted of those given pbs ip and normal, uninjected toads. the toads were anaesthetized. bled, killed and their weights and lengths noted. the parasite-and pbs-injected animals were bled at -day intervals. the uninjected controls were sampled at random intervals throughout the duration of the experiment. in order to detect parasite infection. blood was examined in a similar manner to that obtained from prrinjected animals and the uninjected controls. the sera were isolated and stored at - °c. specificifv. the promastigotes and procyclics of lershmania herfigi her@ and trpanosoma brucei brucei respectively, both kinetoplastid flagellate species related to c'. ~uscic~ulrtu, were used in the antibody assays to examine for possible non-specific reactions in the toad sera. antisera preparation. rabbit anti-toad serum was produced by an immunization schedule as described previously (ingram & alexander. ) and the immunoglobulin tttres of the antisera obtained were estimated by either countercurrent electrophoresis or elisa (ingram & molyneux, a). antigen extract. parasite antigen extracts were produced in two ways for use in the immunological techniques. the choanomastigotes were centrifuged at x g for io min in cold pbs. ph . . the pellet formed was resuspended in pbs and then washed and centrifuged a further three times. prior to use, the cells were subjected to either freezing and thawing (f&t) or sonication (son) treatments. in the former case the pellet was resuspended in chilled pbs, broken up by mixing and the parasites f&t at -min periods for min. the material wsas then centrifuged to remove cell debris and the supernatant protein concentration measured by the lowry method. alternatively, after addition of cold buffer, the pellet was disrupted by ultrasonication for three -min intervals whilst the mixture was kept chilled. the sonicated material was then left overnight at "c to further remove any protein. it was then centrifuged at x g for i min and the amount of soluble protein in the supernatant determined. agglutination assay two-fold serial dilutions of toad sera. inactivated by heating at x"c for min to destroy naturally-occurring complement activity, were preparcd with pbs. ph . (containing x mr+naci). to each dilution wsas added an equal volume of choanomastigotes (i x io cells ml-') and the mixtures incubated at °c for min. the direct agglutination (da) end point titre was regarded as that dilution in which visible agglutination was observed when compared to the pbs/parasite controls. normal toad sera were examined for the presence of natural haemagglutinins against sheep crythrocytes (she) before commencing the iha test as described by weir (iy x). she were tanned with . x it)-' mg ml-' tannic acid and coated with either f&t or son ant&n extract containing .y mg ml-' protein. doubling dilutionr of inactivated sera were made and to each was added the same volume of % tanned and coated she.the test samples were incubated at °c for min followed by{ overnight at c. the samples were then examined and the degree of haemapglutination assessed. untanned, tanned and antigencoated she were used as controls. antigen. toad antisera and either bvs or gpc were mixed together and fixation allowed to occur overnight at "c. sensitized she were then added and the mixtures incubated at °c for min after which the end-points were scored. following further incubation for h at sc, the samples under test were re-examined. the complement-fixing antibody (cfa) titre was taken as that dilution which gave % hacmolysis. ellsa. a modification of the method of chandler. cox. premier & hurrell (i yx ) was used. the concentrations of antigen. rabbit anti-toad antiserum and enzyme-conjugate used for the elisa were determined by chequerboard titration. the f&t and son antigen extracts (containing i mg ml-' protein) were prepared in . m-carbonate/bicarbonate coating buffer ph y. : c'. fi~scicrtlartr was adjusted to . x " cells ml-' in coating buffer and the three antigen preparations were separately dried onto the plates by overnight incubation at °c. after washing with pbs (containing o.os% tween ) ph . , a range of two-fold serial dilutions of control and immune sera were added to the appropriate wells and the plates were reincubated at °c for min. they were washed again. rabbit anti-toad antiserum (elisa titre i : ) at a dilution of : s was added and the plates were incubated as before. after re-washing, sheep anti-rabbit i@ immunoglobulin comugated to urease. diluted i in . was dispensed into the wells and the plates were similarly incubated. after a final three washes with pbs/tween followed by four washes with distilled water, urease substrate (sera-i.ab, crawley. u.k.) was added to the wells. the plate\ were subjected to a final incubation at °c for ~ min and the reaction halted by the addition of i % thiomersal solution (v/v). the end point antibody titre was considered to be that dilution which was visually different from the appropriate reference controls included with each experimental run. the mean toad serum antibody titres, s.e.m. and ranges in the control and c. fasciculuta -injected groups, together with the number of sera-containing detectable antibodies, are given in table . the mean value, range in titres and number of animals in which antibodies were detected were lowest for the cft and highest by elisa. of the control and parasiteinjected toads, and %, respectively contained antibodies detectable by elisa. when all four immunological assays were applied to each individual serum, in all cases antibodies were detected by at least two or more of the methods used. however the background 'positive antibody' titres in the control animals ranged from to -j depending on the technique employed. therefore values higher than -' were considered to be positive for the parasiteinjected toads. furthermore comparisons between control and immune sera for each method revealed significant differences in antibody titres (p< . ; student's t-test). moreover, 'antibody' levels were not significantly different between the two control groups (p> . ). the titres against l. hertigi and t. brucei varied from to -j and from to - in the control and immune sera, respectively. the results for each technique were compared in turn with those for the other methods and the half matrix of the pearson product-moment correlation coefficients (r) calculated for all experimental animals ( table ). the significance of each r value was tested by the t-test. in the case of the control sera, r values ranging from to sy% (p< . ) were calculated whilst overall higher correlations were determined for sera from immune animals which varied from to % (all i'< . ). in order that the results of the present study can be used by other investigators for comparative purposes with different experimental models, regression formulae to convert the antibody titres as determined for each technique to those of another are given in table . the regression equations, based on the rectilinear relationship y = mx + c, were only calculated for the highest mean titre found for each of the four immunological methods. the mean antibody titres against c. fasciculata and the number of control and immune sera containing detectable antibodies were the lowest for da and cft, intermediate for iha and highest for the elisa method (table ) . these findings may reflect the different sensitivities of the immunological techniques used (voller & de savigny, y ). the classical agglutination test has often been used for antibody titration in amphibian immunobiological studies (cooper, ). significant correlations (p < . ) were obtained for both control (range y- %) and immune sera ( -y %) when the da titres were compared to the values found for the other methods. although da is simple to perform, preconditions of the test include antigen-type specificity, the non-immobilization and non-autoagglutination of parasites and usually the use of living cells. in the current study, loss of cell motility was observed in some instances and the possibility of inclusion of nonviable cells in others cannot be ruled out. nevertheless caution must be taken in the interpretation of natural 'antibody' levels in view of the detection of 'antibodies' in normal bvs against l. hertigi and t. brucei with titres within the range found for c. fuscicdata. positive results for normal bvs would suggest the presence of low amounts of specific immunoglobulins induced by a current infection with or previous exposure to c. fasciculata parasites. however, the low levels of naturally-occurring 'antibodies' in normal bvs may also have been stimulated by the environmental presence of micro-organisms or other trypanosomatid flagellates which non-specifically cross react with shared cell wall carbohydrate antigenic determinants (andrews, reilly, ferris & hanson, ; schnaidman, yoshida, gorin & travassos, ; sharabi & gilboa-garber, ). in the present study, the low levels of 'antibody' in sera from the control groups are not likely to be caused by a current infection because no increase in titres were found throughout the o-week duration of the experiment. the lack of detection of c. fasciculata in blood microscopically or by culture coupled with the finding of parasite antigen(s) in blood up to weeks postinjection by the immunoenzyme method suggests that . grids possesses an efficient immune system responsible for the rapid elimination of antigen. therefore it was not possible in the work reported here to correlate the level of parasitaemia and antibody titres. however the exposure of b. grids to c. fasciculata evoked a specific immune response and resulted in significantly increased levels of serum antibody. under normal environmental situations, it is feasible that naturally-occurring immunoglobulins in bvs restrict parasite numbers to below a potential infectious threshold. the finding of serum antibody titres above those of normal background levels in amphibians or other animal hosts in similar habitats or areas endemic for certain diseases would indicate current infection with parasites, other infectious agents or pathogens within a population. no haemolytic anti-complementary activities were detected in normal bvs against the antigen extracts unlike previous reports for amphibians (gigli & austen, ) . whereas the cft is frequently nonspecific and inconvenient for handling many samples, it can utilize crude, soluble parasite antigen extracts. as with da cross reactivity can often lead to false positive results. when the results obtained by cft were compared with each other and with iha and elisa, the lowest range of correlations, although significant and similar to those determined for the da comparisons, were found for control bvs ( - %; igm which is an efficient complement fixer and also a good agglutinin (atwell & marchalonis, ; yamaguchi. kurashige & mitsuhashi, ) . however, the 'antibodies' in normal bvs would be present in limited amounts, fix complement less effectively and hence result in low background cfa values. this could also account for the lowest correlations found when the cfa titres were compared to those of the other methods for the controls. the source of complement seems to be important for the efficient fixation of toad antibodies. the use of toad serum as homologous complement source gave a higher mean value and usually slightly higher individual endpoint titres in both control and immunized animals compared to the antibody levels obtained with heterologous gpc. homologous serum has proved a reliable source of complement for the fixation of immunoglobulins in other anurans (alexander & steiner, ; sekizawa, fujii & katagiri, ) . however the use of commercially available gpc is also known to initiate good fixation in amphibian species (lallone, chambers & horton, : romano, geczy & steiner, ). in the current study, % correlation (p < . ) and "/,, (p< . ) were found for control and immune sera. respectively when the different complement sources were compared. low (so- %, controls) and high ( - x, immunized) but significant (i'< . ) r values were determined in comparisons between iha and cft. iha is prone to lack specificity in some cases and, in contrast to the cft, usually requires highly purified antigen preparations but is easier to perform. elisa gave the highest percentage positive titres in all the samples examined and appears to be the most sensitive of the techniques used to detect anticrithidial antibodies in toad sera. elisa was easy to peform, specific, an important factor which affects the values of the antibody titrcs is the preparation of the antigen extract (crouch & raybould, ; pappas. hajkowski, cannon & hockmeyer. ) . son antigen preparations gave higher titres than f&t antigen extracts. however, the coating of elisa plates with whole cells produced the highest values. in the work reported here, similar batches of antigen were used thus reducing potential variations due to different preparations. it is of interest to note that a comparison between iha and elisa control titres revealed - % (p < . i ) significant correlations and similar numbers of positive animals. this implies that the above two techniques have similar sensitivities in the screening of normal bvs for anti-parasite antibodies. nevertheless the method of antigen preparation ma> not be a salient criterion for antibody estimation in immune sera because correlation values of over % (f'< . ) were found when iha titres were compared to those of elisa. elisa seems appropriate for use in serodiagnostic surveillance programmes. applied to different amphibian species or other aquatic and semi-aquatic lower vertebrates, to detect antibodies stimulated against diverse parasite environmental pathogens. furthermore this technique would be of value in screening lower vertebrates for potential carriers or reservoirs of infective kinetoplastid tlagellates or indeed other pathogenic micro-organisms. such information may reflect the health status of animals within a population and aid in the determination of specific epidemiological and aetiological features of zoonoses and epizootics prevalence. ac~k~f~~*,lef~~.lg~l?~e~~t,s-'i'his work was undertaken whilst one of us (gai) was on an eec funded project contracted to the british council. in part subcontracted to the university of salford, for the development of the faculty of science at yarmouk university. jordan. ga would like to thank dr s. k. abdul-hafez for the use of his laboratory facilities for part of the work, dr n. ishmail for assistance with the bleeding of the animals and dr ellen lee of the university of salford computing centre for advice on the use of the computer for statistical analysis of the results. diseax\ in an amphihian colony the first component of complement from the bullfrog runa cutrsheianu: functional properties of cl and isolation of subcompon-entclq leptospiral agglutinins in sera from southern illinois herpetofauna immune response in xenopua iuevis and immunochemical properties of the serum antibodies. immunology : -l x. key: cord- -qwdxgk z authors: lafaye, pierre; li, tengfei title: use of camel single-domain antibodies for the diagnosis and treatment of zoonotic diseases date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: qwdxgk z camelids produce both conventional heterotetrameric antibodies and homodimeric heavy-chain only antibodies. the antigen-binding region of such homodimeric heavy-chain only antibodies consists of one single domain, called vhh. vhhs provide many advantages over conventional full-sized antibodies and currently used antibody-based fragments (fab, scfv), including high specificity, stability and solubility, and small size, allowing them to recognize unusual antigenic sites and deeply penetrate tissues. since their discovery, vhhs have been used extensively in diagnostics and therapy. in recent decades, the number of outbreaks of diseases transmissible from animals to humans has been on the rise. in this review, we evaluate the status of vhhs as diagnostic and therapeutic biomolecular agents for the detection and treatment of zoonotic diseases, such as bacterial, parasitic, and viral zoonosis. vhhs show great adaptability to inhibit or neutralize pathogenic agents for the creation of multifunctional vhh-based diagnostic and therapeutic molecules against zoonotic diseases. the generation of monoclonal antibody (mab) triggered a revolution in biotechnology. therapeutic mabs belong to the fastest-growing branch of biotechnology. however, the large size of the molecules may hinder efficient tissue penetration. this obstacle can be overcome using fab or single-chain fv (scfv) fragments, as they are three to six times smaller than full-sized antibodies. however, cloning these fragments, consisting of heavy and light chain variable regions linked by disulfide bridges or a linker, is challenging and they are not always efficiently expressed [ ] . an alternative approach to avoid these pitfalls is the use of functional fragments of heavy chain antibodies, which are present in the serum of animals belonging to the camelidae family. they interact with the antigen by virtue of one single variable domain referred to as vhhs, single-domain antibodies (sdabs), or nanobodies ™ (trademark of ablynx). they combine the advantages of both immunoglobulins and small molecules and provide an alternative to conventional antibodies and binders derived from alternative scaffolds. sharks also produce a unique heavy-chain only immunoglobulin that does not associate with light chains, referred to as ignar. v domains of ignar are often applied in biotechnological and biomedical fields (for a review [ ] ). the camelidae family (order: artiodactyla) consists of six species: dromedary (camelus dromedarius), bactrian camel (camelus bactrianus), llama (lama glama), guanaco (lama guanicoe), alpaca (vicugna pacos), and vicuna (vicugna vicugna). animals belonging to this family are well adapted to live in harsh environments, such as deserts or high altitudes [ ] . ungar-warom et al. [ ] and azwai et al [ ] have isolated low molecular weight ig-like proteins from dromedary serum. however, the detailed characterization and demonstration of the potential usefulness of these proteins stemmed from the work of the hamers laboratory in the free university of brussels [ ] . the authors demonstrated that these unusual proteins are "heavy-chain only antibodies'' (hcabs), devoid of light chain. these antibodies form homodimers and interact with antigens by virtue of only one single variable domain, referred to as vhh (vh domain of heavy-chain antibodies) to distinguish it from conventional vh [ ] . in contrast to conventional antibodies, hcabs do not possess the ch domain [ ] . the active antigen-binding fragment of heavy chain antibodies can be cloned and expressed in the form of vhh, which consists of only one domain (fig. ) . the percentage of hcabs in the bloodstream of camelids varies greatly among species. it can reach a relatively high level in camels, ranging from % to %, whereas the maximum level is % in south american camelid species [ ] . despite the absence of light chains, the heavy-chain antibodies exhibit a broad antigen-binding repertoire. they exhibit specific characteristics, such as the substitution of three to four hydrophobic residues (which interact with the vl in conventional antibodies) by more hydrophilic amino acids. vh possess the conserved val , gly , leu and trp while in vhh, these amino acids are often substituted (val phe or val tyr, gly glu, leu arg and trp gly) [ ] [ ] [ ] . furthermore, the complementarity determining regions (cdrs) of vhhs, and especially cdr , are statistically longer than those of conventional vh-vl antibodies [ ] . over the last decades, vhhs have received progressively greater interest from the pharmaceutical and biotechnology industries, due to their specific properties. indeed, vhhs provide the following advantages over conventional antibodies and their recombinant fragments: -vhhs are weakly immunogenic in humans, because the genes encoding them share high sequence homology with genes belonging to human vh families and [ , ] . -vhhs consist of only one domain. they can thus be easily engineered, cloned, and expressed with high yields using various expression systems [ ] . -vhhs are highly soluble and stable, even under denaturing conditions or high temperatures [ ] . -the high variability of the length and sequence of vhhs allows them to recognize a variety of protein epitopes, located not only on the surface of a protein [ ] , but also buried deep in the clefts [ ] . vhhs have been shown to recognize a wide range of epitope types, from small haptens [ , ] to the binding sites of enzymes [ , ] , and can bind epitopes that cannot be recognized by conventional antibodies [ ] . -the small size and basic isolectric point of vhhs allows them to penetrate tissues, pass through barriers, such as the blood-brain barrier [ ] [ ] [ ] [ ] [ ] , and bind intracellular antigens. -vhhs can be efficiently functionalized [ ] [ ] [ ] and are widely used for imaging [ , , ] . there has been an explosion in the number of publications concerning applications of vhhs that cover their use in pharmaceutical development or as biotechnological tools [ , [ ] [ ] [ ] [ ] [ ] . here, we mainly focus on the potential use of vhhs for the diagnosis and the treatment of zoonotic diseases. zoonotic diseases (zds) are infections that can be naturally spread between vertebrate animals and humans. several recent outbreaks, such as ebola and zika have emphasized the serious impact of these diseases on human health [ ] . their expansion is related to global trade, human migration, and climate change. up to now, the outbreaks have been contained by the control of human populations in the affected areas, the use of antibiotics, and the development of rapid diagnostic tests. however, the emergence of zds is still a major challenge and there is a crucial need for new tools for diagnosis and therapy. camelid vhhs could offer an attractive possibility for the development of such tools in the future. campylobacteriosis is caused mostly by campylobacter jejuni or campylobacter coli. poultry are naturally infected, without clinical signs, and it is the leading cause of foodborne gastroenteritis in humans worldwide [ ] . a vhh that binds c. jejuni flagella was isolated by riazi et al. [ ] and engineered for greater thermal and proteolytic stability. hussack et al. [ ] obtained a highly stable vhh through the use of error-prone polymerase chain reaction and disulfide-bond engineering. this vhh, directed against the flagella, can potently inhibit c. jejuni motility and is being studied for the prevention or significant reduction of c. jejuni colonization in the gastrointestinal tract of chickens. recently vanmarsenille et al [ ] described the isolation and characterization of vhhs against multiple campylobacter strains. these vhhs which bind with the major outer membrane protein (momp) interacted with c. jejuni isolates and c. coli isolates. they could potentially be used in therapy and as a diagnostic tool. escherichia coli is a facultative gram-negative bacterium that belongs to the family enterobacteriaceae. although it is normally commensal in nature and animals, many strains are food and waterborne zoonotic pathogens. shiga toxin (stx)-producing e. coli (stec) bacteria (which include enterohemorrhagic e. coli [ehec]) cause no discernible disease in their animal reservoirs; however, diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (hus) are common in humans [ ] . the major virulence determinants of stec are mainly caused by the shiga toxins stx and stx [ ] . vhhs that neutralize stx and/or stx have recently been obtained [ ] [ ] [ ] . the vhh vb specific of stx was unable to give protection against a lethal dose of toxin in mice while a trivalent molecule (two copies of vhh vb and one copy of anti-albumin vhh) presented an extended half-life and was able to neutralize the in vivo effect of toxins in mouse models [ ] . tremblay et al [ ] showed that vhh heterodimers, containing two linked neutralizing vhhs, generally neutralized stx much more efficiently than a pool of individual monomers. moreover co-administration of an effector ab substantially improved the ability of stx toxin-neutralizing vhhs to prevent death or kidney damage in mice following challenge with stx or stx . listeriosis is a bacterial infection caused by the gram-positive bacterium listeria monocytogenes. the major source of infection is contaminated food. the disease primarily affects elderly people, immunocompromised patients, pregnant women, causing abortion, and newborns [ ] . vhhs specific for the invasin, internalin b, of l. monocytogenes have been isolated [ , ] and can inhibit listeria invasion in vitro. a crystal structure between one vhh and internalin b shows that the vhh competes with c-met, the target cell receptor of this invasin, explaining the protective effect [ ] . two vhhs that specifically bind to three l. monocytogenes serotypes ( / a, / b, and b) have been isolated from a non-immune library [ ] . a sandwich elisa was developed using a mab for antibody capture and one vhh, l - for detection, with a detection limit of × colony forming units (cfu)/ml of bacteria in milk. bacillus anthracis, the causative organism of anthrax, is a sporeforming gram-positive bacillus commonly found in the soil of endemic areas. herbivores can be infected while grazing. recently, anthrax was detected in siberia after degradation of the permafrost due to global warming [ ] . b. anthracis is also one of the most important biological warfare agents, because of its pathogenic nature and spore-forming capacity [ ] . the bioterrorist events in the united states in revealed that treatment with antibiotics is not always sufficient to prevent patient deaths, due to the effects of toxins produced by the bacteria. neutralizing mabs may therefore be of great therapeutic value, complementing antibiotic treatment to prevent the toxin-dependent symptoms of anthrax [ ] . anthrax is caused by a toxin consisting of protective antigen (pa), lethal factor (lf), and edema factor (ef). several vhhs directed against the three components of the toxin have been shown to be efficient against anthrax disease [ , ] . gene therapy with an adenoviral vector expressing a bispecific vhh, consisting of two linked vhhs targeting different pa-neutralizing epitopes, was tested in mice, and found to protect them from anthrax toxin challenge and anthrax spore infection [ ] . taeniasis occurs in the human host, after ingestion of undercooked pork infected with cysticerci. cysticercosis is caused by the larval stage of the pork tapeworm taenia solium. humans are the definitive host, harboring the adult tapeworm in the intestine, but both pigs and humans can be infected with the cysticerci [ ] . the mabs obtained thus far are not genus-specific, preventing definitive diagnosis of infection by t. solium [ ] . indeed, specific binders are needed. deckers et al. [ ] have isolated vhhs specific for a glycoprotein of t. solium that do not cross react with other taenia species and a sandwich elisa has been developed. this serodiagnostic test could be helpful in pigs for epidemiological studies and monitoring the efficacy of control programs. african trypanosomiasis (at), or sleeping sickness, is mainly caused by a unicellular flagellated protozoan parasite, trypanosoma brucei gambiense, belonging to the genus trypanosoma. at affects mainly remote rural areas and its distribution coincides mostly with the habitat of the hematophagous insect vector, i.e., the tsetse fly (glossina sp) [ ] . multiple vhhs have already been generated against the parasite (for a review, [ ] ). trypanolytic vhh an disturbs the endocytic machinery of the parasite in the flagellar pocket of the parasite. nontrypanolytic vhh an was made more potent by linking the nontoxic prodrug cephalosporin mustard (ccm) onto the highly toxic pdm at the surface of the parasite [ ] . linking this vhh to apolipoprotein l-i resulted in an immunotoxin that lyses almost all trypanosomes [ ] . another approach is to couple pentamidine, a first-line antitrypanosomiasis, to vhh an to effectively target the drug to the parasite. in vivo, a ten-fold lower dose than the minimal full curative dose of free pentamidin incorporated into this conjugate cured all infected mice, wheras a -fold lower dose cured % of them [ ] . parasite development in the tsetse fly and subsequent spread of the parasite can be controlled through the expression of trypanolytic vhhs in genetically modified tsetse fly symbionts [ ] . in addition, vhhs that target the paraflagellar rod protein of varioius trypanosomes have been described, but are mainly useful as diagnostic markers of trypanosiomasis [ ] . a vhh (nb ) directed against t. congolese aldolase (tcoald) has been developped for a sandwich immunoassay. this vhh is highly specific and did not recognize other trypanosomes such as t. brucei brucei, t. vivax and t. evansi [ , ] . influenza a viruses (iav), members of the rna family orthomyxoviridae, consist of up to subtypes, depending on the variation/combination of the surface glycoproteins, hemagglutinin and neuraminidase. iav are further classified as human, swine (siv), bat, equine, or avian influenza viruses (aiv). siv and aiv are transmitted from pigs or birds to humans, respectively, mostly via direct contact with infected animals. the infection in humans ranges from mild selflimiting respiratory-like illness to death. however, pandemic outbreaks remain unpredictable, as illustrated by the h n virus (also named mexican flu) and h n virus. occasional zoonotic infections with these viruses and their high propensity to reassort with siv have earmarked them as a major pandemic threat [ ] . several vhhs specific for influenza viruses have been raised against the nucleoprotein [ ] and m ion channel protein [ ] of influenza a, and the neuraminidase [ ] and hemaglutinin [ ] of h n . most of these vhhs can neutralize influenza viruses. ploegh et al. [ ] exploited the ability of vhhs to bind the intracellular nucleoprotein protein to block viral replication, leading to the possibility of creating new therapeutic molecules to prevent viral escape due to antigenic variation. an alternative approach has been to create multivalent vhhs to increase their antiviral potential: dimers made by the fusion of two neutralizing vhhs [ , ] or a vhh-fused to an immunoglobulin fc region [ ] . in vitro antiviral potency was increased from to logs relative to the monomeric vhh. the rabies virus (rabv) belongs to the genus lyssavirus of the rna family rhabdoviridae, within the order mononegavirales. despite the availability of a vaccine against rabies virus (rabv), rabies continues to claim , human lives per year, mostly in developing countries in asia and africa [ , ] . the vaccine is mostly used therapeutically as a post-exposure treatment. for infection combined with a seriously bleeding injury, the who recommends complementing vaccination with local instillation of human or equine rabies immunoglobulins (rig) to neutralize the rabv load in situ. there is currently a critical shortage worldwide and the who is exploring alternative approaches, such as cocktails of human or humanized neutralizing mabs [ ] . neutralizing anti-rabv vhhs directed against glycoproteins have been raised from a vhh phage library generated from the immunization of a llama with inactivated rabies vaccine (genotype , sanofi pasteur msd) [ ] . the ic s of the cvs- (genotype ) strain ranged from to nm. these vhhs were fused to an anti-albumin vhh to extend its serum half-life and were able to neutralize the virus at picomolar doses [ ] . a combined treatment based on vhh and vaccine (rabipur, novartis) acted synergistically to protect mice in an intranasal rabies infection model [ ] . however, the principal difficulty of an antiviral approach against rabies resides in the specific neurotropism of rabv, which makes it not readily accessible once it has accessed the cns. at this stage, only molecules capable of crossing the blood brain barrier and penetrating into neurons would be able to inhibit the infection. foot-and-mouth disease (fmd) is a contagious viral disease that affects cattle, swine, sheep, and approximately wildlife species (including llama and camel), with a potential for rapid spread between susceptible animals. the disease has been identified worldwide whereever livestock are raised. in the last years, there have been massive outbreaks of fmd in countries formerly free of the disease, such as the united kingdom in [ ] and taiwan in [ ] . seven antigenically distinct serotypes of fmd viruses have been identified: o, a, c, asia , sat , sat , and sat [ ] . emergency vaccination can be used as an effective control measure for fmd outbreaks in fmd-free regions, such as the european union, but the development of novel antiviral therapies that confer rapid protection against fmd is still needed. moreover, it is important to develop a rapid diagnostic test for identification of the various serotypes of the viruses involved in fmd outbreaks. vhhs directed against serotype o have been raised. they have provided only limited protection to pigs. trimers consisting of two vhhs specific for fmdv and one vhh specific for porcine ig have been constructed to increase their potency and half-life. these trimers provided better protection to pigs and delayed fmd transmission [ ] . specific vhhs raised against fmd asia virus have also been obtained. they have been used to develop diagnostic assays by conjugating them to either quantum dots [ ] or carboxyl-magnetic beads [ ] . in this review, we provide evidence for the possible use of vhhs as valuable biomolecules for the diagnosis and treatment of zds, including bacterial, parasitic, and viral zoonosis. vhhs provide many advantages over conventional antibodies and currently used antibody based fragments. the ease of high-level production, small size, and high stability make vhhs extremely reliable for genetic and chemical modification, such as the production of vhh-based fusion proteins to increase the persistence of vhhs in serum or confer additional functions. construction of multivalent vhhs consisting of two or more linked vhhs targeting various epitopes leads to an increase in their ability to neutralize toxins [ , ] and viruses [ , , , ] , suggesting the development of these approaches for vhhs targeting other viruses not yet tested. presently available anti-viral therapeutic mabs, at various stages of pre-clinical evaluation, mostly target surface antigens that are often diverse or variable in sequence (e.g. hiv gp , influenza ha): their efficiency thus requires challenging protein engineering efforts to obtain antibodies that are either broadly neutralizing or robust against viral escape through antigenic variation (drift). an alternative is the use of "broadly neutralizing antibodies (bnab)", which are antibodies found in infected mammals able to neutralize most strains of a given highly antigenically variable pathogen. bnabs have been isolated from infected humans against hiv [ ] , influenza [ ] , and dengue viruses [ ] . camelids could also be a source of bnabs. indeed antibodies against mers-coronaviruses (mers-cov) [ ] [ ] [ ] , crimean congo hemorrhagic fever virus (cchfv) [ , ] , rift valley fever (rfv) [ ] , toxoplasma gondii, and rickettsia sp. [ ] have been found in the sera of infected camels, whereas antibodies against rabies virus, vesicular stomatitis virus, and fmd virus have been detected in llamas [ ] and could lead to the possible isolation of specific broadly neutralizing vhhs. many neutralizing vhhs that bind to different sites on the same target, including hidden antigenic sites, can be isolated from immunized or infected camelids. these vhhs can be engineered in various ways to improve their diagnostic and/or therapeutic properties and efficacy. in addition, vhhs readily and rapidly penetrate tissues, even the brain, and it is likely that specific vhh-based constructs will be developed that can neutralize agents involved in brain infections, such as influenza [ ] , zika, or rabies virus. overall, vhhs or vhh-based molecules are potentially valuable diagnostic and therapeutic reagents to treat zds. expression of anti human il- and il- scfvs in transgenic tobacco plants antibody repertoire development in 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reduces foot-and-mouth disease transmission between pigs characterization of single-domain antibodies against foot and mouth disease virus (fmdv) serotype o from a camelid and imaging of fmdv in baby hamster kidney- cells with single-domain antibody-quantum dots probes specific detection of foot-and-mouth disease serotype asia virus by carboxyl-magnetic beads conjugated with single-domain antibody broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals tackling influenza with broadly neutralizing antibodies recognition determinants of broadly neutralizing human antibodies against dengue viruses antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies to hemorrhagic fever viruses in domestic livestock in niger: rift valley fever and crimean-congo hemorrhagic fever crimean congo hemorrhagic fever among the one-humped camel (camelus dromedaries) in central sudan a transversal study on antibodies against selected pathogens in dromedary camels in the canary islands serologic survey of viral antibodies in the peruvian alpaca (lama pacos) key: cord- -odly fwg authors: madic, j.; magdalena, j.; quak, j.; van oirschot, j. t. title: isotype-specific antibody responses to bovine herpesvirus in sera and mucosal secretions of calves after experimental reinfection and after reactivation date: - - journal: veterinary immunology and immunopathology doi: . / - ( ) - sha: doc_id: cord_uid: odly fwg abstract isotype-specific antibody responses to bovine herpesvirus (bhv ) were measured in sera, nasal, ocular and genital secretions of calves that were reinfected with bhv and weeks later treated with corticosteroids to reactivate putative latent virus. after reinfection and after reactivation, no bhv -specific igm antibody response was detected. the serum iga response was only transiently detectable after reinfection and again appeared rapidly after reactivation in most calves. most calves showed an increase in nasal and ocular iga titres after reinfection and reactivation; some calves also had iga antibodies in genital secretions. a salient finding was that after reinfection and reactivation more calves showed a serum iga response than virus shedding or an increase in serum igg or igg titres. this suggests that the serum iga response would be the most sensitive indicator to detect bhv reinfection and reactivation. no correlation was found between nasal iga titre at the time of reinfection or corticosteroid treatment and the period of virus shedding, suggesting that nasal iga does not play a major role in protection against reinfection with bhv . bovine herpesvirus (bhvl) can cause the disease entities: infectious bovine rhinotracheitis, infectious pustular vulvovaginitis and infectious balanoposthitis. because the immunity induced by a primary bhvl infection is relatively shortlasting, cattle may be reinfected with bhvl. cattle infected with bhvl are lifelong carriers of the virus in a latent, non-infectious form, mainly in the trigeminal ganglia. as a result of stress, simulated by the administration of corticosteroids, bhvi can be reactivated and again be shed into the environment. because the isotype-specific antibody responses after reinfection with and reactivation of bhvl have only been fragmentarily studied (rodak et al., ; guy and potgieter, a,b; osorio et al., ; edwards et al., ) , we extensively investigated these responses. in this report we describe their kinetics in sera, nasal, ocular and genital secretions of experimentally reinfected and corticosteroid treated calves. the isotype-specific antibody responses after primary infection have been recently reported (madic et al., ) . in a previous experiment (madic et al., ) , calves were randomly divided into eight groups of four calves. seven of these groups were intranasally infected with one of seven bhvl strains and one group served as control (table ) . two calves inoculated with the iowa strain died. in the present experiment the remaining calves were all (re)inoculated by intranasal spray, ml into each nostril, containing a total of lo tcid,, of the virulent iowa strain of bhvl (kindly provided by dr. j.m. miller, national disease center, ames, iowa), weeks after the primary infection. the titre of the inoculum was verified by virus titration on the day of inoculation. none of the calves developed clinical signs characteristic of bhv , except the primary inoculated control calves. six weeks after reinfection all calves were treated intramuscularly with . mg of dexamethasone (opticortenol os%, ciba) kg-' body weight for consecutive days. after this treatment none of the calves developed moderate or severe clinical signs. blood samples were collected on post-reinfection days (prd) , , , and and on post-treatment days (ptd) , , , , and , where ptd marks the beginning of the corticosteroid treatment. serum was obtained from whole blood by cenuifugation and stored at - °c until tested. nasal, ocular and genital secretions were collected on , , , , , , , , , and and on ptd , , , , , , , , and . the mucosal secretions were collected with cotton tips (medical wire & equipment co. (bath) ltd., potley, corsham, uk) and suspended in ml of phosphate buffered saline solution, ph . , clarified by centrifugation and stored at - °c until tested. between and ~ of nasal or ocular secretions, and - ~ of genital secretions were absorbed by a cotton tip. thus, the dilution (log,,) factor for nasal or ocular secretions was between . and . , and for genital secretions between . and . . blood contamination of secretions was checked with the sangur-test (boehringer gmbh, mannheim, germany). for virus isolation, nasal swabs were collected on prd o- , , and and nasal and ocular swabs on ptd o- , , and . virus was isolated from these swabs as described previously (kaashoek et al., ) . ' + . appearance of anttbodies or a four-fold or higher increase in titre when the pre-sample was positive. "number of days of virus excretion. these were performed exactly as previously reported (madic et al., ) . an igm antibody response was not detected in sera or secretions of any of the reinfected calves. the control calves, which were infected for the first time, developed an igm antibody response in serum, nasal and ocular secretions that was first detected on prd and lasted until prd . after corticosteroid treatment none of the calves developed detectable igm antibodies against bhvi in sera or secretions. twenty-one of the calves reacted after reinfection with a detectable iga response in serum ( table ). the mean prd of first detection was . . the control calves all developed iga serum antibodies with a mean prd of first detection of . the iga antibodies in reinfected calves peaked around prd - ( approximately days after their first detection. the mean prd of last detection was . for the reinfected calves and . for the control calves. in nasal secretions a four-fold or higher increase of iga antibody titres was found in all but one reinfected calves (table ) . this increase occurred between prd and , with a mean prd of first detection of . . in the control calves iga was first detected in nasal secretion on prd . the iga antibody peaked between prd and and gradually decreased thereafter (fig. ) . in all but three calves, the iga antibodies persisted until the day of corticosteroid treatment (ptd ). a fourfold or higher increase in ocular secretions was seen in all but reinfected calves (table ) between prd and , with a mean prd of first detection of . . all control calves developed iga antibodies in ocular secretions with a mean prd of first detection of . the mean titres in ocular secretions were lower than in nasal secretions (figs. and ). in of calves iga persisted in ocular secretions until ptd . the individual iga antibody titres in secretions varied considerably. in of reinfected calves iga antibody was detectable at low levels in genital secretions for a few days (table ) . iga antibodies were not detected in genital secretions from control calves. after corticosteroid treatment all but one calf developed serum iga antibodies (table ) . on average, these appeared in the blood on ptd . . the serum iga antibodies remained detectable for a period varying from to days. in animals they were still present on ptd , which marked the end of the experiment. in all but two calves a fourfold or higher increase of iga antibodies was noted in nasal secretions after corticosteroid treatment; in ocular secretions such an increase was found in all but three calves (table ) . the mean ptd of this increase was . (range - ) for nasal secretions and . (range - ) for ocular secretions. the peak of the mucosal iga titres was around ptd and reached values comparable to those after reinfection (figs. and ) . the individual mucosal iga antibody titres fluctuated considerably and persisted until the end of the experiment. in nine calves iga antibodies were detected in genital secretions; five of them also had genital iga antibodies after reinfection (table ) . ocular iga . , in of the calves, reinfection did not lead to a significant increase in igg antibody titre in serum; in the remaining five calves a fourfold or higher increase was detected between prd and . in all control calves iggl antibodies were first detected on prd . the course of mean serum igg titres per group is given in fig. . nine calves, two of which were controls, had minimal iggl antibody titres (mean log,, titre of . ) in nasal secretions and reinfected calves in ocular secretions (mean log,, titre of . ) for a short period. in none of these secretions were erythrocytes detected. the genital secretions were negative (table ) . after corticosteroid treatment of calves showed a significant increase in iggl antibody titre in serum, usually on ptd . the igg antibody titres of the other remained stable. the course of mean serum iggl titres per group is given in fig. . in nasal secretions of of calves low levels of iggl antibodies (mean log,, titre of . ) were detected and in ocular secretions of of calves (mean log,, titre of . ), during a period varying from to days. in approximately one-third of the nasal secretions, - erythrocytes pull ' were demonstrated. erythrocytes were not detected in ocular secretions. two calves had igg antibody in genital secretions, which were free of erythrocytes (table ) . the reinfection did induce a fourfold or higher rise in igg serum antibody titre in of the calves between prd and . all control calves induced an igg serum response between prd and . the development of mean serum igg titres is shown in fig. . three calves showed low levels of igg antibodies (mean log,, titre of . ) in nasal secretions after reinfection, and none had these antibodies in ocular or genital secretions after reinfection (table ) after corticosteroid treatment the serum igg antibody titre significantly increased in of calves, between ptd and . the development of mean serum igg titres is shown in fig. . thirteen of calves had detectable levels of igg antibodies (mean log,, titre of . ) in nasal secretions, and of calves in genital secretions; none had igg antibodies in ocular secretions (table ) . the number of days of virus excretion per individual calf after reinfection and after corticosteroid treatment is given in table . after reinfection bhvl was shed between prd and , with a mean prd of first detection of . . after corticosteroid treatment virus excretion was usually detected between ptd and , with a mean ptd of first detection of . . there was no correlation between nasal or ocular iga antibody titre at the time of reinfection or corticosteroid treatment, and the respective virus excretion periods in the individual calf, e.g. some calves that had no nasal iga at reinfection shed bhvl for a shorter time than some calves that had a high nasal iga titre at reinfection (data not shown). calves in which no virus excretion after reinfection and/or corticosteroid treatment was detected yet developed an antibody response against bhvl, mainly of the iga isotype. the primary infections had induced a solid immunity, because intranasal reinoculation with a high dose of a virulent bhvl strain prevented virus shedding in of calves. in the remaining calves, reinfection led to l- days of minimal virus excretion, whereas the primary infected controls had the typical virus excretion patterns (kaashoek et al., ) . in spite of this minimal virus replication, all calves did respond with antibody formation to the reinfection. after corticosteroid treatment virus replicated longer and to higher titres in nasal and ocular mucosae, which resulted in a more pronounced antibody response. a bhvl-specific igm antibody response was not detected in any of the reinfected or reactivated cattle in either sera or mucosal secretions. after intranasal re-exposure with bhv , guy and potgieter ( a) observed a slight increase in igm antibody titre, whereas after intra-amniotic inoculation a rapid increase was detected after abortion. in addition, they detected a secondary igm antibody response after reactivation (guy and potgieter, b) . these discrepancies may be explained by the difference in igm detection method or by differences in experimental design, including breed of cattle, strain and dose of virus, route and duration of corticosteroid treatment. osorio et al. ( ) could not detect an igm antibody response after bhvl reactivation either. after reactivation of herpes simplex virus, an igm antibody response is seldom observed and then associated with severe encephalitis (kalimo et al., ; van loon et al., ) ; however, reactivation of herpes zoster virus often leads to an igm response (tedder et al., ) . after reinfection of calves with bovine respiratory syncytial virus (brsv) igm was not detected in sera, but was detected in nasal and ocular secretions (kimman et al., ) . however, the pathogenesis of a brsv infection is different from that of a bhvl infection: the latter giving rise to clinical signs of the upper respiratory tract, whereas a brsv infection mainly affects the lower respiratory tract. one of the most salient findings of this study is that in most calves a temporary iga serum response was demonstrated both after reinfection and after reactivation. the serum iga response in reinfected or reactivated calves was much earlier detectable than that in primary infected calves, indicating a memory response. rodak et al. ( ) and edwards et al. ( ) could not detect any serum iga response in bhv -reinfected calves. eleven calves in which no virus excretion could be detected after reinfection and/or reactivation even showed a clear systemic iga antibody response. an explanation for this unexpected tinding may be that in latently infected calves that are given corticosteroids non-infectious physical rather than infectious bhvl particles may be produced (pastoret et al., ) . these physical particles stimulate an antibody response. however, the discrepancy between virus excretion and antibody response may be due to our failure to detect infectious virus particles, because these were not excreted via nasal or ocular secretions or because only one nasal or ocular swab was taken per h. however, it may be the serum iga response seems to be a much more sensitive indicator for reinfection with or reactivation of bhvl than detection of nasal and ocular virus shedding. in the case of brsv infection the secondary serum iga response was not transient, but persisted for more than . months (kimman et al., ) . not only was there a vigorous iga response in serum, but also a secondary type iga antibody response was observed in nasal and ocular secretions in most reinfected and reactivated calves. the mucosal iga response after reinfection was more rapid and stronger than in the primary infected calves (figs. and ) , indicating the existence of mucosal memory and thus confirms earlier reports on brsv and bovine corona virus infections (kimman et al., ; heckert et al., ) . after the primary infection (madic et al., ) , iga antibodies were not detected in genital secretions, whereas, probably as a result of priming, after reinfection and reactivation genital iga antibodies were found in of calves. the increase of nasal iga antibodies followed the onset of the virus shedding period with several days, suggesting that existing nasal iga antibodies, which can neutralise bhvi (madic et al., ) ) did not play a major role in curtailing the amount of virus shedding after reinfection or reactivation. this is further substantiated by the finding that no correlation existed between nasal iga titres at reinfection and onset of corticosteroid treatment on the one hand, and virus shedding periods on the other. whereas of calves had virus in nasal secretions, only five had an increase in serum iggl titre and in serum igg titre after reinfection. guy and potgieter ( a) also found that the antibody increase after reinfection was more frequent in the igg than in the iggl isotype. after corticosteroid treatment, virus-positive nasal secretions were detected in of reinfected calves, and a significant increase in serum iggl and igg antibody titre in and calves, respectively. there was no correspondence between virus shedding and antibody titre increases (table ). the inability of some calves to elicit a booster iggl or igg serum antibody response may partially be due to the high antibody titres that already existed after reinfection and corticosteroid treatment. in any case, more calves reacted with a systemic iga response after reinfection and corticosteroid treatment than with a systemic iggl or igg response. this further supports the systemic iga response to be a sensitive indicator of bhvl reinfection and reactivation. particularly after reactivation, but also after reinfection, iggl antibodies were seen in nasal and ocular secretions. igg antibodies were only detected in nasal secretions in fewer calves than iggl antibodies. most of the igg containing secretions were free of erythrocytes, excluding leakage of blood as a cause of the igg and igg presence. whether transudation or selective transport from serum or mucosal production is responsible for the mucosal igg cannot be conclusively determined, but the iggl /igg antibody ratio in serum usually did not correspond with that in nasal secretion (data not shown). this indicates selective transport of iggl and igg rather than transudation from serum. the igg isotype was never detected in ocular secretions after primary infection (madic et al., ) , after reinfection and after reactivation, suggesting that this isotype may not or rarely pass the conjunctival mucosae. this finding is also in favour of selective transport of iggl through ocular mucous membranes. iggl and igg antibodies against bhv were detected in genital secretions in a few calves, only after corticosteroid treatment. there were clear differences in periods of virus shedding after corticosteroid treatment between the groups of calves primary infected with different bhvl strains, e.g. calves given the cooper strain had by far the longest virus shedding periods from nose and eye. these differences in virus shedding did, however, not result in marked differences in isotypespecific antibody responses between the groups of calves. to our knowledge, this is the most extensive study performed on isotype-specific antibody responses to bhvl after reinfection and reactivation. we may conclude that the igm isotype is absent in secondary and tertiary antibody responses to bhvi and that the serum iga response is a more sensitive indicator of bhvl reinfection and reactivation than virus shedding or the increases in iggl or igg serum titres. in addition, nasal iga antibody does not to appear to be involved in virological protection against reinfection or reactivation. the virulence of british isolates of bovid herpesvirus in relationship to viral genotype bovine herpesvirusinfection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus kinetics of antibody formation after the reactivation ofbovine herpesvirus- infection in cattle a conventionally-attenuated glycoprotein e-negative strain ofbovine herpesvirus type is an efficacious and safe vaccine solid-phase radioimmunoassay of human immunoglobulin m and immunoglobulin g antibodies against herpes simplex virus type capsid, envelope, and excreted antigens local and systemic antibody response to bovine respiratory syncytial virus infection and reinfection in calves with and without maternal antibodies isotype-specific antibody responses to bovine coronavirus structural proteins in serum, feces, and mucosal secretions from experimentally challenge-exposed colostrum-deprived calves isotype-specific antibody responses in sera and mucosal secretions of calves experimentally infected with bovine herpcsvirus detection ofbovine herpesvirusl-specific igm using a capture enzyme immune assay with isotypespecific monoclonal antibodies effect of repeated treatment with dexamethasone on the re-excretion pattern of infectious bovine rhinotracheitis virus and humoral immune response a study of the dynamics of the production of class-specific antibodies to infectious bovine rhinotracheitis (ibr) virus in calves using a solid-phase radioimmunoassay detection of antibody to varicella-zoster virus by competitive and igm-antibody capture immunoassay diagnosis of herpes encephalitis by elisa the authors thank h. paal and k. weerdmeester for skilful technical assistance. key: cord- -cynpob b authors: godakova, svetlana a.; noskov, anatoly n.; vinogradova, irina d.; ugriumova, galina a.; solovyev, andrey i.; esmagambetov, ilias b.; tukhvatulin, amir i.; logunov, denis y.; naroditsky, boris s.; shcheblyakov, dmitry v.; gintsburg, aleksandr l. title: camelid vhhs fused to human fc fragments provide long term protection against botulinum neurotoxin a in mice date: - - journal: toxins (basel) doi: . /toxins sha: doc_id: cord_uid: cynpob b the bacterium clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (bont) and characterized by damage to the nervous system. in an effort to develop novel c. botulinum immunotherapeutics, camelid single-domain antibodies (sdabs, vhhs, or nanobodies) could be used due to their unique structure and characteristics. in this study, vhhs were produced using phage display technology. a total of different monoclonal vhhs were selected based on their comlementarity-determining region (cdr ) sequences. different toxin lethal dose (ld( )) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. we demonstrated that modification of neutralizing vhhs with a human immunoglobulin g (igg) fc (fragment crystallizable) fragment (fusionbody, vhh-fc) significantly increased the circulation time in the blood (up to days). at the same time, vhh-fc showed the protective activity times higher than monomeric form when challenged with ld( ). moreover, vhh-fcs remained protective even days after antibody administration. these results indicate that this vhh-fc could be used as an effective long term antitoxin protection against botulinum type a. botulinum neurotoxin (bont) is the strongest organic poison for humans and animals. it is produced by the anaerobic, gram-positive, spore-forming, rod-shaped bacterium clostridium botulinum [ , ] . the estimated human lethal dose is about nanograms per kilogram of bodyweight if the toxin is inhaled and one microgram if it is taken orally [ , ] . the most common forms of natural botulism are food-borne, wound, and infant [ ] . food-borne botulism occurs through contaminated food ingestion, and the case fatality rate is now about - % in developed countries (as opposed to - % before ). wound botulism occurs when an open wound is exposed to c. botulinum spores, and the case fatality is approximately - % of patients even with aggressive treatment. infant intestinal botulism occurs with spores ingested and spread over the digestive tract, as infants lack the protective flora of adults, with case fatality rate estimated to be le,ss than - %. inhalation botulism does not occur naturally and may occur in the context of a bioterrorist attack [ , ] . among the four major human pathogenic bonts (bont/a, b, e, and f), bont/a poses the most serious challenge for medical treatment due to its extremely high potency and extraordinary persistence in human patients [ ] . according to the world health organization [ ] , an antitoxin, usually based on equine antitoxin, should be administered as soon as the diagnosis is made. however, side effects, such as allergic reactions, fever serum sickness, and anaphylactic shock, often occur. in the past, at-risk persons and populations have been vaccinated with a chemically inactivated penta-serotype bont/a-e toxoid [ ] ; however, its use has been discontinued due to declining potency [ ] . in other affected individuals, botulism treatment is mainly supportive, with mechanical ventilation being the only effective life-saving treatment [ , ] . a good alternative to antitoxin serum with minimal or no side effects is treatment with monoclonal antibodies (mabs) to bont, which can be prduced in vitro. along with conventional antibodies, single-domain antibodies (sdab), also referred to as vhhs (variable domains of heavy-chain only stibodies) or nanobodies, have been widely used since their discovery in camelids along with classical immunoglobulins (igg) [ ] [ ] [ ] . the absence of light chains in igg and a lack of constant domain (ch ) in the heavy chain are the key characteristics of heavy-chain antibodies (hc-abs). therefore, an antigen-binding site of hc-abs is formed only by a single domain, which is linked directly via a hinge region to the fc (fragment crystallizable) domain. hc-abs recognize the antigen with only one special variable domain, referred to as vhh. the structure of the vhh domain resembles the vh igg domain [ ] . the complementarity-determining region (cdr ) of these hc-abs possesses the extraordinary capacity to form long finger-like extensions, which can extend into cavities on antigens, as the cdr is often much longer than that of conventional vh domains [ , ] . despite their small size (~ kda), sdabs maintain affinities and antigen-binding specificities comparable to those of full-size mabs [ ] . clear advantages include the ability to recognize hidden antigenic sites that are inaccessible for conventional antibodies due to their structure; stability over a wide range of temperatures and ph; high solubility, as well as economical and facile expression and production in large quantities in microorganisms [ , ] . several studies were conducted to test the potency of vhhs as inhibitors of viral infections [ ] and different toxins, produced by such plants and microorganisms as ricinus communis [ ] , mycoplasma hominis [ ] , clostridium tetani [ ] , clostridium difficile [ ] , crotalus durissus terrificus [ ] , and shiga toxigenic escherichia coli (stec) [ ] . it has been demonstrated that the antigen-binding region of vhhs produced by camelids showed strong anti-bont activities in animal models [ , ] . due to their unique features and high efficacy, sdabs are currently in clinical phase trials for the treatment of a wide range of diseases, including cancer, inflammation, hematology, and respiratory diseases [ ] . for instance, cablivi is a nanobody-based medicine, which has recently been approved by the food and drug administration (fda) for acquired thrombotic thrombocytopenic purpura (attp) treatment [ ] . however, the advantages of vhhs may also become their impediments. their small size enables good penetration into tissue and rapid distribution, but their short half-life in the blood circulation limits the time for interaction with hard-to-reach epitopes and for crossing endothelial barriers in sufficient amounts [ , , ] . nevertheless, due to their relatively simple structure, vhhs can be optimized by genetic engineering to obtain desired properties, including extended half-lives [ ] . one approach was to add an albumin-binding peptide to the c-terminus of vhh anti-botulinum, which increases the serum half-life of the vhh to - days [ ] . another approach was to generate virus vectors encoding chimeric proteins with one or more vhhs fused in frame to a cdna encoding the red blood cell membrane proteins glycophorin a or kell. in vitro studies and stem cell transplantation research has demonstrated that the half-life of these vhhs could be extended to several weeks with retained functionality [ ] . the vhh could also be fused to the fragment crystallizable (fc) region of an igg. such modifications, termed fusionbodies, increase the total protein complex size, as well as vhh-fc interaction with neonatal fc-receptor (fcrn), making it impossible to clear by the renal filtration system [ ] . furthermore, the attached fc fragment can promote effector functions, such as phagocytosis and cytotoxicity, thereby enhancing the neutralization of pathogen entry and replication [ ] . in this study, we focused on screening specific bont/a neutralizing alpaca vhhs from a vhh antibody immune library by phage display using bont/a and bont/a treated with dithiothreitol (bont/a-dtt) as antigens. two clones (b and g ) with high neutralizing potency at lethal dose ( ld ) were obtained in phage forms. these clones were produced as vhhs, which were then modified as dimers or fused with human igg fc-fragments (vhh-fc, fusionbody). the vhh-fc modification greatly increased their neutralizing potency and serum half-life. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations to generate an immune library of single-domain antibodies (figure a ). after and days post-immunization, blood was collected, and the bont/a specific antibodies titer was measured by enzyme-linked immunosorbent assay (elisa). the immune serum showed a clear response to bont/a (figure b ). before immunization, all sera showed the background levels of antibody titers. the final titer of toxin-specific antibodies in the alpaca serum was more than / , as the result of the five-fold immunization scheme. to generate a panel of single-domain antibodies to bont/a, we constructed an alpaca immune vhh library for display on the bacteriophage surface by cloning the nucleotide sequences coding for vhh repertoires of the immunized alpaca into an expression phagemid vector. after the transformation of recombinant phagemids into competent e. coli cells, a library, with a size of × phagemids, was obtained. phage display and two rounds of selection were performed for acquiring phage libraries. purified bont/a was used as an antigen for the first round. two independent second rounds were performed separately, with bont/a and bont/a-dtt used as antigens. dtt reduction was used for fragmenting toxins into two constituent parts-the heavy chain (hc) and light chain (lc) [ ] -for more uniform absorption of chains on the immunoplate's surface and to evenly distribute the epitopes on both chains. the polyclonal phage library titer for all rounds was approximately cfu (colony-forming unit)/ml (figure a) . after two rounds of selection, the specificity of each library was determined by elisa ( figure b ). to test the neutralizing potency and specificity of the polyclonal phage libraries obtained in the second round of selection, an in vivo toxin neutralization assay was performed ( figure c ). balb/c female mice were divided into groups and received one intraperitoneal injection with ld or ld bont/a after h incubation with phage libraries. previously obtained phages specific to tetanus neurotoxin (tent) were used for controls as non-specific phages. all mice in the positive control group survived, while all mice in the negative control group and the tent group died. challenging two experimental groups with ld and ld provided full and partial protection, thus allowing further selection of monoclones. a total of clones showing elisa readouts higher than . ( figure a ) and negligible reactivity with bovine serum albumin (bsa) were selected for sequencing. a total of clones with different cdr amino acid sequences were selected for further research. to test the neutralizing potency of the selected clones, an in vivo monoclonal phage neutralization assay was performed in which mice were administered the corresponding phage clone (figure b ). each group received one intraperitoneal injection with ld bont/a previously mixed with the corresponding phage clone at cfu. only the four clones (b , c , b , g ) that fully protected the mice against a ld challenge were chosen to test their protectiveness with a ld challenge. mouse groups, which received clones b and c premixed with bont/a, were partially protected, while two other groups, which received clones b and g premixed with bont/a, showed % protection. thus, clones b and g were chosen for further research as the most protective. it should be noted that the clones obtained by selection on bont/a-dtt showed a greater diversity in the cdr sequence and demonstrated neutralizing activity, unlike the clones, which were selected on bont/a. the most protective clones, b and g , were titrated in series down to cfu and introduced intraperitoneally into mice simultaneously with ld of the toxin. the neutralizing potency of both clones was comparable, with the lower threshold being cfu (figure c ). finally, both clones were tested for cross-reactivity protectiveness with another toxin serotype, bont/b. each group of mice was challenged with ld of the toxin and administered cfu/ml phages. both clones failed to protect the mice, demonstrating that the two protective clones are specific to bont/a. to increase the protectiveness of the selected clones by increasing the antibody circulation time in the blood and/or their additional functionality, two modified constructions were synthesized. one construction was a dimer form of each clone (b -dimer and g -dimer) held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. the second construction was a monomer of each clone linked to a human igg fc fragment (b -fc and g -fc) (figure a ). fc-modifications have been used to increase the antibody circulation time in blood. human fc-fragments cross-react with murine fc-receptors and are capable of binding mouse fcrn [ , ] . human fc was chosen for these constructions to be used in further research and clinics. the dimers were produced and purified from the bacterial periplasmic fraction and verified by sds-page to be~ kda. vhhs fused with fc fragments were produced in chinese hamster ovary (cho-s) cells and verified by sds-page to be~ kda under reducing conditions (figure b ). to determine the polypeptide chain of the toxin that bound specific antibodies, it was denatured by dtt into its hc and lc. sds-page was performed in denaturing conditions followed by western blot. b -fc and g -fc clones were tested, and anti-human igg (fc-specific)-peroxidase antibodies ( : ) were used as detection reagents ( figure c ). these antibodies were associated with the hc ( kda) of the toxin, which corresponds to the receptor domain. an immunoblot with bont/a and bont/a-dtt (under reducing conditions) were performed as control (figure d ). we investigated the affinity of clones b and g via surface plasmon resonance (spr). amine coupling was used to immobilize bont/a and bont/a-dtt on the sensor chip. the kinetic binding on-and off-rates between the antibodies and the toxin were determined from sensorgram analysis and used to calculate the equilibrium dissociation constant (kd) ( table ). table . kinetic parameters of antibody interactions with the toxin obtained by spr (surface plasmon resonance). association (on-rate, k a ), dissociation (off-rate, k d ), maximum analyte binding capacity (r max ), equilibrium association constants (k a ), equilibrium dissociation constants (k d ), and chi for the chosen vhhs (variable domains of heavy-chain only antibodies) binding to botulinum neurotoxin (bont)/a or bont/a-dtt (dithiothreitol). bont/a-dtt k a ( /ms) for the in vivo protection analysis, modified forms of vhhs, different amounts of monomers, dimers, and vhhs fused with fc fragments were premixed with ld bont/a and injected intraperitoneally into mice ( figure a ). monomers had only partial protectiveness even at the highest amount, µg, with protectiveness gradually decreasing and failing at µg. the dimers showed a better result on average, with full protectiveness of the b -dimer at µg and partial protectiveness at lower amounts, failing at µg, as well as partial protectiveness of the g -dimer at - µg amounts, failing at µg. the best results were demonstrated by vhhs fused with fc fragments, with g -fc failing to fully protect the mice at . µg and b -fc protecting the mice at amounts as low as . µg. all forms of vhhs partially protected the mice at high amounts, whereas vhhs fused with fc fragments could protect the mice even at the lowest amounts tested ( . - . µg). to assess the circulation time of various antibody modifications in the blood after a single injection, elisa was used to measure the concentrations of g , b , g -dimer, b -dimer, g -fc, or b -fc in mouse blood taken at different time points after injection. the concentrations of g , b , as well as b -dimer and g -dimer, detected at h post-injection were only about % of the initial level observed at h. however, b -fc and g -fc modification constructs had a relatively long serum half-life. the concentrations of b -fc and g -fc h post-injection were approximately % of the initial level. moreover, two weeks after injection, the concentrations of b -fc and g -fc antibodies were approximately % of the initial level. these data are in agreement with reports on other vhhs with monomeric and fusionbody formats [ ] (figure b ). vhhs fused with fc fragments demonstrated the best protection, and the mice treated with these preparations were alive two weeks after the end of the experiment. based on the analysis of b -fc and g -fc clones' circulation time in the serum (presence of antibodies days after injection), we decided to conduct an experiment on the survival of these mice, which previously received a single injection of the vhhs with the fc fragment, with a repeated administration of only the lethal toxin dose days after the original administration. these mice were challenged with bont/a ld to examine their protection over time (figure c ). all mice that previously received - µg of vhh-fc were still fully protected. the mice which previously received - µg of the vhh-fc were partially protected ( - %) (clone b -fc) or not (clone g -fc). after two weeks, µg of the preparation had no protectiveness. we also tested the prophylactic efficacy and possible treatment of the toxin by administering the two selected fc-fused clones one hour and three hours before and after toxin challenge with ld (figure d ). both clones fully protected the mice before the toxin challenge and one hour after. three hours after toxin administration, the clones lacked protectiveness. overall, we obtained numerous clones after two rounds of biopanning; we selected clones for initial analysis based on their cdr s, chose two clones (b and g ) with the best pre-mixed results in phage form in vivo, produced them in protein form, and modified their structure and characteristics by dimerization via a (gly ser) linker and fusion to a human igg fc fragment to enhance their protective activity. we demonstrated that modification of neutralizing vhhs with an fc fragment (fusionbody) significantly increased the circulation time of antibodies in the blood. at the same time, fc fusion significantly increased the protective activity of vhh clones to more than times compared to monomeric forms. moreover, clone b -fc increased the protective activity at least , times compared to the monomeric form, with % protectiveness even at . µg. the estimated molecular ratio of the ld to . µg pre-mix was : . bont is the most potent and lethal known toxin. therefore, the development of new therapeutic agents for exposure prevention and treatment is essential. therapeutic approaches have been summarized in previous publications [ ] . along with serum therapy, specific antibodies are a promising tool for neutralizing bonts. approaches for the generation, selection, combination, and modification of mabs against bont/a have been described and tested [ ] [ ] [ ] [ ] [ ] . camelid vhhs are a popular tool for constructing recombinant antibodies to detect and neutralize a range of targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in particular, it has been shown that hc-only antibodies or vhhs derived from hc-only antibodies, produced by camelids demonstrate strong anti-bont activities in animal models [ , , ] . the main differences of vhhs from conventional antibodies include specific conserved amino acid substitutions in framework region (fr ) that make contact with the lc of classical antibodies, as well as a longer length, more variable amino acid composition in the cdr s, and a folded back loop [ , , ] . specific vhhs can be isolated from vhh libraries. one type of such libraries-immune libraries-can be produced based on peripheral blood lymphocytes isolated from camelids that have been immunized with an antigen of interest in a prime-boost strategy [ ] . in our study, we used a toxoid consisting of bont/a toxin and small amounts of hemagglutinin (ha) as an antigen and screened for vhhs against bont/a and bont/a-dtt using phage display technology. the neutralizing clones in our work were selected on bont/a-dtt. it should be noted that the neutralizing clones were later found in the bont/a library as well. however, there were fewer of them, so a greater number of clones had to be analyzed. overall, approximately clones from bont/a-dtt and more than clones from bont/a libraries were examined. the approach of splitting the toxin into its hc and lc enriches the neutralizing clones in the library. based on their neutralizing activity when pre-mixed with different toxin ld doses, two clones were selected. interestingly, all clones with neutralizing potency did not have the highest elisa signals. therefore, the od (optical density) of the elisa signal does not always mean high neutralizing activity and in vivo protective activity of these vhhs. the next step was to produce and purify vhhs in protein form to test their neutralizing potency in vivo. however, their protection was weak. even when pre-mixing a µg dose of these vhhs with the toxin, they had a - % protection efficiency. administration of lower doses of the preparation was non-protective, with µg lacking effectiveness. therefore, two clones-b and g -were modified to increase their half-life and protection efficacy. increasing the vhh size by oligomerization-the coupling of two or more vhhs via a specific linker-increased their protectiveness; however, even bivalent constructs were rapidly cleared [ ] . another approach was vhh fusion to an fc region of the igg molecule. constructs with fused fc fragments were previously made to extend the antibody half-life to neutralize the middle east respiratory syndrome coronavirus (mers-cov) [ , ] , target the c-x-c chemokine receptor type (cxcr ) to prevent human immunodeficiency virus (hiv- ) strain entry and replication in vitro [ ] , and increase the fc effector functions to neutralize rotavirus [ ] as well as the influenza virus ha [ ] . a recent study of modified vhhs dimers and vhhs fused to fcs targeting unique epitopes on the immunogen, composed of a portion of the central delivery domain and the entire combined repetitive oligopeptides (crops) domain of clostridium difficile type b, showed modest and much greater toxin inhibitions, respectively [ ] . it should be noted that a combined approach for efficient serum clearance of bont/a has been developed before. sepulveda et al. [ ] used single-chain variable fragments (scfvs) as protein binding agents pre-complexed with one or two single anti-tag mabs with an fc domain and tested this construction in vivo for protection and pharmacokinetics. when three or four types of such constructions were given simultaneously, mice were protected at high ld s, and bont/a was rapidly cleared from the sera. protection against bont/a light chain was observed when scfvs fused with fc fragments (scfv-fc) obtained from a macaque immune library were tested ex vivo [ ] . furthermore, the substantial contribution of the homologous fc fragment to the potency of three individual anti-botulinum mabs in antibody preparations has been demonstrated [ ] . in our work, two chosen clones were dimerized via a (gly ser) linker or fusion to human fc fragments to stabilize the molecules and slow down their clearance. these modifications led to improved protectiveness of the preparations. the dimers demonstrated protectiveness at lower doses compared to vhhs, with - % protection efficiency at and µg. clone g -dimer failed to protect the mice at µg, like the vhhs, while clone b -dimer showed partial protectiveness and failed at µg. therefore, the effective dose (ed ) for clone b -dimer was µg, and for the clone g -dimer, was µg. the best results were obtained after the fusion of the vhhs to fc fragments, which is consistent with previous research. both clones fully protected the mice at doses as low as . µg. the protection provided by g -fc was half at . µg, and the clone failed to protect the mice at . µg. b -fc demonstrated full protection down to the lowest tested dose of . µg. the dose at which its protectiveness begins to decrease was not reached. therefore, both clones with fc fragments showed at least a -fold improvement in protectiveness compared to conventional vhhs and dimers. the ed dose for clone b -fc could not be determined, while it was . µg for clone g -fc. besides, the pharmacokinetics analysis of various antibody forms showed that clones b and g containing the igg fc fragment were detected in the mouse serum days after a single injection, which is confirmed by full or partial animal protection from the challenge with the toxin days after administration of various b -fc and g -fc doses. testing the prophylaxis and possible treatment of the toxin by administering b -fc and g -fc one hour and three hours before and after toxin challenge with ld showed that both clones fully protected the mice before the toxin challenge as well as one hour after, but failed to protect the mice three hours after toxin administration, suggesting that these clones could be used for prophylaxis and emergency therapy immediately after toxin administration. this corresponds with the results obtained previously by sepulveda et al. [ ] showing that mice receiving scfvs pre-complexed with anti-tagged mabs with fc fragments were protected only two hours after intoxication, while four hours after, toxin administration lethality was only delayed. the acquisition of antibodies against bont/a and confirmation of their neutralizing potency in vivo in phage forms when pre-mixed with the toxin has provided a new method to screen for neutralizing vhhs before obtaining them in protein form, which can efficiently reduce time and material costs for their production and testing. vhhs, along with their modifications as oligomers and fusions to fc fragments of the igg, increase the range of options for bont/a targeting and neutralization by improving the blood circulation time and therapeutic potential. alpaca immunization and blood collection were performed on one clinically healthy -year-old male alpaca (vicugna pacos) on the "russian alpacas" farm, private land located in pokhodkino, moscow region, russia. this sample collection did not involve endangered or protected species, and no specific permissions were required for these locations/activities. six-week-old female balb/c mice (weighing - g) were purchased from "pushchino breeding facility" (pushchino, moscow region, russia) accredited by association for assessment and accreditation of laboratory animal care (aaalac international) and maintained at the central animal facility at the gamaleya research center of epidemiology and microbiology. mice were kept at a constant temperature ( ± • c) and relative humidity ( %) with h of artificial light per day. they were housed in individual cages ( per cage). mice were fed with dried food and water ad libitum. mice were observed every two hours post-injection except during the night for one week. the animals with characteristic symptoms of botulism, including muscle paralysis and respiratory difficulty, were euthanized by cervical dislocation. bont/a from the c. botulinum strain a was obtained from the gamaleya research center, moscow, russia collection. bont/a is very toxic; therefore, appropriate safety precautions were taken during these experiments. the neurotoxin was handled at a class biosafety cabinet. the antigen was a toxoid consisting of bont/a toxin and small amounts of ha, and . % formaldehyde was used for toxoid preparation. the treatment was carried out for seven days at • c [ ] . mice received intraperitoneal injections to determine residual toxicity, which showed its absence. before use in alpacas, the antigen was purified through a . µm filter. the final concentration was µg/ml. before immunization, the neurotoxin was inactivated by . % formaldehyde at ph = during - days. bont/a preparations were used with freund's adjuvant without pre-adsorption. alpaca immunization was performed using five sequential injections with an interval of days between the first and second immunizations and days between all subsequent immunizations. for the first injection time, µg ( ml) of the antigen and complete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : were administered. the four subsequent immunizations were performed with µg ( . ml) of the antigen and incomplete freund's adjuvant (sigma, st. louis, mo, usa) at the v/v ratio of : . small blood samples ( - ml) were collected before immunization, as well as after the third and fifth immunizations as a control. five days after the final injection, ml blood sample was collected and placed into a sterile vacuum collection tube with lithium heparin to prevent blood clotting. mrna isolation, pcr amplification, and library construction were performed, as described elsewhere [ , ] . briefly, camelid vhhs from peripheral blood b-lymphocytes (about cells/ml) of the immunized alpaca were cloned into a phen expression phagemid vector [ ] . the primer set used for pcr amplification of antibody genes appended the sfii (neb) restriction site at the -end and the noti (neb) site at its -end (table ) . recombinant phagemids were introduced into freshly prepared competent suppressor tg e. coli cells (lucigen, middleton, wi, usa). using this method, a library of × individual clones for biopanning and isolation was obtained. the bacteria from the vhh library were added to × yt medium (sigma, st. louis, mo, usa) (with µg/ml ampicillin and % glucose) and incubated at • c in a culture shaker at rpm to an od = . . km helper phages (patrick chames "antibody therapeutics and immunotargeting team" of the cancer research center of marseille) were added to the bacteria (multiplicity of infection, moi = ) and left without shaking at • c for min. the culture was centrifuged at rpm for min at • c, and the cell pellets were resuspended in × yt medium (with µg/ml ampicillin and µg/ml kanamycin) and cultured overnight at • c, in a culture shaker at rpm. the next day, the culture was centrifuged, the supernatant was purified and concentrated by % polyethylene glycol (peg) , . m nacl precipitation, and the pellet was resuspended in phosphate-buffered saline (pbs) with % glycerol. bont/a-dtt was treated with mm dtt for at least min at • c. microtiter plate wells were coated with µg of bont/a for the first round and with bont/a and bont/a-dtt for the second round in . m nahco buffer (ph = . ) at • c overnight. after rinsing three times with pbs with . % tween (tpbs), the plate was blocked with blocking buffer (tpbs with % non-fat dried milk) at • c for h. a total of~ phages were added to each well and incubated at • c for h. unbound phages were removed by washing times with tpbs. the bound phages were eluted by trypsin with a final concentration of mg/ml. tg e. coli cells at od = . were infected with the eluted phages and incubated without shaking at • c for min. after culturing the mixture in × yt agar plates (with µg/ml ampicillin and % glucose) at • c overnight, the cells were scraped. recombinant phages were obtained by packaging with km helper phages, and their titers were determined. a total of two panning rounds were performed. for a polyclonal elisa, an immunoplate (maxisorp, nunc) was coated with ng of bont/a and bont/a-dtt with . m nahco buffer (ph = . ) at • c overnight. the plate was rinsed three times with tpbs, blocked with blocking buffer at • c for h, and a total of~ phages from the starting library or the first or second rounds were added to each well and incubated at • c for h. the unbound phages were removed by washing times with tpbs. the plate wells were detected by horseradish peroxidase (hrp)-conjugated anti-m antibodies ( : ) (abcam, cambridge, uk), followed by the addition of peroxidase substrate , , , -tetramethylbenzidine (tmb) (bio-rad, hercules, ca, usa). the reaction was stopped by m h so , and the absorbance at nm was read with an iems reader mf (thermo labsystems, waltham, ma, usa). a monoclonal elisa was performed following the same protocol, with particular clones bound to bont/a and bont/a-dtt being selected, and the phage plasmids were isolated for sequencing. antibody genes in phagemids were sequenced with a primer set used for pcr according to the protocol of big dye terminator . cycle sequencing kit for the genetic analyzer applied biosystems (waltham, ma, usa). the electrophoretic dna separation was performed in cm capillaries with pop polymer. the selected vhhs expressed in phen plasmid were transformed into e. coli bl cells (neb, ipswich, ma, usa) for expression and purification. an overnight culture was obtained at • c, in a culture shaker at rpm. the next day, the cells were harvested by centrifugation, lysed by bugbuster protein extraction reagent (novagen, madison, wi, usa), and vhhs were purified from the lysate by talon superflow containing co + agarose (ge healthcare bio-sciences ab, uppsala, sweden). the eluted fraction was subjected to dialysis with visking dialysis tubing (mwco - ) (serva, heidelberg, germany). vhhs were separated by sds-page (bio-rad, hercules, ca, usa) under denaturing conditions and had an expected molecular weight of~ kda. antibody affinity was determined by surface plasmon resonance (spr) using a biacore instrument (ge healthcare bio-sciences ab, uppsala, sweden). bont/a and bont/a-dtt were immobilized on the surface of cm sensor chips in the amount of µg each in mm sodium acetate buffer ph = . using the amine coupling kit recommended by the manufacturer (ge healthcare bio-sciences ab, uppsala, sweden). vhhs ( -fold dilutions from µg down to µg) were captured on the sensor chips and submitted at a constant flow rate of µl/min with hbs-ep ( . m hepes ph . , . m nacl, mm edta, . % v/v surfactant p ) as a running buffer at • c with an injection time of min and dissociation time of min. after each injection, the chip surface was regenerated with mm tris-hcl, ph = . for s at a flow rate of µl/min. calculations were performed using biaevaluation software (ge healthcare bio-sciences ab, uppsala, sweden) with reference-subtracted fitting. the selected vhhs were held by a glycine-serine linker (gly ser) and expressed in the pet plasmid. protein expression and purification were performed as for vhhs. nucleotide sequences of vhh genes fused to the human igg fc-fragment were synthesized and cloned into the plasmid pshuttle-cmvfuse (stratagene, la jolla, ca, usa). the cho-s cell culture (thermofisher, waltham, ma, usa, r ) was transiently transfected with pfuse plasmid using the cho gro system (mirus bio, madison, wi, usa), according to the manufacturer's protocol. a plasmid carrying the green fluorescent protein gene in a % amount was used as control. the efficiency of transfection was assessed using axio imager z (carl zeiss, oberkochen, germany) and determined by the number of fluorescence cells compared to their overall amount, which was %. cells were cultured in shake flasks at rpm, % co , % humidity, at • c during transfection and • c h after the transfection for days. starting from day three, cellboosts a ( %), b ( . %) (hyclone, san angeo, tx, usa), and % sigma bioreactor feed (sigma, st. louis, mo, usa) were added each day. after days of cultivation, the culture was clarified by centrifugation at × g and cleared through a . µm filter. the antibodies were purified using protein a affinity chromatography on an akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden), with a mabselect sure ml column (ge healthcare bio-sciences ab, uppsala, sweden), according to the manufacturer's protocol. to determine which bont/a polypeptide chain binds vhhs, the toxin was denatured by dtt into its hc and lc. sds-page was performed using a mini-protean tgx stain-free precast gel (bio-rad, hercules, ca, usa) in denaturing conditions. the separated bands were transferred onto a nitrocellulose membrane using a trans-blot turbo system (bio-rad, hercules, ca, usa). the membrane was blocked with a blocking buffer at • c for h. vhhs were diluted in the blocking buffer, added to the membrane, and incubated at • c for h. after rinsing the membrane three times with tpbs, anti-his-tag-hrp-mouse antibodies (genscript, piscataway, nj, usa) diluted : in blocking buffer were added, and the membrane was then incubated at • c for h. the membrane was rinsed three times with tpbs, followed by the addition of clarity western ecl blotting substrates (bio-rad, hercules, ca, usa) for detection. the membrane was visualized on an amersham imager (ge healthcare, buckinghamshire, uk). the neurotoxin as a multi-oligomeric complex toxin with has and ntnha (non-toxic non-hemagglutinin) ( kda) was used for immunization. toxins were prepared and purified as follows. the strain was cultivated under anaerobic conditions for five days. bacterial cells were separated by centrifugation at × g for min at • c. the proteins from the culture filtrate were concentrated by acid precipitation at ph = . for min. the precipitate was separated by centrifugation at , × g for min at • c and dissolved in mm citrate-phosphate buffer with ph = . . gel filtration s and ion-exchange chromatography on akta start chromatography system (ge healthcare bio-sciences ab, uppsala, sweden) on de cellulose (pharmacia, uppsala, sweden) were then carried out. the toxin ( - %, kda) was purified by additional de cellulose chromatography (pharmacia, uppsala, sweden) in borate buffer ph = and eluted by mm nacl. non-sorbed material containing specific antigenic and biological activity was used as bont complex with ha. the specific antigenic activity was a positive reaction with monospecific antibodies to bont/a, ha, and ntnha (gamaleya laboratory of clostridiosis and commercial preparation of scientific centre for expert evaluation of medicinal products russian federation). phages with titers - cfu/ml were prepared, as described above, and mixed with standard saline solution. vhh proteins at different amounts ranging from µg to . µg were prepared, as described above, and mixed with standard saline solution. balb/c - g female mice were divided into groups of four, including the positive and negative control groups. phages or vhh proteins were premixed with the appropriate toxin ld ( ld~ pg bont/a) and incubated for h at • c. all mice received one intraperitoneal µl injection of phages or proteins premixed with various ld s of the toxin and were observed once a day for one week. a specific pathological pattern was observed in sick mice, which is the pathological pattern with dystonia's abdominal muscles (waistline increasing) and death at - pg that is neutralized by monospecific antibodies to bont/a. the positive control group received the rabbit antitoxin igg (gamaleya laboratory of clostridiosis), and the negative control group received a standard saline solution. a group of five six-week-old female balb/c mice was intravenously (i.v.) injected with µg g , b , g -dimer, b -dimer, g -fc, or b -fc into the tail vein. blood was collected from the facial vein at , , , , , , , , h time points. sera were separated and stored at − • c until further use. concentrations of the injected antibody molecules in the above-collected samples were measured by elisa. for elisa, bont/a was coated on microtiter plates (nunc) overnight at • c at ng/well in mm bicarbonate buffer. after washing three times with tpbs, plates were blocked with % dry milk in pbs for one hour at • c. then, × diluted sera were added to the wells, followed by a one-hour incubation. mouse anti-his-tag antibody [hrp] ( : ) (genscript, piscataway, nj, usa) was used to detect monomers and dimers of g and b antibodies in the mouse sera. goat anti-human igg bacterial toxins-a table of lethal amounts clostridial neurotoxins botulinum toxin as a biological weapon-medical and public health management a camelid single-domain antibody neutralizes botulinum neurotoxin a by blocking host receptor binding the effect of formalin on botulinum toxins a, b and c notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid 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coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov nanobody-fc constructs targeting chemokine receptor cxcr potently inhibit signaling and cxcr -mediated hiv-entry and induce antibody effector functions intracellular antibody-bound pathogens stimulate immune signaling via the fc receptor trim universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin neutralization of clostridium difficile toxin b with vhh-fc fusions targeting the delivery and crops domains efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents development of neutralizing scfv-fc against botulinum neurotoxin a light chain from a macaque immune library role of homologous fc fragment in the potency and efficacy of anti-botulinum antibody preparations monoclonal antibodies to type a, b, e and f botulinum neurotoxins selection and identification of single domain antibody fragments from camel heavy-chain antibodies basics of antibody phage display technology multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -bdpp qmf authors: lanzavecchia, antonio title: dissecting human antibody responses: useful, basic and surprising findings date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: bdpp qmf human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target‐agnostic approach based on high‐throughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host–pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non‐ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. human memory b cells and plasma cells represent a rich source of antibodies that have been selected in response to human pathogens. in the last decade, different methods have been developed to interrogate the human memory repertoire and isolate monoclonal antibodies. i will discuss how a target-agnostic approach based on highthroughput screening of antibodies produced by cultured b cells and plasma cells has not only provided potent and broadly neutralizing antibodies against a range of pathogens, but has also advanced our understanding of basic aspects of the immune response, from host-pathogen interaction to the role of somatic mutations in affinity maturation and in the diversification of the antibody response. most surprisingly, this approach has also revealed a new mechanism of diversification based on templated insertion of non-ig dna into antibody genes that we discovered in the context of the immune response to malaria infection. a trove of antibodies m emory b cells represent the repository of the immune experience of an individual. they are selected in the germinal centres, where they undergo a process of somatic mutations and selection by antigen and t helper cells. memory b cells persist for a life time and can rapidly respond to a booster immunization by generating large numbers of plasma cells that are transiently present in the blood and localize to surviving niches in the bone marrow as long-lived plasma cells that are the main source of serum antibodies. the interrogation of memory b cell and plasma cell repertoires of humans offers unique opportunities. as compared to mice, humans are naturally exposed to their own pathogens, often through recurrent infections, and are therefore a better source of antibodies that target the most relevant antigens and can reveal mechanisms of host-pathogen interaction. furthermore, given the genetic variability of the human population and the idiosyncrasies of the immune response, some individuals may generate antibodies with unusual potency or breadth. finally, studies of the antibody response in humans can shed light on basic aspects of the immune response, such as the role of somatic mutations and the mechanisms that underpin the generation of broadly neutralizing antibodies or autoantibodies, and possibly uncover examples of such mechanisms that may be uniquely developed or better identified in humans. a target-agnostic approach to identify functional antibodies in the last decade, several methods have been developed to isolate human monoclonal antibodies. in principle, they come down to two basic approaches. the first approach consists of the isolation of single antigen-specific b cells using a fluorescently tagged antigen as a bait, followed by cloning of immunoglobulin genes and expression of the recombinant antibodies in a cell line. this method is very effective, but requires that the target antigen is known and available in a purified form. in my laboratory, we developed an alternative approach based on high-throughput screening of the antibodies produced by cultured b cells or plasma cells (fig a) . memory b cells from selected donors are immortalized with high efficiency (> %) by combining epstein-barr virus (that delivers signal and signal ) with a tlr or tlr agonist (that delivers signal required for potent activation of memory b cells) (traggiai et al, ) . the immortalized b cells proliferate and secrete large amounts of antibodies in the culture supernatant. antigen-specific plasma cells recovered following a booster immunization have lost proliferative capacity, but can be kept alive in cultures supplemented with il- and stromal cells. in these cultures that mimic the survival niches of the bone marrow, a single plasma cell secretes antibodies at a constant rate of pg/day (corti et al, ) . to facilitate the isolation of specific antibodies and the cloning of the vh/vl genes, both immortalized b cells and plasma cells are seeded in clonal conditions and cultures are handled with an automated liquid handling system. there are distinctive advantages in screening the repertoire of secreted antibodies. the first is that the approach is target-agnostic, since screening can be done using functional assays, such as virus neutralization, without a priori knowledge of the target antigens, using even whole bacteria or parasites. second, it is well suited to identify antibodies that cross-react with different antigens, since parallel screenings can be performed against multiple targets. third, it bypasses the need to sequence and express large numbers of antibody sequences since cells of interest are selected based on the initial screenings. a relevant example of the utility of the target-agnostic approach comes from a study of the antibody response to human cytomegalovirus (hcmv), a complex herpesvirus expressing different surface glycoproteins that causes serious pathology in the foetus and in immunocompromised patients. by screening for the capacity to neutralize the wild-type virus, we isolated antibodies that were , -fold more potent than antibodies to the fusion protein gb and identified their viral target as a pentameric complex formed by gh, gl, pul , pul and pul a. we then produced a soluble form of this pentamer and found that it can elicit, in mice, neutralizing antibody titres that exceeded by more than -fold those induced in humans by natural infection (kabanova et al, ) . these studies illustrate a general approach of "analytic vaccinology" where the neutralizing antibodies are used for the identification of the target antigen and for the quality control of the recombinant vaccine. the possibility of performing parallel screening against multiple targets was instrumental for the identification of broadly reactive antibodies capable of neutralizing multiple viruses. these include the antibodies fi and fy , which bind to conserved epitopes in the stem region of influenza hemagglutinin (ha) and neutralize all influenza a viruses (corti et al, ; kallewaard et al, ) , and mpe , an antibody that binds to a unique conserved site on the pre-fusion form of the f protein and neutralizes four different paramyxoviruses, including human rsv and mpv (corti et al, ) . these antibodies are promising candidates for prophylaxis and therapy of infections since they trigger effector functions and target conserved structures, thus limiting the possibility of selecting escape mutants. in addition, they represent useful tools for the design of stabilized pre-fusion glycoprotein vaccines. a straightforward application of the method lies in the rapid isolation of neutralizing antibodies against emerging pathogens, such as ebola virus, sars (traggiai et al, ) and mers coronaviruses. in the case of mers, it was possible, in only months, to go from a sample of memory b cells to preclinical validation of a neutralizing antibody and the generation of a cell line producing the antibody in grams/l amounts. similar studies on dengue and zika viruses defined different classes of antibodies endowed with neutralizing or infection-enhancing activity. the interrogation of the memory b-cell repertoire offers the opportunity to investigate, in biologically and medically relevant settings, fundamental aspects of the human immune response, such as the relative contribution of germ line v-gene polymorphism, vdj junctional diversity and somatic mutations to antigenic specificity. an interesting example comes from the study of antibodies that neutralize group influenza viruses and that are produced by most individuals following infection or vaccination. as reported by several laboratories, these "public" antibodies use vh - and bind, exclusively through the vh, to a conserved region in the ha stem. by isolating a large number of sister clones and by performing a genealogical analysis of different clonal families, we found that the unmutated common ancestors (uca) have low affinity for group ha and do not neutralize infection (fig b and c) . remarkably, the first branch points acquire through a single p aa mutation in hcdr , high affinity and neutralizing activity, comparable to that of the antibodies isolated from memory b cells that carried up to - amino acid substitution (pappas et al, ) . these findings demonstrate that affinity maturation can be achieved rapidly, through a single mutation, and that numerous and redundant somatic mutations continue to accumulate, providing an extensive diversification within the proliferating clone. interestingly, in these antibodies, a critical contact residue, f , is germline-encoded, but this position is dimorphic, with different alleles carrying either f or l. consequently, individuals who lack a f -encoding allele cannot produce the public vh - antibody response, while individuals that have a f allele have a high frequency of precursors, with the only additional requirement being a -amino acid hcdr with a y that makes a critical contact site with the antigen. these findings underline the impact that vh-gene polymorphism can have on the antibody response and suggest that certain alleles may play the role of immune response (ir) genes and become positively selected in the population exposed to a given pathogen. the genealogical analysis of the two paninfluenza neutralizing antibodies, fi and fy , showed that the corresponding ucas have already high binding affinity and neutralizing activity, but only on group viruses and acquire the capacity to neutralize group viruses through somatic mutations (kallewaard et al, ) . these findings are consistent with a model where pan-influenza neutralizing antibodies arise from the priming of high-affinity precursors by a group virus (in this case h n ), followed by somatic mutations and generation of a variant with broader reactivity to group viruses that can be boosted by a group virus such as h n (fig d) . importantly, the isolation of cells producing the identical fi antibody for consecutive years in response to vaccination or infection indicates that secondary antibody responses are to a large extent independent from germinal centres, a factor that allows the antibody to retain the original specificity. the genealogical analysis of the neutralizing antibody mpe revealed a similar mechanism, with the uca possessing high affinity for rsv only and mpe acquiring cross-reactivity to mpv through somatic mutations (corti et al, ) . the extensive antibody diversification observed in humans can increase antibody affinity and promote breadth, but comes at the price of generating many new specificities that may cross-react with self-antigens. the notion that autoantibodies can be generated by somatic mutations is supported in humans by the finding that the ucas of autoantibodies specific for desmoglein or gm-csf found in patients with severe autoimmune diseases do not bind to the respective self-antigens (di zenzo et al, ; piccoli et al, ) . thus, it is tempting to speculate that these autoantibodies may have been generated by somatic mutations in b-cell clones responsive to a foreign antigen in the absence of structural mimicry. importantly, the mechanisms of deletion or anergy that restrain naïve autoreactive b cells may not apply to activated/memory b cells, which have different triggering requirement and can traffic to peripheral tissues. as mentioned above, the target-agnostic approach is especially suited to dissect the antibody response to complex pathogens in the absence of a detailed knowledge of the target antigens. we were interested in identifying antibodies that could broadly recognize the variant surface antigens (vsas) of plasmodium falciparum, the parasite causing malaria, which are expressed on the surface of infected erythrocytes (ies). vsas mediate adhesion of ies to endothelia and are targets of antibodies that control disease, but their high number (> genes), their extensive polymorphism and their clonal expression provide the pathogen with a powerful chameleon-like escape strategy. out of a large kenyan cohort, we initially selected two individuals with serum antibodies that cross-agglutinated erythrocytes infected by different p. falciparum strains. from their memory b cells, we isolated a panel of broadly reactive monoclonal antibodies using staining of infected erythrocytes as a screening strategy. surprisingly, all the broadly reactive antibodies had a unique structure, since they carried a large insert between the v and the dj segments. the inserts, of approximately bp, comprised the exon encoding the extracellular domain of the collagen-binding inhibitory receptor lair /cd , encoded in the leucocyte receptor cluster on chr. (tan et al, ) . given the insertion between v and dj, the lair domain is positioned at the tip of the hcdr (fig a and b) . the target antigens of lair -containing antibodies were identified in some members of the rifins, a family of polymorphic vsas expressed on the surface of ies (fig c) . importantly, the inserted lair domain was both necessary and sufficient for binding to rifins, as demonstrated by its insertion into an irrelevant antibody or by the production of lair -ig fusion proteins. to investigate how frequently lair containing antibodies are produced, we screened two additional cohorts from mali and tanzania and found that up to % of the individuals have such antibodies in the serum (pieper et al, ) . this study led not only to the isolation of more lair containing monoclonal antibodies, but also to the finding of a new insertion modality where the lair exon with and intronic sequences is inserted into the immunoglobulin switch region. this insertion leads, by exon shuffling, to the production of the original antibody and of a bispecific antibody containing the lair domain precisely placed at the elbow between the vh and ch domains (fig a and b) . in one case, the lair insertion in the switch region was accompanied by the genomic deletion of vh and ch , generating a "camel-like" lair containing antibody. in each individual analysed so far, the lair -containing antibodies dominate the response to ies and are produced by a single b-cell clone that contains a unique insert and undergoes extensive expansion and somatic mutations, thus demonstrating an extraordinary fitness. somatic mutations in lair were more frequent when the insertion was at the v-dj junction, which is continually targeted by aid in germinal centres, and less frequent when the insertion was in the switch region, which is only transiently targeted by aid during switch recombination. interestingly, somatic mutations in the lair domain eliminate collagen binding and modulate affinity and cross-reactivity with different rifins. the finding that collagen binding was always lost, even when the somatic mutation mechanism was less effective, suggests that loss of self-reactivity is a prerequisite for expansion of b cells making lair -containing antibodies. the insertion of non-vdj sequences encoding structured domain into ig genes is a new and potentially general concept. our finding was facilitated by the huge expansion of lair -containing b cells and by the unique properties of the antibodies made. in an initial attempt to determine the generality of this phenomenon, we found frequent insertions in the switch region of memory b cells from european blood donors. interestingly, the inserts largely originate from genes that are transcribed in b cells and are encoded in virtually all chromosomes and, in rare cases, comprised exons with frames compatible with expression ( fig d) . malaria infection can cause chromosomal instability and translocations involving the switch region, suggesting that it may also play a causative role in the generation of templated insertions. however, the finding of frequent templated insertions in european blood donors is more consistent with a general mechanism that remains to be molecularly defined. the sites of insertion suggest a mechanism of patch-repair of dna double-strand breaks induced by either rag or aid during v-dj recombination or class switch. the integrity of the genomic lair loci in b-cell clones producing lair containing antibodies suggests a copy-andpaste mechanism. in view of the finding that the inserts contain intronic sequences and are derived from expressed genes, we favour the hypothesis that the templated dna may originate from the resolution of stalled replication forks caused by a clash between transcription and dna replication. our findings suggest that the insertion of templated dna represents a novel and general mechanism of antibody diversification that can provide a broad range of protein domains to be further diversified by somatic mutations. as exemplified by lair , the insertion of a domain encoding a pathogen receptor can generate public antibodies that are effective against the pathogen. distinctive advantages of these receptor-based antibodies lie in the broad recognition of all pathogen variants, leaving no room for the selection of escape mutants, and in the possibility of diversifying the receptor domain through somatic mutations to increase binding to pathogen and decrease self-reactivity. the new antibodies are reminiscent of ehrlich's "side-chain" theory of antibody production, where the antibody was essentially a secreted form of the receptor for the pathogen. we predict that other examples of receptor-based antibodies will be found in response to a variety of human pathogens. besides lair , other candidates for the generation of receptor-based antibodies to p. falciparum are icam , an adhesion molecule that binds to ies, and lilrb , an inhibitory receptor that, like lair , has been shown to bind to rifins. similarly, insertions of icam or slam domains may generate receptor-based antibodies that neutralize rhinoviruses or measles virus. as a final remark, we realize that, by uncovering the ingenuity of nature, we have learned new ways to engineer antibodies and, possibly, to edit antibodies in primary b cells using the endogenous aid activity. we also discovered how strong antibody responses can be generated by a single b-cell clone, raising a case for adoptive b-cell therapy. a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins cross-neutralization of four paramyxoviruses by a human monoclonal antibody pemphigus autoantibodies generated through somatic mutations target the desmoglein- cis-interface antibody-driven design of a human cytomegalovirus ghglpul l subunit vaccine that selectively elicits potent neutralizing antibodies structure and function analysis of an antibody recognizing all influenza a subtypes al is scientific founder of humabs biomed, has substantial interest in antibodies developed by humabs and is a shareholder of vir bio. key: cord- -jtvn tlm authors: cimolai, nevio title: a minimalist strategy towards temporarily defining protection for covid- date: - - journal: sn compr clin med doi: . /s - - - sha: doc_id: cord_uid: jtvn tlm until either efficacious therapy or vaccination for covid- is achieved, there will be a need to regain world economic stability while yet controlling the pandemic with current approaches. for those infected thus far, there is a prevailing perspective that devising recognition for protective immunity will progressively allow segments of society to return to some functionality more than is existing. at this time, the best correlates with protection from natural coronavirus infections are systemic neutralizing antibody and mucosal iga. serum neutralizing antibody more easily fulfills the latter requisite, but current live virus methods for neutralization prevent large-scale application. it is conceivable that the exposure of previously infected individuals can allow for the definition of protective thresholds of neutralizing antibody. thereafter, many other antibody assays will be able to screen for surrogate protection after correlations with protective neutralizing antibody are made. specificity of common antibody tests would benefit from confirmatory blocking systems or confirmatory immunoblotting fingerprints with well-defined antigen(s). the opportunity for the scientific community to make these assessments is evident in the current context of the covid- epidemic given the large numbers of infected individuals worldwide. such information will also be vital to guide vaccine development and/or immunotherapy. the clinical burden of covid- in the current pandemic is undoubtedly considerable, but the socioeconomic burden must equally weigh in determining how the world will move forward until either an effective chemotherapy and/or vaccine is devised [ ] . pending that such success in treatment and/or prevention are achieved, the continuous or relapsing lockdown of societies and hence economies has the potential to cause more damage than may be initially apparent [ ] . indeed, it could be prognosticated that the relative economic standstill may cause more damage to humanity than the disease itself. many countries have attempted to partially restore pre-pandemic functions only to experience yet second waves of increasing infections during july and august, . to some, the answer may be to tolerate the expectations of herd immunity with lesser in the way of resistance to infection spread [ , ] . to others at the other end of the spectrum, a more cautious approach has been to create the best environment for disease prevention while patching holes for or propping up failures in the economy as they appear [ ] . somewhere in the midst of this maelstrom, there will need to be practical strategies for achieving success with both the pandemic and the economies simultaneously. contexts of disease and economy will no doubt vary along a spectrum and may require somewhat different approaches in their detail. in this review, a minimalist strategy is proposed to in part provide some solutions towards regaining economic focus while preventing disease. these steps are a modest beginning from the perspective of devising recognition for protective immunity that will progressively allow segments of society to proceed with their lives as they once were or nearly so. such a strategy will thereafter be enhanced as treatment or vaccine developments arise. studies with passive immunity are highly suggestive that antibody has a significant role in protection of coronavirus infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the latter includes passive administration of anti-sars-cov- human monoclonal antibody in animal systems [ ] . these findings are critical to our theme since direct antibody in some way may be used as the correlate with neutralization if not directly then by association. therefore, the prospects of finding a serological assay based on antibody detection that defines in some way neutralization and then after disease prevention are considerable. infection with coronaviruses generally protects against reinfection [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . analogous to the role of passive immunity, pre-existing immunity can potentially be defined with the right measures. both parenteral neutralizing antibody and secretory iga (siga) are associated with protection in model systems [ , , [ ] [ ] [ ] [ ] [ ] . siga has logistical problems with collection and analysis, although technological advances are quite likely capable of overcoming the latter. therefore and in the interim, serum neutralizing antibody measurements will generally be applied as the standard, and other correlates of the latter could be put into common use. for mers, mild infection was associated with decreased antibody levels [ ] [ ] [ ] . severe infections are associated with long-lived neutralizing antibody. increased neutralizing antibody is associated with decreased viral shedding, but there have not been enough natural infections to allow for study and analysis of natural protection as would be desirable. likewise for sars, field studies for the practical protective effects of neutralizing antibody could not be studied due to the short-lived spread of the virus. in studies for sars-cov- , of over control sera from uninfected patients, no neutralizing antibodies were found [ ] . severe disease was associated with earlier generation of anti-sars-cov- antibody, and peak neutralizing antibody was measured at approximately ½ weeks. stereotypic antibody responses to sars-cov- led to hypotheses that there should be cross-reactivity with sars-cov in the constant rbd [ ] . in both humans and animals, such cross-reactivity was suspected to be due to non-neutralizing anti-s antibody again likely relating to a conserved region [ ] . crossneutralization was however uncommon, but conceivably there could be other reasons for any such non-specificity [ ] [ ] [ ] . among humans, s-specific antibody dominates and may correlate better with neutralization [ , , ] . furthermore, the correlation between antibodies for different antigens (e.g., s and n proteins) may not be as initially conceived [ , ] . human monoclonal antibodies that neutralize virus or pseudovirus especially recognized the rbd [ ] . collaborative groups have found a strong association between neutralizing antibodies and igg antibodies to the rbd [ , ] . others have found strong correlations between neutralizing antibodies and eia-detected antibodies to various sars-cov- antigens [ , ] .some have found diversity in immune responses contingent on the nature of presenting disease [ , ] . the latter would be consistent with the variable antibody responses now determined for the spectrum of pediatric disease [ ] . for eia-based antibody studies otherwise, we have learned so far that the type and severity of disease and age may have bearing on the quality and quantity of antibody responses [ , [ ] [ ] [ ] [ ] . gender may also possibly affect antibody responses or other relevant immune responses [ , ] . does neutralization antibody ensure protection? at this time, we can reasonably conclude that the existence of neutralizing antibody and its quantitation associate with protection against coronavirus infections. what we cannot do, however, is guarantee that the presence of all such neutralizing antibody, as measured by conventional live virus neutralization methods in vitro, will ensure protection. that is, laboratory methods measure neutralization as so defined but not protection directly. therefore, it can be held that not all neutralization methods duly measure the same effect that leads to that neutralization outcome [ ] . in essence, what creates an in vitro neutralization may or may not truly block viral entry, prevent intracellular events, or ameliorate disease. one potential and theoretical approach to vetting sera that have a higher likelihood of indication for protection is to choose those with higher neutralizing titres. there are several limitations with the application of neutralization as a surrogate for protection [ , ] . infections with coronaviruses do not always lead to a major increase in such antibody. previously acquired antibody is not always protective for subsequent infection, but low levels of antibody better correlate with susceptibility. resistance is best to the homologous coronavirus, but susceptibility can be had to heterologous strains. there is also the prospect that subsequent changes in viral genome may be accompanied by changes in epitopes that afford neutralization with an accompanying change in neutralization capacity of post-infection or post-vaccination sera [ ] . the latter could be the prelude to neutralization escape mutants should they occur [ ] . the latter may also be the prelude to emerging reports of possible sars-cov- repeat infections [ ] . the traditional approach to determining antibody responses is likely to dominate the search for protective immunity, but due consideration and open-mindedness should be maintained for measuring t cell post-infection immunity and its correlates [ ] [ ] [ ] [ ] . with the availability of viral antigen, most scientists in the know-how would be able to fashion a test for antibody determination in short order and most would likely choose an enzyme immunoassay (eia) (or nearly equivalent non-enzymebased assay) for its potential of automation and widespread use. the latter is especially likely in the current context where a majority of serodiagnostic tests are of that methodology. the expeditious derivation of an eia or equivalent is a common goal when serodiagnosis is deemed to be of potential value. whereas the creation of an eia is seemingly a relatively easy task, the application of any such is where the test of the matter lies. historically, whether for antibody or antigen detection, eia approaches had a number of pitfalls that were repetitively realized for nearly every such system that was subsequently developed [ , ] . as would naturally be desirable, an assay with very high sensitivity at the appropriate timing is one evident target. furthermore, an assay with a highly specific result is another evident target. the reality of serodiagnostic systems, however, is that there is generally a gray or indeterminate zone where sensitivity and specificity collide [ ] . there is an intersection of true positive results and false positive results more or less in almost every system with few exceptions. for whatever maneuver is conceived to diminish that overlap, eia or an equivalent for antibody detection can be modulated to enhance predictive values in one direction or another, but commonly realizing that there will be a trade-off. the application of a serodiagnostic assay for antibody postinfection or vaccine is furthermore complicated in its evaluation. typically, a standard set of known or presumed "negative" sera are tested as is a standard set of known or presumed "positive" sera. many such analyses, however, do not thereafter assess the assay in the context of the working world prospectively. the jeopardy here is that the prevalence of true disease in the population of patients from whom sera are acquired can vary considerably. there is an inherent bias in such assessments to ensure that the tested serum pool has a considerable number of true positive samples. biases in this regard can be greater than those engendered by convenience sampling [ , ] . given the functionality of these assays, the predictive values of positive or negative tests vary considerably depending on the prevalence of the infection in the population so studied [ ] . for example, even for a given high sensitivity and specificity, a reduction in prevalence from to % can lead to a situation where the false positive assays nearly equal the number of true positive assays. the latter scenario can occur when assessments are performed on preselected sera rather than on sera from patients prospectively collected in a larger population. therefore, unless there is considerably widespread infection leading to a proportionately high seroprevalence, serodiagnostic tests will likely suffer with their predictive values. how would this dilemma then be potentially overcome? one such approach to improving specificity would be to conduct blocking assays (direct or competitive) for positive sera in order to reduce the number of false positive tests [ ] . this is a time-honored approach that is often forgotten when new serodiagnostic assays are initially created. the approach was also germane to some antigen detection eia versions. effectively, the specificity is enhanced when it can be shown that the antigen of choice significantly reduces the assay quantitation after absorption. the implementation of a blocking step can be entered into an automated procedure in tandem from the start or may be performed as a confirmatory test. the latter is especially suited to a scenario where the proportion of positive tests is low. the simplicity of a blocking test mandates only a brief turnaround time if it is performed in an algorithmic second step. there may be various approaches to the definition of antigen used in the blocking test, but it would be best in the inaugural stages to choose antigen(s) that are deemed inherent to the neutralization effects seen in postinfectious sera. although it could prove that one antigen, e.g., rbd or spike protein sequence otherwise, may alone suffice, pilot studies would be better to work backwards after establishing the antigen-neutralization correlates as described above. purity of the blocking antigen is essential to avoid non-specific competition of non-viral elements if virus is acquired from tissue cultures. another approach to a blocking assay would be the competition between antibody in the human serum specimen and a known monoclonal antibody with affinity to sars-cov- . choice of a specific monoclonal antibody, or several for this purpose, requires specific characterization. there are many commercial assays for sars-cov- antibody that have emerged, but for a survey sample of several so assessed thus far, none has blocking assays as potentially part of the procedure [ ] [ ] [ ] [ ] [ ] [ ] [ ] . developmental assays also commonly do not include blocking assays [ ] [ ] [ ] . igm assays are particularly an issue with non-specificity since apparent igm rises may relate in part to solid phase (e.g., polystyrene) absorption or non-specific igm antibody (e.g., rheumatoid factor). of note, early reports have now emerged of blocking assay variations for systems relating to either mers-cov or sars-cov- [ ] [ ] [ ] . as suggested above, antigen detection can also suffer from non-specificity, and this dilemma has now been found in some systems [ ] [ ] [ ] . again, a blocking assay in any such configuration for antigen detection has potential to resolve this critical issue especially when raising the threshold for the enhancement of sensitivity is being considered. a second approach, alone or alongside an eia-blocking assay, could be immunoblotting. again, it could prove that one antigen substrate may suffice, but it would be prudent to work backwards after establishing the antigen-neutralization correlates. as experienced pointedly with lyme disease or hiv infections, the immunoblot response can be considerably variable, but a confirmatory fingerprint or several fingerprints can be defined as minimal diagnostic thresholds. a higher threshold stringency can enhance serodiagnostic specificity. guan and others provided some key insights in this regard with sars-cov immunoblotting, and it is only highly probable that similar findings will be had with sars-cov- [ ] . not all antigens identified in immunoblotting may have functions in neutralization, and likewise, there may be more antigens that are capable of inducing neutralization than are identified by resolving antigens in denaturing electrophoresis procedures [ ] . given advances as detailed by olvera et al. and several others, the fingerprint patterns could also conceivably be assessed with peptide sets rather than larger antigens which may bear non-specific epitopes [ , , ] . the protein micro-array concept bears relevance to the ingenuity of how a complex recognition pattern could occur and be thereafter automated [ , ] . furthermore, we should not restrict ourselves to common antigens that are currently thought relevant since novel key antigens may yet arise [ ] . if serological methods of choice later prove to be a surrogate for infectious virus neutralization, e.g., viral pseudoparticles, blocking test components can also be built into those processes [ ] [ ] [ ] [ ] [ ] . the latter approaches too are capable of automation design or large batch processing. if conventional neutralization assays yield titres in lower dilution than pseudotype virus neutralization assays (e.g., s protein-bearing pseudoviruses with murine leukemia virus), the latter may set higher thresholds as the correlation dictates [ ] . if neutralizing antibody correlates with protection, is there a threshold at which the prediction can be confident? whereas there are no guarantees, if the presence of neutralizing antibody correlates with protection, it can be reasonably hypothesized that more protection correlates with higher titres. such a hypothesis is easily testable in the current pandemic given preliminary consensus for the type of neutralization assay to be used. it is proposed that the : - : standard of conventional neutralizing antibody be used as the minimal threshold in inaugural studies. the threshold could be raised or lowered depending on the outcome of field assessments. prospective follow-up of study subjects with the established minimum neutralizing antibody and its potential change in titre and/or protection would be determinable over time. there is then a role for other measures of antibody thereafter. as the initial neutralizing antibody threshold for protection is established, correlates with other tests as surrogates can then begin. in the latter, it would also be best to choose higher thresholds of positivity rather than any positive test per se. such thresholds could thereafter vary pending correlations that become apparent. such an approach provides an abundance of precaution rather than the converse. the test of a minimalist strategy and thereafter its refinement is currently feasible in the milieu of the current pandemic. given the magnitude of infected individuals throughout the world, the numbers required to test for protection could be easily acquired from a multi-centered approach with highly endemic foci or with even a regional study again in a highly endemic area. there will be concern in allowing previously infected, neutralizing antibody-positive individuals to be naturally exposed once again in the community, but such an approach has little difference to the broad testing of candidate vaccines. indeed, the answers may come sooner than the answers of whether vaccine candidates will succeed. infection exposures in the community for those previously infected with or without such antibody markers will also slowly answer other questions relating to those with low level neutralizing antibody (e.g., below threshold), those that do not have any measurable antibody, anamnestic responses, and susceptibility to changing virus. furthermore, the minimalist strategy will potentially set the goal posts for vaccine evaluations. reports of repeat sars-cov- infections have already begun to emerge as the pandemic continues [ , [ ] [ ] [ ] . undoubtedly, much more similar information will be collated anecdotally or in larger series and observations. an analysis of these is relevant in considering any minimalist strategy. gousseff et al. present a case series of healthcare workers and community patients who appeared to have a relapsing pattern of covid- [ ] . the establishment of re-infection within such a short period of time is tenuous at best. early and later positive diagnostic samples could be assessed with viral sequencing. serological profiling with both igg and igm and correlation with viral neutralization are additional tools of interest. the persistence of viral genome after acute infection, and as determined with genetic amplification technology, is well-known and can be measured in weeks. accordingly, comparison of diagnostic cycle thresholds can be of value as was done. co-determination of other pathogens, especially viral for co-infection, is essential. that some patients may clinically relapse with a complicating respiratory illness after an initial non-complicated illness is in keeping with many other respiratory infections. viral culture to confirm live virus provides an additional step in confirmation but evidently is difficult to achieve without support from reference level laboratories. as the authors suggest, are these examples simply ones of persistence or re-infection? bentivegna et al. describe a -year female with possible re-infection in which repeat rt-pcr positive samples of the respiratory tract were obtained in the context of four negative samples in between over a - -week period [ ] . immunoglobulin g serology was reactive on each occasion. igm serology was reactive only late in the second putative infection. blocking assays for the second serology could have potential use if devised. neutralization tests would have been of value to correlate with putative protection. an established fingerprint of immunoblotting if devised could be sought for in both early and later blood samples to further characterize the quality of immune reactivity. this case report emphasizes the role for a better understanding of applicable diagnostic and confirmatory serology. tomassini et al. highlight a case series of six patients who were possibly re-infected [ ] . igm serology, blocking for igg serology, neutralization correlates, and comparison of diagnostic ct values all have their potential merit. to and colleagues describe a repeat infection in a -year male in which two distinct isolates were believed to have been identified by whole-genome sequencing [ ] . the episodes occurred some months apart. measurable igg was not detected within days of the first episode. igm diagnostics, blocking for igg serology, and neutralization correlates all have their potential merit. the authors initiate relevant dialog about the relevance of genetic drift and possible re-infection. van elslande et al. provide some evidence for a repeat infection after a -month interval [ ] . a comparison of diagnostic samples suggested repeat infection with phylogenetically distinct strain detections. diagnostic serology with blocking studies, neutralization correlates, and comparison of diagnostic ct value all have their potential merit. these authors repeat the theme on genetic drift and its implications. in any of the aforementioned, sample collection and recollection validation shortly after the initial diagnostic specimen test positive can also contribute to overall accuracy of determining re-infections. while such an approach to defining protective immunity in the interim reaches our goals if that is possible, the otherwise sensible approaches to disease prevention should continue to be enforced. given the current evidence on person-person direct transmission, person-person aerosol transmission, and environment-person indirect transmission, adherence to the basic principles of abrogating infection spread should be maintained as is practical. facets of physical distancing, appropriate decontamination and disinfection, and contextappropriate use of masking all have their roles in minimizing risk. it will be the study populations where some of these preventions may be less stringent in order to test the hypotheses of natural protection after infection or vaccine prevention. nevertheless, if we do define a highly probable and preventative serodiagnostic correlation, the modes of prevention may eventually be seen in alternative fashion or stringency. the overall approach to disease control will be fluid and will adapt as the potential arises. the global economic outlook during the covid- pandemic: a changed world after less than months, the simulations that drove the world to strict lockdown appear to be wrong, the same of the policies they generated herd immunity or suppression strategy to combat covid- misinformation and de-contextualization: international media reporting on sweden and covid- . glob health prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome 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studies and the path forward adjusting coronavirus prevalence estimates for laboratory test kit error antibody tests for identification of current and pat infection with sars-cov- development of urease conjugated enzyme-linked immunosorbent assay (elisa) for the detection of igm and igg antibodies for the detection against mycoplasma pneumoniae in human sera evaluation of six commercial mid to high volume antibody and six point of care lateral flow assays for detection of sars-cov- antibodies antibody response against sars-cov- spike protein and nucleoprotein evaluated by automated immunoassays and elisas performance characteristics of the abbott architect sars-cov- igg assay and seroprevalence in performance characteristics of four high-throughput immunoassays for detection of igg antibodies against sars-cov- comparison of different serological assays for sars-cov- in real life evaluation of sars-cov- serology assays reveals a range of test performance humoral immune response to sars-cov- evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against sars-cov- sars-cov- -specific elisa development a highly specific and sensitive serological assay detects sars-cov- antibody levels in covid- patients that correlate with neutralization sars-cov- assays to detect functional antibody responses that block ace recognition in vaccinated animals and infected patients competitive elisa for the detection of serum antibodies specific for middle east respiratory syndrome coronavirus (mres-cov) plasma from recovered covid- subjects inhibits spike protein binding to ace in a microsphere-based inhibition assay comparison of automated sars-cov- antigen test for covid- infection with quantitative rt-pcr using nasopharyngeal swabs including from serially followed patients low performance of rapid antigen detection test as frontline testing for covid- diagnosis another false-positive problem for a sars-cov- antigen test in japan use of viral lysate antigen combined with recombinant protein in western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome sars-cov- consensus-sequence and matching overlapping peptides design for covid- immune studies and vaccine development protocol and reagents for pseudotyping lentiviral particles with sars-cov- spike protein for neutralization assays development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against sars-cov- . genes dis a simple protein-based surrogate neutralization assay for sars-cov- neutralizing antibody and soluble ace inhibition of a replicationcompetent vsv-sars-cov- and a clinical isolate of sars-cov- longitudinal dynamics of the neutralizing antibody response to sars-cov- infection development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein clinical recurrences of covid- symptoms after recovery: viral relapse, reinfection or inflammatory rebound? new igm seroconversion and positive rt-pcr test after exposure to the virus in recovered covid- patient setting the criteria for sars-cov- reinfection-six possible cases symptomatic sars-cov- reinfection by a phylogenetically distinct strain publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments this review is dedicated to those healthcare workers and researchers who have had the commitment to protect the populace and energetically pursue the science. key: cord- -zf tcwe authors: ge, shikun; xu, long; li, ben; zhong, fagang; liu, xiang; zhang, xiaoying title: canine parvovirus is diagnosed and neutralized by chicken igy-scfv generated against the virus capsid protein date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: zf tcwe canine parvovirus (cpv) can cause acute and highly contagious bloody enteritis in dog. to obtain antibodies against cpv, hens were immunized with virus-like particles (vlp) of cpv-vp . the igy single chain fragment variables (scfv) were generated by t phage display system and expressed in e. coli system. the titer of the primary scfv library reached to . × ( ) pfu/ml, and % of the phages contained the target fragments. the cpv-vlp and cpv-vp protein showed similar reaction values to the purified scfv in the elisa test, and the results of elisa analysis using igy-scfv toward cpv clinical samples were consistent with commercial immunochromatographic assay (ica) and pcr detection, the scfv did not show cross reactivity with canine distemper virus (cdv) and canine coronavirus (ccv). igy-scfv was successfully expressed in crfk cells, and in the virus suppression assay, % of cpv infections were eliminated within h. docking results demonstrated that the number of amino acids of the binding sides between scfv and vp were aa and aa , respectively. this study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian igy-scfv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine. canine parvovirus (cpv) was first identified in [ ] ; it has a single-stranded dna genome of negative polarity, about bp in length. the cpv capsid is a nm diameter icosahedron containing three structural viral proteins (vp , vp and vp ) and two non-structural proteins (ns and ns ), among them, vp accounts for % of the viral capsid and represents the major determinant of host range and virus-host interactions [ ] . although the conventional attenuated and inactivated cpv vaccines have been successful in reducing the disease outbreaks, the genus of parvovirus still causes severe epizootics worldwide and leads to severe economic losses in dogs [ ] . antibody based approach is promising in cpv disease diagnosis, therapy and prevention, and the relevant hyper immunoglobulin g (igg) and full-length monoclonal antibody (mabs) have been routinely applied in the veterinary practices [ ] . at present, most commercially available mabs are produced in mammalian system using hybridoma technology. functional antibody fragments (i.e.: scfv, fab) generated by phage-display technology have been not yet routinely evaluated and applied in the veterinary medicine despite it has been well confirmed that such engineered antibodies offer a series of advantages over polyclonal and full-length monoclonal antibodies. antibody phage display technology is to display polyclonal antibodies on the surface of the phage shell protein and to screen the specific monoclonal antibodies open access *correspondence: zhang@bio.uminho.pt department of biology, centre of molecular and environmental biology, university of minho, campus de gualtar, - braga, portugal full list of author information is available at the end of the article by bio-panning procedure [ ] . phages are more stable and can be stored for years at °c; it can be re-produced rapidly, successively and inexpensively with a confirmed sequence in the prokaryote or eukaryote systems without hybridoma and immunization procedure. furthermore, higher affinity mutants of scfv can be generated through site directed mutagenesis which is much easier and simpler to be performed [ ] . chicken (gallusb gallus domesticus) igy antibody generated by igy-technology is another conventional approach in generating large amount of high specific antibody and have been used for broad biomedical purposes owning to a series of advantages such as higher productivity, better animal welfare, higher immunogenicity to mammal conserved proteins and lower cross-reactivity, as compared to the generation and application of mammalian serum igg [ ] . our previous work confirmed that the generated polyclonal igy targeted to cpv-vp could apply into the immunotherapy and immunoprophylaxis for the cpv infection [ ] . in recent years, as an interesting tendency in igy technology, generation of igy-scfv in order to better combine the biological superiorities of both igy and recombinant antibody fragment, is gaining increasing attention and application [ , ] . our previous studies demonstrated that igy-scfv can be generated and applied in different immunoassays for the detection of small molecules gentamicin [ ] and large molecules pifn-γ [ ] in the veterinary practice. as an attempt to understand the feasibility of chicken igy-scfv in diagnosis and therapy of veterinary diseases, this study aimed to construct and characterize chicken sourced scfv against cpv-vp virus like particles (vlp) using phage display technology, and to evaluate the specificity, sensitivity and virus inhibition ability of the obtained scfv. twelve-week old white leghorn hens were immunized intramuscularly with cpv-vlp (provided by dr. shiqi sun [ ] ) mixed with freund's adjuvant (sigma-aldrich, st. louis, mo, usa) at four different sites of breast muscles. cpv-vlp protein ( µl, mg/ml) in equal volume of phosphate buffered solution (pbs, . m, ph . ) was emulsified with freund's complete adjuvant (fca; sigma-aldrich, st. louis, mo, usa) in the first immunization, and four booster immunizations were followed up with freund's incomplete adjuvant (fia; sigma-aldrich, st. louis, mo, usa) at -week intervals. all experimental animal protocols were reviewed and approved by the institutional ethics committee for the use of laboratory animals. the hen's spleen was collected to extract the total rna by total rna kit (tiangen biotech, beijing, china), and the first-strand cdna was synthesized by hiscript q select rt supermix for pcr (+gdna wiper, vazyme biotech, nanjing, china). the heavy variable fragment (v h ) and light chain variable fragment (v l ) genes were amplified by pcr with primers ( table ). the v h and v l were assembled with primers hf-ecor i & lr-hind iii by overlap pcr. the products of overlap pcr (scfv) were purified through gel extraction kit (omega, norcross, ga, usa). this protocol was performed as described in [ ] . the scfv gene products were digested with ecor i and hind iii restriction enzyme and ligated to t select - b ( . pmol; novagen, darmstadt, germany) by t dna ligase (takara biotechnology, dalian, china) in a work volume of µl at °c overnight. the ligation products were directly added to t package extract ( µl) and the mixtures were incubated at °c for h in vitro to create the primary library by add luria-bertani medium ( µl) to stop the reaction. the primary scfv library was amplified by liquid lysate amplification refer to the t select system manual. the titers of the primary and amplified library were evaluated by plaque assay. this protocol was performed as described in [ ] . the amplified library was subjected to four rounds of bio panning on microplates for the enrichment of the specific scfv phages refer to the t select system manual. the microplates were coated with cpv-vlp overnight ( µg, µg, µg and µg per well in each round) at °c, and washed with tween-pbs (pbst, tween . %; µl/well) and pbs, respectively. after blocking with skimmed milk ( %) in pbst, the amplified phages from the initial library of each round of panning were added to the microplates and incubated at °c for h, and the bound phage was eluted with sds ( %) in each round. in the first three rounds, eluted phages were amplified by infect bl bacterial culture (novagen, darmstadt, germany). the enrichment of specificity was determined from the input/ output ratio of the phage. a total of random clones of on the plates were selected from the fourth round to identify the positive rate by using a pair of universal primers ( table ). the amplified library was added with % chloroform and stored at °c. this protocol was performed as described in [ ] . the phage-scfv with the highest affinity was selected to ligate with pet- a (+) vector use t dna ligase (takara biotech, dalian, china). the recombinant plasmids were transformed into bl (de ) competent cells, and the scfv expression was induced with isopropylβ-d-thiogalactopyranoside (iptg). the bacterial cells were collected by centrifugation and sonication, and the supernatants and pellets were used for sds-page analysis, respectively. protein was purified by histrap hp histidine-tagged protein cultured columns (ge, pittsburgh, pa, usa). this protocol was performed as described in our previous reports [ ] . the cpv-vp proteins were separated by sds-page ( % spacer gel and % separation gel) and blotted on nitrocellulose membranes ( western blot was performed as described in [ ] . to determine the accuracy of scfv, a total of clinical dog stool samples were collected using sterile swabs in an animal hospital (xinger, xi'an, china). among them, samples were confirmed cpv positive and were negative by the hospital using commercial colloidal gold test strip (ica). the samples were homogenized ( %, w/v) in pbs ( ml, ph . ) and centrifuged, the supernatants were used for indirect elisa and pcr. in order to verify whether scfv has specificity in clinical practice detection, canine distemper virus (cdv) and coronavirus (ccv) were used to detect the cross-reaction by elisa. the crfk cells (lmai bio, shanghai, china) were seeded into -well plates with optimum culture medium and transfections were performed when the cell monolayer density reached %. the transfection mixture (containing the pcmv- -scfv) was added into each well and incubated for h at °c in moist % co . at h post-transfection, the cells were fixed with paraformaldehyde ( %), permeabilized with triton x- ( . %) and incubated with bsa (beyotime, shanghai, china) to block nonspecific binding sites. they were incubated with diluted his-tag antibody ( µl; affinity biosciences, oh, usa) for h then washed with pbs. goat anti-mouse igg (h + l) fluor -conjugated ( : ; affinity biosciences, oh, usa) was added. the stained cells were visualized by use a nikon eclipse i fluorescence microscope (nikon, sendai, japan). total protein of the cells transfected h were extracted for western blot; his-tag antibody and goat anti-mouse igg hrp (biosharp, hefei, china) were used as primary and secondary antibodies, respectively. ica was performed as described in [ ] . normal crfk cells ( docking was performed by discovery studio . software (biovia, san diego, ca, usa) and the antibody model cascade program in discovery studio software was used to build the molecular model of the scfv. then, to obtain the docking conformation, the scfv molecular model was docked with vp using the zdock molecular dock program. finally, the residues contact frequency (rcf) algorithm was used to analyze the predicted results, and the bind surfaces of amino acids in scfv and vp were obtained. the lengths of vh-linker, vl-linker and scfv were approximately bp, bp and bp, respectively ( figures a and b) . the titer of the primary anti-cpv scfv library ( figure c ) and the amplified library were . × pfu/ml, and . × pfu/ml, respectively. there were % of the phages containing the target fragments in the primary scfv library. after rounds of "bind-elute-amplify" bio-panning procedure, the data of input and output phages in each round indicated that the phage library had been enriched times ( figure and table ). a total of scfv genes showed relatively high binding capacity to cpv-vlp in the reactivity detection of phages, and the sequencing confirmed that all the scfv genes have complementary determining region (cdr ) that was the main mutation region in both vh and vl. there were few clones having limited mutations in framework region (fr, figure ). the phage-scfv (no. the solubility analysis showed that scfv mainly existed in the form of inclusion body ( kda; figure a , lane ). the denaturation and purification of scfv inclusion body protein showed a single protein band with high purity ( figure b ). the vp protein could bind to scfv with a single binding strip ( kda; figure c ). the immunoreactivity of scfv against soluble vp was examined by elisa. scfv bound in a dose-dependent manner to the soluble vp ( figure ). the minimum antibody concentration for the detected antigen was ng/µl. the coincidence of elisa ( figure a ) and pcr (figure c) with ica (data not show) was % and . %, respectively. the scfv showed no cross reactivity with cdv and ccv ( figure b ). the sequencing results showed that the scfv and pcmv- vectors were successfully connected (figure a) , ifa confirmed that scfv was significantly expressed in crfk cells ( figure b ). immunoblotting further confirmed that the scfv protein was correctly folded and modified in the cell ( figure c ). the crfk cells expressing scfv showed a small amount of cytopathic effect (cpe) after cpv infection; the cells without scfv expression were significantly broken away from the bottom wall, became round, and some cells even broke up ( figure d ). virus tcid at different time points was determined; the growth rate of virus in cells expressing scfv was significantly lower than that in cells not expressing scfv ( figure e ). the inhibition rates of scfv on virus growth at h, h, and h were %, % and %, respectively ( figure f ). the main structural domains of scfv were vlcdr , vlcdr , vldr , vhcdr , vhdcr , and vhcdr , with antigen-binding sites on scfv ( figure a ). the dimensional scfv model was subsequently constructed ( figure b ). a total of highly similar vp homologous sequences were simulated by software ( figure c ), the vp molecular stereo model was established by combining the characteristics of each sequence ( figure d ). after analysis on scfv and vp binding mode ( figure e ), the interacting amino acids at the binding sides of scfv (aa ) and vp (aa ) were confirmed ( figure f and g). the mabs have been widely applied in the biomedical areas owning to their high specificity and homogeneity. however, mabs produced by mammals may have the side effects of immunogenicity, thrombocytopenia and hypersensitivity reactions, etc., which has greatly limited their application in target detection and treatment [ , ] . with the development of antibody library technology and humanized antibody modification technology, humanized recombinant antibodies have been gradually developed and entered clinical trials [ ] . diversified antibody generation strategies could be a future tendency in antibody engineering in order to better combine the characteristics and advantages of antibodies from different sources. as a notable example, brolucizumab (beovu) is the first fda approved rabbit-derived scfv used as vascular endothelial growth factor (vegf) inhibitor for the treatment of exudative (wet) age-related macular degeneration (amd), diabetic macular oedema and macular oedema secondary to retinal vein occlusion, which could better overcome the possible side effects (discomfort and increased tears in the affected eyes, itchy or watery eyes, dry eyes, swelling of the eyelids, etc.) of murine-derived igg-fab fragment (lucentis) [ ] . in avian igy, similar attempts have also been made. for instance, the snake venom contains neurotoxic proteins, the urgent administration of hyperimmune serum from horse used to be the most efficient treatment, which can recognize many different antigenic determinants. however, generation of equine anti-venom is costly and associated with several potential side effects. chicken igy-scfv has been generated against glutaraldehyde-attenuated daboia russelii formosensis (drf) venom proteins for passive immunization, which can identify and neutralize the toxic activity of the venom components, with only small quantity of antigens required to induce a significant antibody response in hens, and can be also used as a rapid diagnostic tool for wound secretions to determine snake types [ ] . as summarized by previous authors, owning to their unique structure, phylogenetic distance and tmechanisms of molecular diversification, igy antibodies provide a series of important advantages over mammal igg, including the stronger immune responses of chicken system to the proteins conserved among mammals, decreased/no cross-reactivities (i.e.: rheumatoid factor, human anti-mouse igg antibody, complement system, fc receptors) in the mammal systems [ , ] , and more convenient design of primer against igy-scfv (table ) , as igy only has one isotype and lacks hinge region [ ] . furthermore, it is noteworthy to address, recent studies confirm that from the glycobiology point of view, recombinant igy antibody could be a potentially promising immune-therapeutic candidate after proper antibody engineering and expression as igy is more heavily glycosylated [ ] , and has higher sialic acid content [ ] as compared to mammal igg. recombinant functional antibody fragments remove or reduce irrelevant structures, while retain the specificity and main biological activities of natural antibodies, which offers a wider application prospect than natural antibodies [ ] . recombinant igy-scfv could combine the advantages of both igy molecular and functional antibody fragment [ ] . designing on chimeric antibody could be the next step for igy-scfv study in order to provide better compatibility of the antibody in the host system, and to recoup the possibly decreased specificity and affinity of antibody fragments as compared to full length antibody. recent study confirmed that mammalian igg and avian igy shared compatible v-c region interfaces, which may be conducive for the design and utilization of mammalianavian chimeric abs [ ] . in our study, the high consistency of elisa analysis to pcr and ica on clinical samples confirmed the specificity of the obtained igy-scfv (figure ) , which offers the potential using igy-scfv for rapid detection of cpv. scfv neutralized the virus ( figure d ), inhibited cpv replication with significantly reduced growth rates of the cpv observed in the crfk cells ( figure e , f), which provides the value of further therapeutic investigation of obtained igy-scfv. as alternative to viral components, cpv-vp -vlp was used as immunogen in this study. with increasing applications in vaccine design and immunization, vlp has been recognized as safe and effective particle to stimulate adequate immune responses for both viral and non-viral diseases by inducing lymphocyte proliferation and specific antibody with high titer [ ] . the docking of scfv-vp shows that there were antigenbinding sites on scfv, with high binding force to vp . according to residues contact frequency (rcf) algorithm analysis, binding amino acids on scfv and binding amino acids on vp were involved in the binding (figure ), these results provide us confidence that a well-designed cpv-vlp can be used as a potent immunogen to induce qualified specific antibodies. in conclusion, we demonstrated that specific igy-scfv can be generated with high specificity and significant inhibition to cpv growth. as a preliminary evaluation, our work revealed the potential of igy-scfv as a novel approach in veterinary diagnosis and therapy. ccv: coronavirus; cdv: canine distemper virus; cpv: canine parvovirus; ica: immunochromatographic assay; igg: immunoglobulin g; igy-scfv: chicken igy single chain variable fragments; iptg: isopropyl-β-d-thiogalactopyranoside; mabs: monoclonal antibody; scfv: single chain fragment variables; vlp: viruslike particles. canine gastroenteritis associated with a parvovirus-like agent. the can veterin the three-dimensional structure of canine parvovirus and its functional implications a survey of diagnosis and treatment of pet canine parvovirus disease in china generation and validation of canine single chain variable fragment phage display libraries antibody phage display: overview of a powerful technology that has quickly translated to the clinic recombinant antibody fragment production use of igy antibodies in human and veterinary medicine production of egg yolk antibody (igy) against recombinant canine parvovirus vp protein chicken monoclonal igy antibody: a novel antibody development strategy insights into the chicken igy with emphasis on the generation and applications of chicken recombinant monoclonal antibodies preclinical studies and clinical evaluation of compounds from the genus epimedium for osteoporosis and bone health generation and characterization of chicken-sourced single-chain variable fragments (scfvs) against porcine interferon-gamma (pifn-gamma) convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over m website views per year • at bmc self-assembly of virus-like particles of canine parvovirus capsid protein expressed from escherichia coli and application as viruslike particle vaccine construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scfv) antibody against staphylococcus aureus developed with the t phage display system preparation of a chicken scfv to analyze gentamicin residue in animal derived food products antibody validation by western blotting immunofluorescent cell assay of infectious pancreatic necrosis virus the safety and side effects of monoclonal antibodies therapeutic antibodies, vaccines and antibodyomes phage display technology for human monoclonal antibodies agents for the prevention and treatment of age-related macular degeneration and macular edema: a literature and patent review single chain antibody fragment against venom from the snake daboia russelii formosensis chicken egg yolk antibodies (igy-technology): a review of progress in production and use in research and human and veterinary medicine egg yolk antibodies (igy) and their applications in human and veterinary health: a review the impact of n-glycosylation on conformation and stability of immunoglobulin y from egg yolk publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations dr. xy zhang acknowledges the adjunct position and supports offered by the university of guelph, canada. authors' contributions xyz, fgz and skg conceived this project. skg, lx and bl performed the experiments. skg, lx, xl and xyz wrote the manuscript. all authors read and approved the final manuscript. this work was finically supported by the grand of china natural science foundation ( , ) and the incubation project on state key laboratory of biological resources and ecological environment of qinba areas (slgpt kf - ). all data supporting our findings are included in the manuscript. all experimental animal protocols were reviewed and approved by the institutional committee of shaanxi university of technology for the use of laboratory animals (approval number - ). not applicable. the authors declare that they have no competing interests. key: cord- - sqbqjm authors: nan title: monday: posters date: - - journal: vox sang doi: . /j. - . . .x sha: doc_id: cord_uid: sqbqjm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases ed- - standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be g (fda criteria). however, actual measurements have found a mean hemoglobin content of . ± . g per unit, with variability between manufacturers. percent of units may contain less than . g of hemoglobin while percent may contain more than . g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be . to percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of . g/dl. for pc transfusion, about percent of transfusions achieve the targeted dose of - ¥ platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of g/dl rather than g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of % yielded the same outcomes as one of %. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below % were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of g/dl and unlikely to be necessary at a hb above g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: ( ) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; ( ) the quality of retrieved studies is assessed systematically; ( ) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and ( ) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. ed- - biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf- /cxcr pathway has been emphasized, either by down regulation of cxcr , changes in sdf- gradients, or by disruption of sdf- /cxcr signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf- /cxcr , disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla /vcam- pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt- l, il- ) or chemokines (i.e. il- , gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf- molecules (i.e. ctce ), or cxcr antagonists (i.e. amd ) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. ed- - the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by - fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by %. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. ( ) ann med : - ). determination of the coding sequence of all the genes giving rise to antigens within the blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl ( ) human blood groups, nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. ( ) transfusion : - ), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. ( ) transfusion : - ). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. ed- - the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd + t cells and class ii molecules present exogenous antigens to cd + t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 's and 's -protect oncology patients from clinically relevant bleeding in more than % of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below per microliter (or in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol ; : - . additional data on lumbar puncture are reported in ann hematol ; : - . another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about % to about % of patients. ed- - ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over % in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: . studies of interventions comparing ffp with no ffp; . studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; . studies of interventions comparing ffp with a different blood product; . studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range - patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication ) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in . first in the commission agenda was a recommendation on donor selection criteria, given in . then came the european blood directive / /ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over donations per inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of nd august which came into force as of january st . this act was then updated in november according to eu directive / /ec and came into force as of january th . this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of nd august , in force since january st , has been supplemented by decrees: . procedures for external bts audits; . requirements for donor selection; . requirements and procedures for organization and safe management of blood transfusion in hospitals; . requirements for implementing of national and regional donor registers; . employment criteria for bts personnel; . training requirements for hospital personnel involved in blood and blood product administration; . national, uniform price list for blood and blood products; . organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age years, range - ; median weight kg, range . - . kg) with solid tumors at onset or on relapse underwent collections with the p y kit of the fresenius hemocare com.tec blood cell separator. our cd + cells target was ¥ e /kg. collections were started if a peak of at least . e /l cd + cells ( per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio : - : was combined with heparin u/kg. in children with < kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in % albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of ml (range . - . ml) was processed in a separation time of min (range - min). the median product weight was g (range - g) and yield of cd + cells was . ¥ e /kg body weight (range . - . ¥ e /kg body weight). three poor mobilizing patients (peripheral blood cd + peak of - cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose ( . and ). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under kg. m-pa- alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in , aubuchon and birkmeyer published a paper (jama ; ( ) : - ) where they concluded that the cost was usd per qaly, which is far above the 'acceptable limit' of usd . this estimate was adjusted to usd . mill. per qaly in a letter to jama (jackson, jama ). in , riedler et al. published (vox sang : - ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp for neonates and gbp for patients aged . the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = ) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas ) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination ( . mmol/l), maximum storage time of whole blood before separation ( °c, h), maximum storage time of mb plasma before freezing ( h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+ . %), fibrinogen (clauss) (- . %), factor v (- . %), factor viii (- . %), factor xi (- . %) and protein c (- . %). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha- -antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment + , thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at °c for months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of < . mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within hours (usually - hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but : plasma-containing transfusions is the most commonly cited figure. in % of patients, recovery is well underway within hours, and leads to complete resolution. death occurs in - %. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: ( ) antibody-mediated and ( ) -hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than % of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in % these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since . since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past years, cases of trali and cases of possible trali have been confirmed by critical review of each questionnaire. of cases of definite trali, donor specimens were obtained in cases. of cases, anti-leukocyte antibodies were detected in cases ( %) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (< %). of cases of antibody positive donors, anti hla antibodies were detected in cases, anti hna antibodies were detected in cases, and both were detected in cases. of cases of positive anti hla antibodies, class i antibodies were detected in cases, class ii antibodies were detected in cases, and both were detected in cases. on the other hand, the anti-leukocyte antibodies were detected in % of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion ( %). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of adverse events analysed by the serious hazards of transfusion (shot) scheme ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , ( %) were haemolytic transfusion reactions (htrs). were due to incorrect blood component transfused (ibct): / were abo incompatible and / caused by other red cell antibodies. a further cases were reported as acute htrs (ahtrs; i.e. occurring within hours of transfusion) whilst were recognised more than hours after transfusion and reported as delayed htrs (dhtr). / ( %) patients died and suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. / ahtrs occurred in group a ( / ) or group b ( / ) patients given group o platelets. of the ahtrs related to red cell transfusion, were due to errors, were in patients with auto-antibodies, in only of whom alloantibodies had been adequately excluded/identified. at least / dhtrs were potentially avoidable; in cases the antibody was detectable retrospectively in the pre-transfusion sample; in cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. % htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in of patients ( %) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations - allogeneic from matched sibling healthy donors and autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf ( - g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was - ¥ e /l (autologous setting) or on the th day - hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was . - . l, and . l for one pbsc transplantation in average. results: using a minimal target dose of cd + cell count ( ¥ e /kgbm), performing one mnc-apheresis procedure for % recipients sufficient number of pbscs were obtained. the mnc yield was . ¥ e /kgbm in allogeneic and . ¥ e /kgbm in autologous setting in average. the mean cd + yields for allogeneic and autologous transplantations were . ¥ e /kgbm and . ¥ e /kgbm, respectively. hematopoietic reconstitution was achieved on the . th day for leukocytes and the . th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd + cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late s but it was only in the s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive / /ec off. j. european union. . . :l / -l / ) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa- years of shot data - : a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to deaths and cases of major morbidity in the uk between and . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that % of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for % of all pre-transfusion testing but % of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. % of hospitals reported that they participated in shot in , but only % of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa- passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b ) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds ( depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa- evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for days. red cell quality was determined by measuring the in vivo red blood cell recovery hours after re-infusion of the -day stored red cells. storage hemolysis and atp were also determined. methods: units of blood ( ml) were collected from normal volunteers into the leukotrap wb system containing cp d/as- anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within hours at room temperature (rt units), or after hours at - °c (cold units). the prion filter set was sterilely connected to the units on day , and the units were filtered and stored for days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with -cr radioisotope, re-infusion, and determination of the -hour in vivo rbc recovery. a donor sample was also labeled with m-tc to allow for red cell mass determination. thus, both single-and double-label -hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on units. the mean single-label -hour recoveries were . % and . % for the rt and cold units, respectively. the mean double-label recoveries were . % and . % for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the -hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than % of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp d/as- (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~ ¥ e platelets in ml of % plasma and % intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria ( , , cfu) were added to the double units. each double unit was divided into two identical products containing , , or cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process ( mm amotosalen + j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days , and of storage. samples ( ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at and cfu per product, strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and strains (e. coli, c. perfringens) tested negative after days of platelet storage, respectively. k. pneumoniae tested positive after - hours of culture when sampled on day of platelet storage for both and cfu per product. at cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after days of platelet storage. the anaerobic cultures of p. acnes became positive after hours of culture when sampled on day of platelet storage. of the strains studied, only s. aureus consistently tested positive after - hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire -day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv- and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from to showed a significant decrease from / and / before nat implementation to / and / after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is / , five times higher than hiv one and times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples / , / , and / , in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of days, ultrio assay is projected to close the wp by an average of days on undiluted samples, days in minipools of samples, days in minipools of samples and only days in minipools of samples. the projected yield calculated on the basis of . million donations collected per year in france, would be . unit per year for minipool-nat and to units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: of clinical samples ( . %) typed identically with this system and cat, and of the discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average genome equivalents(geq)/ml early in pregnancy to about geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t / being only minutes. in we have shown that this cell free fetal dna could be used for rhd genotyping. in the last years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over %, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only - % of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. ( ) transfusion ( ), ]. this system utilized low-speed centrifugation of -well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for . % of samples, compared to . % for cat (p > . ). when samples were tested for unexpected alloantibodies, fc determined the correct result for . % of samples, as compared to . % for cat (p > . ). this novel method was better than cat at detecting weak anti-a (p < . ) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (< bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = . ) but a better correlation was observed with the langmuir equation (mean r † = . ). in maternal sera, the mean ka of anti-d was . ¥ to the m- (from . to ¥ to the m- ). in the sera from immunized donors, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ) and in lots of anti-d ig, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ). the comparison of anti-d affinity measured in cases of hdn in which infants presented a fetal anemia and in cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was . ¥ to the m- whereas in the cases of postnatal anemia the mean value of ka was . ¥ to the m- . conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa- alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa- alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, ; anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, and anti-hpa- b, ). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges . - . ). interestingly, we used lyophilized platelet with a high expression of cd bearing the hpa- system and we have detected anti hpa antibodies among sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa- system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between and g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between and g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for cases of cardiac bypass surgery. in addition, exclusive use of fuwb for burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa- transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention . the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from centres ( patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < . ) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < . ). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults ( ml/kg in premature; ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < ¥ cells/l if bleeding is observed; (b) a plt count < ¥ cells/l without bleeding; (c) a plt count < ¥ cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in children ( premature infants and at term newborns). the weight of premature infants ranged from - g and at term newborn from - g. gestation age of premature infants ranged from - weeks and of at term ones, of course, from - weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of - ml/kg, with a plt count < ¥ cells/l. results : in the study period, plt transfusions have been performed: children have been only transfused one time, while multiple plt transfusions (ranged - ) needed for children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome ( cases) , haemorrhagic syndrome without sepsis ( cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome ( cases). after hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all little patients. the highest increase in plt count was ¥ cells/l, while the lowest ¥ cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. % of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies fusion society of turkey (bbtst) in with contribution of blood transfusion centers. according to these figures % of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the blood banks performing apheresis, were university hospital blood banks. another blood banks were producing both productive and therapeutic, produce only productive and produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only centers reported to prepare random platelets as well. each center had apheresis machines between - . a total of centers was carrying out around < procedures, around - , at centers about - , at a further centers around - procedures a year (one center was excluded). of the responders to the survey , all procedure were done at blood banks, whereas at of them all were carried out by the hematology clinics. at other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: centers < , centers - , centers - and centers > units have donations a year. around % of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are % lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the hospital based transfusion services, which are financed within the budget of the whole hospital. the hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to % ( blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population ( in the north, in the central and in the south region). each sub region will have one standard mobile blood collection team to collect blood units daily, i.e. annually. the blood units per teams provide the blood units ( %) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p- years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in . since and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in , / . codex of voluntary blood donation and health care staff has also been established; blood donors donates blood annually. in the past years, there was over million blood donations, performed in accordance with who regulations. over transfusion medicine specialists and technicians specially trained for the work in blood transfusion service (n = ), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with . million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from . now, they have been regulated by tender. in , test to lues was introduced, to hbsag in , to a-hiv in and to a-hcv in . information system was introduced in . nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso implementation. in the current reform of transfusiology system we are aiming for percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde . the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in in stations of blood transfusion and in blood transfusion departments at big hospitals. amount of donors in was equal to , voluntary donors being . % of them. the average number of whole blood donations in relation to the general population is per inhabitants, and on average percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is . ml per inhabitant and ml per one bed. an average volume of one blood donation is ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about . % of collected blood is used for procurement of blood components and preparations, . % of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv- / , hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is . %. % of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: % albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first . cross-matches last year (in months) were found incompatible due to dat positivity of blood donors' red cells ( . %). dat positive [( +)-( +)] samples were only igg positive in cases, only c d positive in and igm positive and c d positive in case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c c, -c d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the donors that participated in the survey ( . %) answered the questionnaire, ( . %) men and ( . %) women. that the survey was useful thought % and % that it was not. opinions were elaborated by . %. that the questionnaire should be completed before each blood donation was the opinion of . %, % thought it should be filled out only the first time blood is donated and . % that the questionnaire should not be completed at all. the answers given were sincere in . % of blood donors, % were not and . % were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years ( ) ( ) ( ) ( ) ( ) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from . . till . . . statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total donors in nitm and ( . %) deferrals. . % of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in ( . %) blood donors, use of drugs - ( . %), low blood pressure - ( . %), high blood pressure - ( . %), infections - ( . %), cardiovascular diseases - ( . %) and others. relation voluntary/family donors is almost equal ( . : . ). in the last two years the number of voluntary blood donors is increasing ( : ), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~ %, which is result of insufficient data and it is increasing in the last two years (> %). reasons for deferral are predominantly from temporary character ( . %). permanent deferrals are only ( . %), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of % macedonians, % albanians and % others (serbs, gypsies, turks). over % of blood donors are voluntary non-remunerated and ~ % are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from till . results: there were blood donations for the mentioned period. predominant blood donors are employed and high school students in %. family blood donors are ~ %; between them % are from albanian population. the ratio between blood donors macedonians vs. albanians is : . woman blood donors are presented with %. first time blood donors are %, and regular donors arẽ %. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than %. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donat- conclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype b of hepatitis c virus is the most frequent in blood donors d, from a to f, from a to k, a and a. these are differently distributed in the world: types and are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from . to % of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may to december , blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: blood donors have resulted hcv-rna positive with identification of the following genotypes: a = cases ( . %); b = ( . %); a + b = ( . %); a/ c = ( . %); = ( . %); = ( . %); none was a or a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv- a has showed a double prevalence in men ( cases, . %) respect in women ( cases, . %), while genotype b is more frequent in women ( cases, . %) than in men ( cases, . %), moreover genotypes and do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt > iu, our results show that . : donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype b has resulted the most frequent in blood donors' population, followed by type , while the others have showed a very low prevalence. the high frequency of genotype in blood donors is explained by the observation that hcv is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype is principally linked to a not transfusion transmission way; genotype is linked to old age, to female sex and to post-transfusion transmission; genotypes and are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes and are still little known because extremely rare in europe. p- kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel . the kel (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family - ( . %) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger - ( . %). motives for their donation are the following: their wish to help the ones that need blood - ( . %), concern that some day everyone can be a potential recipient of blood - ( . %), because of offered benefits - ( . %), for a friend or relative - ( . %), care for their health - ( . %), because of citizen duty - ( . %), because the others donate - ( . %), curiosity - ( . %). they want to be invited every months - (%) students, every months - ( . %), every months - ( . %) and ( the mean age of case group was / ± / and the mean weight of them was / ± / , / % was male and the mean number of blood donation was / ± / . the mean age of control group was / ± / and the mean weight of them was / ± / . / % of them was male and the mean number of blood donation was / ± / . the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < . ). the rate of vasovegal rx was higher in blood donor (p < . ) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < . ) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than - and - whole blood units per year, respectively. we present studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. mg and mg daily combined with mg ascorbic acid over months (males) or months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (> mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period - . methods: the donation of whole blood and aphaeresis platelets from donors of age from to (max. years for each class) were count for the period of five years ( ) ( ) ( ) ( ) ( ) . the percentage of the man´s donation was calculated for each target class ( ) ( ) ( ) ( ) ( ) . results please see tables and . in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from to decreased during objected years. unfavourable state of total number of births in the czech republic ( birth in republic ( birth in , birth in ) and its decreasing tendency ( birth in !) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of women from the district of limassol (the second largest urban center of cyprus) between the ages of and . using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. % of women do not donate blood and attribute their reluctance to do so to health-related problems, while about % of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to % of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively and beat per minutes and blood pressures (bp) were both in the normal range ( / and / mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to / and / mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to / mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the - data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period ( ) ( ) , . percent of persons who presented for donation were deferred; . % were temporary deferrals ( % due to laboratory test results, among others low hemoglobin, . % due to risk of acquiring a transfusiontransmissible infection) and . % were permanent ( % due to the infectious diseases markers, . % due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption : percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure ( . % of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact - % of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia ( . % of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future ( - % of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn , abbott laboratories, usa), in a blood donor setting. methods: the nbm- device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of subjects ( females, males) aged - . subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm- device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device ( donors). hb levels were considered normal when readings were equal to or > . g/dl. results: venous hb measurements ranged from . - . g/dl. the mean nbm- hb level was . ± . g/dl, only . g/dl lower than the mean hb result obtained by venous sampling, which reached . ± . g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ± . g/dl. the mean absolute error (mae) of their difference was . g/dl. when checked against the cell-dyn in the usa center, where subjects had hb of . g/dl or lower, the nbm- and hematastat devices showed comparable sensitivity results. the nbm- using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the december to march were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: donors -control group: donors). computer records were evaluated during the next years ( march to march to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure ( %), deferred for medication ( %) and colds and/or sore throats ( . ). non deferred donors were a little more likely than donors with sttds to return over the next years ( . % vs. . % pv = . ) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers - ( %), while all other ethnic groups are present with only %. the most prevalent is the group of adults aged - ( - . %), with high school education - ( . %) and mostly those who donated blood - times ( - %). conclusion: having in mind that % of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with % in relation to the total number of blood donors surveyed in the period - . this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta -microglobulin (b -mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta -microglobulin (b -mg) is a low molecular weight protein ( daltons) and found in all biological fluids it is light chain of histocompatibility class -human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class -hla antigen plays a role in the elimination of the virus. method & samples: beta -micro globulin was measured in serum drawn from hbs ag positive blood donors include ( . %) female and ( . %) male in age between - years, and healthy ( %) female and ( %) male in the same age we detected serumic b -mg by enzyme immunoassay (ela). results: our studies showed b mg level increased in ( . %) hbs ag positive donor that was significant differences with healthy control (p = . ). conclusions: it seems that serum b mg is a good marker for hbs ag replication. the role of b mg in monitoring of response therapy needs to be more evaluated. and ( . %) were contributed by vd, rd and dd respectively. over the last / years, voluntary donations have shown a rising trend from . % to . %, where as rd ( . % to . %) and dd ( . % to . %) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd ( . % vs. . % vs. . %) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable ( . % vs. . % and . % vs. . %). hiv antibodies was found more frequently in vd as compared to dd [ . % vs. . % (p < . )] whereas, vdrl reactivity was lower in formal as compared to latter [ . % vs. . % (p < . )]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between and . results: during this period people volunteered to donate blood, out of which were allowed to donate blood, while were declined. out of the total number of blood donors were male and female donors. the reasons for declining potential donors were the following: . % had low levels of hb, . % were taking antibiotics, . % were ill, . % had low blood pressure, . % had high blood pressure, . % for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in and , but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. . the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. . the lack of motivation of the studied lot is another cause. the questionnaire was applied on a lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to , which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vÖrÖs*, c bercsÉnyi † , o petrÓ † , r jÁger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the . . in hungary. as the consequence of reorganization till . . , blood banks had been established instead of existing before, under direction of the hhnbts. the working profile of the regional blood centers and local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about inhabitants and hospital beds). local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually donors donate blood in this region. this donation activity covers about the % of all inhabitants. the acceptance ratio of the donors is good ( - % of the donors were deferred). there are hospitals in our region. the regional demand on rbcc is - . u/year, on ffp is . - . u/year and on pc is - . u/year. the poster shows the donor data and the details of blood application of this region since . p- implementation of rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pÉlissier and jl beaumont efs ile de france, paris, france background: single donor rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for rbc collection (weight ≥ kg, height ≥ cm, hb ≥ . g/dl, ferritin ≥ ng/ml for repeat rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the recruited donors, all donors were male, % were regular whole blood donors, % were regular whole blood or apheresis donors and % were new donors. the medical staff was trained on rbc collection with the sdr protocol and disposable set ln pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as to months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table . six percent of the donors had a ferritin level below ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: hematomas and citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of min). the results of the survey were very favorable as more than % of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: ( ) possible risk of infectious disease in seronegative study; ( ) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than % blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in (c = . ; p < . ) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs + apparatus were used for platelet apheresis. apheresis were performed between / / and / / . including criteria of donors are that estimated process time is smaller than minute and estimated postapheresis platelet count is higher than ¥ /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls ( procedures on patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count > ¥ e /l or blast count > ¥ e /l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was . %. tp-treatments ( procedures on patients with symptomatic thrombocythemia and/or platelet count higher than ¥ e /l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction ( . % in average) and with clearly noted clinical improvements, subsequently. tes ( procedures on patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from . % till . %. rbcx treatment ( procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with % albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of collections (range - ). the inlet blood flow ranged between . and . ml/min (median . ml/min). the median blood volume processed was ml (range - ). leukapheresis lasted a median of min (range - ). the median total nucleated cell yield was . ¥ e /kg (range . - . ), mononuclear cell (mnc) yield was . ¥ e /kg (range . - . ) and cd + cell yield was . ¥ e /kg (range . - . ). the median of mnc collection efficiencies was . % (range . - . ). in ( . %) patients, in only one apheresis procedure more than ¥ e cd + cell/kg were collected. during ( . %) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than ¥ e /l in cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately / pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a -year-old pregnant woman, alloimmunized to the hpa- a (p a , zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt . /mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was . ml in a short time procedure ( minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected ( . ¥ e ) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing . ¥ e each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from . /mmc to . /mmc and after a week the child had plt . /mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to - minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm ( - seconds), thrombin time ( - seconds), the dilute fibrin monomer complex ( - mg%), coagulation time ( - min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was - % of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of - procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: pts ( females, male), median age . years ( - ), affected with agvhd grade ii ( ) and extensive cgvhd ( ) gtx with median total granulocyte doses of ( - ) ¥ per gtx corresponding to . ¥ granulocytes/kg in children and . ¥ granulocytes/kg in adults. the wbc counts increased from baseline values of . ( - . ¥ ) g/l for both pediatric and adult patients to peak values of . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at one hour after gtx and to . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at hours after gtx. in out of patients ( %), the crp levels significantly declined ( ( - )%; p£ . ) during the granulocyte transfusion period; in almost all cases ( / ; %) after the initial or nd transfusion. thirty-eight patients ( %) were alive at day + after termination of neutropenia and gtx. patients without crp response to gtx ( / , %) and patients with severe viral infections / ( %) were not among the day + survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: of them with mcs p and with mcsplus (haemonetics), according to automatic standard protocols a p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was : ). most of the donors ( % men and % women) were patient´s relatives. half-hour before the initiation of the procedure, mg of calcium ( tablet cal-c-vita) were administered to each donor. the mean platelet yield was . e /unit. the overall rate of the donor related adverse events was . %. feeling faint was the most frequent event, which was occurred in . % of donations. hypotension and citrate related rates were . % and . %, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was . %. other adverse events were venipuncture related ( . %), machine related ( . %) and miscellaneous complications ( . %). ( ) plateletpheresis using the mcs p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. ( ) with the used acd-a-to-blood ratio ( : ) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. ( ) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis . from the donors immediately with the initiation of the procedure (citrated whole blood) and . from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap- c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs p device (p = . ). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a year old pregnant woman in the th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially mg/kg bw prednisolone for weeks and subsequently mg/kg bw for weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table ) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was . ml per apheresis. after day plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. again lead to a recovery of the patient which held on until day . conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: procedures were carried out with mnc program and procedures with autopbsc on patients with cgvhd. both hemoglobin increased from . ± . to ± mg/tu, k+ from . ± . to . ± . mmol/l, level of glucose decreased from . ± . to . ± . to . ± . mmol/l, ldh from . ± . to . ± . ukat/, lactate from . ± . to . ± . mmol/l, ph from . ± . to . . . the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was . ± . ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage and , in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/ - . background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the . to -fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in cases with bone marrow volume over ml, and cases with bone marrow volume under ml. fifteen of these cases were allogeneic, and were autologous procedures; a software uploaded with cobe pbsc coll vers . and (catalog no: - - ) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: - - ). results: the mean volume reduction was . % ( . - . ) for volumes over ml, and . % ( . - . ) for volumes less than ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd + cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of donors who donated platelets by apheresis procedure from january till april . procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than ¥ /l and the volume of the processed blood was not less than ml. according to histogram, the most frequent donors' initial platelet value ranged from ¥ /l to ¥ /l ( %). final concentration of the samples of tested donors ranged from . ¥ to . ¥ in the average volume of ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < . . leukocyte contamination of the final concentrate prepared without the filter ranged from . ¥ /l to . ¥ /l. presence of red blood cells in the final concentrate ranged from . ¥ /l to . ¥ /l. p- therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of diseases into categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in croatian hospitals in the period - , respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in patients suffering from various diseases. in ( %) patients ta was performed by membrane filtration, while in ( %) separation by centrifugation was used (table ) . according to the ctg, s of the aabb/asfa guidelines, ta was performed in ( %) diseases from ctg i, ( %) ctg ii, ( %) ctg iii, and ( %) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis ( %), collection of pbpcs ( %), sy. guillain-barré ( %), and plasmacytoma ( %). in ctg ii frequent indications were: poisonings ( %), systemic lupus erythematodes ( %), and rapidly progressing glomerulonephritis ( %), and in ctgs iii and iv: cytoreduction-polycythaemia ( %), thyroid storm ( %), gvhd ( %), and reumatoid arthritis ( %). ( ) the time spent for resolving h / , ( ) mtp / , ( ) discarded blood units / . iii group: wrong data input / , donor replacement / , marking errors / , error in determining blood group at the first blood taking / , errors in input medical consulting / and disregard of prohibitions / . the consequences are: ( ) the time spent for resolving h / , ( ) mtp . / . , ( ) discarded blood units / . conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso parameters (soon to be updated with iso ). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p- software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from to more than . indeed, bacterial detection is performed for pools of to platelet samples and to red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt . in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of different reports can be generated on the website as a pdf file. for this study, reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or , -dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) -phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis nd. edition. academic press, new york ; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from ml to a total volume of ml and tests were performed in microtiterplates. results: samples were analysed in hplc and modificated commercial testkits, another samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range - mm atp) and statistical analysis demonstrated a given linearity (r = . ). variability has been calculated as . % (intraassay; n = ) and . % (inter-assay; n = ). the hand-on-time calculated for samples has been decreased from . hours to minutes. at least the costs of atp-determination have been reduced from € . to € . per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: ( ) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. ( ) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about whole blood units were collected and processed by mda national blood services in [ ] [ ] [ ] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some different types of dm were defined. out of reports sent to the corresponding manufacturers for investigation, responses ( %) were received and analyzed. about % of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly % of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table analysis and characteristics of t ( ) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. ( ) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. ( ) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. ( ) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in / / and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso : standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of . million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of %. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of %. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of %, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min . %-max %). but because with vista any procedure brings between . and . products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between € and € (mean value €) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx- filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of units of red cells, measurements of volume, platelet concentration and platelet count/pool of ld platelet pools produced by a team of technologists and of ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by . % of participants. the remaining . % of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in . % and . % of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. % of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso -quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on / iso -quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into levels; -most severe, potentially harmful to patient. -severe, damage to process and result. -moderatly severe, damage to process only. -benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years - , events were detected and recorded in the blood bank, they comprised . % of all tests performed ( ). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level & , whereas technical events were mainly of severity levels & . analysis of event recurrence in the quality system revealed that % of events were resolved, whereas only %- % of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of . %. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: . administrative for defining the it security organization, the information security policy, and information security awareness and training. . physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. . technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: < . ¥ /l, leukocytes: < . ¥ /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv- group m arna, hiv- group o arna, hiv- arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv- group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing % of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives / /ec and / /ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since . blood samples are classified according to adr in division . under un as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. . of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso : standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso : standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and , dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on erythrocyte suspension (es) samples during days of storage at oc on days , , , and . for whole blood (wb) samples during days of storage, same parameters were measured on days , , and . the measurement of the complex impedance of blood samples were performed in the frequency range from khz to mhz. by using the a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and , dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and , dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and , dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that , dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of plateletpheresis procedures performed using two cell separators (cs baxter, usa, mcs p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs was . ± . ¥ and in mcs p, it was . ± . ¥ per unit, however, only - % of sdps showed wbc levels < ¥ . six of donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in , the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in , centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = . ), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured times in order to determine within-run imprecision. coefficient of variation (cv) calculated from consecutive measurements was . %. to investigate between-day imprecision of the hemocue method one sample was divided in aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was . %. during the period of validation measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were . % (low), . % (medium) and . % (high). in order to investigate possibility of batch testing, supernatants of different rbc products were divided in aliquots and measured periodically during -month period. cvs calculated from measurements of each sample were in range . - . %. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a . mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer ( wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the -year period ( - ) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w ' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in . purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is years, of which at least are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: . organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. . collection, processing, storage and transport of blood and blood components . laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) . clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is -year specialization in transfusion medicine, established years ago (over specialists finished this specialization till now). we have postgraduate studies in tm started years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of theoretical and practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory -month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished -mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at - % of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of - %). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than time in - years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost % from the general number of patients of hospitals with positive results at % from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to , whole blood was comprising the majority of transfused units (more then %) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized symposiums, national courses, national and international congresses since . due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to %. in most of the university hospitals and training hospitals this is around %. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was ± and postoperative ± mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived ( ± ) and survived patients ( ± ) was not significant (p = . ). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il- and anti-inflammatory cytokine il- in relation to clinical outcome and complications after cardiac surgery. methods: in participating patients blood samples were taken before and after surgery. using elisa il- and il- were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il- and il- were low. mean postoperative level for il- was ± and for il- ± . compared with patients without mods and without infections and survived patients only the il- was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il- were higher in patients receiving more then units blood transfusions compared to transfusions. conclusion: there is an association between mods and mortality and il- , not with il- . ld has no influence on mean levels of il- and il- . there is a correlation between transfusion of more then units and il- , not with il- . these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july to december . patients were enrolled within h of icu admission. follow-up time was days, hospital discharge or death, whichever occurred first. results: a total of patients ( male, female, mean age . ± . years, range - ) were included in the study. the mean hemoglobin (hb) level at baseline was . ± . , which level was descending during the study. overall . % ( / ) of the patients received one or more rbc units while in the icu stay (mean . ± . units per patients). the mean pretransfusion hb was . ± . g/dl and the mean time to first icu transfusion was . ± . days. more rbc transfusions were given in the first week of the icu stay ( units vs , , units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = . , p < . and r = . , p < . respectively). baseline hb level was significantly related to the number of rbc transfusion (r = . , p < . ), but was not an independent predictor risk factor of length of stay or mortality (r = . , p > . and r = . , p > . respectively). the mean baseline apache ii and saps scores were . ± . and . ± . respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of < g/dl ( . ± . vs . ± . and . ± . vs . ± . respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < . ). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population ( . mill) the trauma team performed initial assessment and resuscitation of patients, % males, during the first six months of . the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: units of erythrocytes (er), of thrombocytes (thr) (buffy coat preparations from donors) and of s/d-treated whole plasma (op) (octaplasÒ) were transfused in uuh during this period. . % of er were given to surgical, . % to medical and . % to gynaecological and obstetric patients. % of er were given to patients above years. er units were given to patients (mean . units/patient; range - ). mean age of trauma patients was ± , median , range - years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb < g/dl, irrespective of age and sex. eighty-eight patients ( . %) received er, of them as massive transfusion ( er units in < hours) ( . %), of these died ( %). altogether trauma patients received units of er ( . % of total uuh consumption), . thr ( . % of total) and units of op ( . % of total). massive transfusion consumed er ( %), . thr ( . %) and op ( . %) units. fourteen adult trauma patients ( . % of those transfused) received or er units only. lowest pre-transfusion hgb was . - . g/dl, median . , mean . ± ; highest post-transfusion hgb . - . g/dl, median . , mean . ± . five patients received er transfusion at higher pretransfusion hgb level than g/dl, but in er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for > days, while only % were issued before days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year . in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of - mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved health institutions across the country. as many as deliveries and abortions were reported in . the institutions reported the usage of doses of mg rhd gamma globulin or doses/ deliveries + abortions. we compared our data with those obtained from similar surveys conducted in , i.e. before the issuance of the guidelines. in that year deliveries and abortions were reported, and doses of rhd gamma globulin or doses/ deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of - beds, general hospitals with the capacity of - beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/ deliveries + abortions in the first category was - with the median being ; in the second category there were - doses with the median being , in the third category - doses with the median being , while in the fourth category the range was - doses with the median of doses. the number of pregnancies which should have been protected with rhd immunization in year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = . ¥ . = . . if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in , doses of rhd gamma globulin or mg/ deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in , the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in a study was carried out regarding the usage of plasma derivates. as many as health institutions took part having the capacity of acute beds out of a total of . the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of - beds and general hospitals with the capacity of - beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in . the consumption of albumin was . kg or kg/million inhabitants in and consumption per bed for the first category institutions was in range . - . kg with the median being . , for the second category the range was . - . kg with the median being . , while for the third category the range was . - . kg with the median being . . in in croatia the consumption of albumin was . kg or kg/million inhabitants. the consumption of i.v. gamma globulin was . kg or . kg/million inhabitants in and the consumption per bed for the first category institutions was in range . g- . g. with the median being . , for the second category was in range . - . g. with the median of . and for the third category was in range . - . with the median . g. in in croatia the consumption of i.v. gamma globulin was . kg or . /million inhabitants. the consumption of the concentrate of factor viii was . iu or . iu per inhabitant in . . iu, i.e. . %, was a recombinant factor viii. in the consumption of factor viii was . iu or . iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased . times and the use of the concentrate of factor viii increased . times if compared with the results in . self-sufficiency has not been reached in plasma derivates in croatia we needed l plasma for gamma globulin and l plasma for concentrate of factor viii for the level of usage of these plasma derivates in . significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than % following transfusion with rhd+ rbc's and as many as % in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital ( beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby . rhd-patients, from to , were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the . rhd-patients we found out identified anti-d, % ( ) detected before any transfusion in our hospital. of the left, nine were excluded because no information was available during some years. had history of pregnancy related to the immunization. patients were exposed to rhd+ blood components: were transfused only with rhd+ platelets (including two women of childbearing age); with rhd + rbc's. the remaining had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period ( ) ( ) ( ) ( ) ( ) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total hospitalized patients who have been analyzed, ( . %) received wb and bp, and there have been transfused units wb and bp. every patient, on an average, has been transfused with . units wb and bp. at the same time ( . %) units have been transfused as a wb, ( . %) have been transfused as red blood cells (rbcs) and ( . %) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during - and to compare them with other studies. one hundred sixty five ( ) unselected patients of whom ( %) were women and ( %) were men with a mean age of years (range - years) were studied retrospectively. seventy six ( %) patients had intracapsular fracture and ( %) intratrochanteric fracture. the mean hb concentration on admission was . g/dl (range . - g/dl) and the mean hb concentration at discharge was . g/dl (range . - . g/dl). a total of units of rbc were transfused (a mean of . units per patient). blood transfusion occurred in patients ( . %), ( - % in other studies) with a mean of . units per patient (range - units), ( . - . in other studies). patients with preoperative hb values < g/dl were transfused more often than those with hb values > g/dl ( % versus %) p: . . women were transfused more often men ( . % versus . %) p: . . patients aged > years were transfused more often than those aged < years ( % versus %) p: . . finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures ( . % versus %) p: . . conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb < g/dl, in patients older than years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of . we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine %, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) . % and trauma/surgery . %, cancer . %. summary/conclusions: the use of ffp corresponds to . % of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from to in our hospital. results: our data are presented in table . conclusion: after , wb usage rates systematically decreased from % to . %. however, in the period of - , the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about % of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since . background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® pre and post a platelet transfusion (n = ) and was compared with healthy controls (n = ). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = ). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < . ). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years ( ) ( ) ( ) , units of wb were collected, among them ( . %) units were collected for pre-storage filtration and in ( . %) of these qc was done. within hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at g for minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume ± . ml ( % corresponds with the guidelines of council of europe), hematocrit . ± . ( %), hemoglobin . ± . g/unit ( %), number of residual leukocytes . ± . ¥ ( %), hemolysis . ± . % ( %), the test of sterility was % negative. during filtration of wb, wbc were removed in approximately . %, the duration of filtration was ± minutes and the loss of hemoglobin was . ± . %. the pre-storage filtration of wb is highly efficient, . % of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from , when the cardiosurgery department was founded in our hospital, to . the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from to were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs - and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about - . % were produced with the use od these filters, and platelets from - % with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts ( ¥ cabg and/or valve; ¥ re-cabg and/or valve; ¥ valve with or without cabg). oncologic surgery patients were also included in three rcts ( ¥ colorectal cancer; ¥ gi-oncology or vascular surgery; ¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in or more of the rcts. multivariate analyses were performed on: in-hospital mortality, -day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from surgery patients were collected ( onco; cardiac; vascular; orthopedic, other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = . ) and at days post-surgery (p = . ). no association between randomization and stay in hospital (p = . ) or stay on icu (p = . ) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = . ), and 'hospital' to be far stronger associated (p < . ). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = . ) and the trial arm became more strongly associated with infections (p = . ). in the analyses of surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = ) and portugal (n = ). in november a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was % (uk) and % (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by % in wales (n = ) and % in england (n = ), but only % in scotland (n = ), % in northern ireland (n = ) and % in eire (n = ). in mainland europe, variation was also observed: % in denmark (n = ), % in portugal (n = ) and % in other countries (n = from countries). fewer laboratories selected c-blood for pmfs: % in england and northern ireland, % in scotland and eire, rising to % in wales. mainland europe showed similar variation: % in denmark, % in portugal (all ccee matched) and % in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged < years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a year old female (with pre-existing antibodies other than anti-k), from % ( ), % ( ) to % in . in this survey, k + blood was selected for a female aged (no antibodies) by %, whilst % selected a k + unit for a female aged (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the and year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data ( ) suggested that > % laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec- ) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c fresh showed ± and after hours storage ± % expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib- ), low (gpib- ) and in-between (gpib- ). the binding and phagocytosis of plt showed an increase of ± % which implicates an indirect and negative relation to gpib- , and direct relation to gpib- expression. anti-human pselectin (cd p) delayed ± % the binding and annexing v ± % the phagocytosis of stored plts, after -hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a -year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured g/l. he received ml . % nacl and ml ringer solutions. after that, he was transfused with units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for hours. two days after the patient was operated. after surgery he was transfused with units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for minutes at °c with fmlp, lps or pma. monoclonal antibodies against hna a and hla-i, or patient serum were incubated with whole blood for min. surface cd b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna a monoclonal antibodies induced neutrophil activation, which were also inhibited by p map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: out of transfusion reactions were febrile, were anaphylactic, were due to circulatory overload, out of transfusion reactions concerned the transfusion of incorrect blood component. out of transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)- beta, il- and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il- has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il- from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from blood donors and frozen at - °c. upon thawing, the level of the il- beta, il- and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il- beta was at the test detection limit in all donor plasma samples. in ( . %) bd, the level of il- was . pg/ml, exceeding the test sensitivity limit of . pg/ml. in ( . %) bd, the level of tnf-alpha was within the range of - pg/ml, with a test sensitivity limit of pg/ml. tnf-alpha levels > pg/ml were measured in plasma samples of ( . %) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il- and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao = mmhg, o saturation = %), hypotension (bp = / mmhg) and fever ( °c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp ( cm h o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for hours the patient improved significantly, was extubated and returned to the surgical unit on the th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a -year old female with a history of multiple abortions whereas the prc unit was from a -year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years - there were . blood components issued in slovenia and atrs were reported ( in blood components issued), in of them the severity grade was ( in . blood components issued). in the year there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from in to atr in blood components issued. . % of all atr were not classified. conclusions: although the number of reported atrs was increased by % and % a year in and respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, . years old, with average weight of kg were used. the animals were dewormed by albendazole ( my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. ( ) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed ( . %) out of ( . %) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa- a in a homozygous hpa- b patient. in a minority of cases the offending antibodies were directed against hpa- b, - a, - b, a, - a, - b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a -year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a -year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a -year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa- a, anti hpa- b and anti hpa- b, respectively. the platelets genotype of the first patient was hpa- b/ b, of the second hpa- a/ a and of the third patient, hpa- a/ a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in . the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: ( ) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. ( ) to use the information acquired to determine the morbidity of transfusion. ( ) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. ( ) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: ( ) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. ( ) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. ( ) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: ( ) the total number of reported cases has steadily increased since the introduction of the programme. ( ) the number of participating healthcare institutions has also increased to % (n = ). please refer to table entitled 'summary of the haemovigilance report - ' . ( ) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. ( ) there has been very good participation from the participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. ( ) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing ( %) were rated for seriousness. of these, ( . %) were rated as grade (moderate to serious morbidity) or worse. ( . %) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in ( . %) and as 'possible' in ( . %). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use ( . per the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested > months following the implicated unit. results: between between - suspected pth cases were reported: ( %) were pth-b, were pth-c ( %) and in patient both hbv and hcv infections were reported. investigation was completed in / ( %) cases, with % of the involved donors ( / ) being retested > months after the implicated donation. hbsag was not detected in any of the retested donors. anti-hbc was detected in donors involved in pth-b cases of which were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 'anti-hbc+ only' donors, and none was found positive. only in / donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in . in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in % ( / ) of the cases where the patient received up to blood components. conclusion: in the past years an average of cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in % of the reported cases. so far, there was no clear evidence of hbv transmission. in cases (out of . million blood units collected nationwide during - ) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. - . , p < . ). the seroprevalence for a-hiv in the bd population was . % (se: . ; ci: . - . ; p < . ) whereas in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-hiv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was . %. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was . , which indicates that the number of bd a-hiv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was . % (se: . ; ci: . - . , p < . ), in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-htlv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was . %. the rp for a-hbc as regards a-htlv was . , which indicates that the number of bd a-htlv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. . despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. . our data show that the real anti-hcv prevalence of our donor population is very low ( . %). . the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. . given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. . donor re-entry in the pool of donors is an issue for further discussion. . the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: . to recognize the epidemiology of hepatitis c and how it differs geographically; . to investigate the risk factors for presence of anti-hcv antibody in blood donors; . to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january , using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was . % out of blood donations. the average prevalence of hcv infection by riba was . %, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was , with cases in males and cases in females. most infections were found among older persons ( % were aged - , and % aged - ). the seropositivity was higher in family/replacement donors ( %) than in volunteers ( %). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion %, hospitalization %, immigrants %, occupational %, sexual transmission %, injection drug use %, household contacts %, other %, tattooing %, unknown %. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested sexual partners and family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using % trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in ) the risk of bacterial contamination, ) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and ) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year . results: in the attached table the estimated risk reduction through % trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by %, the risk of known viral infections by %, and transfusion acquired bacterial infections by %. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly ( : ) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table shows the distribution of the blood donors in different professions. administrative clerks with ( . %) and the workers with ( . %) registered in the group of hbsag(+) blood donors, as well as workers with ( %) and administrative clerks with ( %) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = and p > . there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = . and p < . shows a significant difference, where the workers with ( . %) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table . in the group of hbsag(+) blood donors the number of those who work in production profession- ( . %), is dominant over the number of those that has non-production profession- ( . %). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c = . and p < . there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from confirmed hbsag(+) blood donors and confirmed hbsag(-) blood donors as control group. results: the table shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group ( %) donors that live in a flat dominate compared to ( %) of those that live in a house. having in consideration the presented distribution (table ) for c = . and p < . there is a significant difference, that comes from the bigger number of blood donors ( ) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table ) , we can show that ( . %) of the hbsag(+) donors have less than m living space, in compared to ( %) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between and m , and between and m of living space per person, obviously are not very big. for u = and p > . there is no significant difference in the number of the donors in the two groups, when the available living space in m is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in from cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table shows the distribution of blood donors concerning the use of condoms. table : in the group of hbsag(+) blood donors those who have not used condoms dominate with number of ( . %), in compared to those ( . %) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom- ( %), compared to ( %) who have not. the given distribution of blood donors concerning the use of condoms for c = . and p < . , shows significant difference, which is due to the prevailing of the number of blood donors ( ), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the th- st day and th- th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of - °c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, specimens ( . %) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were . %, . %, and %, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and of them were found positive for one of the three viruses (hiv- , hcv, hbv). with the elisa/axsym assay of the blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those blood units units were given for transfusion following our blood centre protocol and the remaining units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those that were found positive by the procleix ultrio assay was positive for anti-hcv and were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day or and, in case of negative result, prolongation of their storage time to days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to days at to °c with end-over-end agitation. other plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day , and . both aerobic and anaerobic culture bottles were inoculated with a -ml platelet sample. culture bottles were incubated at °c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of plt concentrates were cultured and bacterial contamination was assessed in each unit at day , and after collection. on of storage day two out of ( . %) plt units were found to be positive for bacterial growth. cases of unconfirmed positive results were noted at the beginning of the study. out of the other units which were negative on day and continued to be cultured for the next days, the assessment at day found no other positive. after further storage, at day , defined as the end of the prolonged incubation period, out of the plt concentrates ( . %) grew bacteria although testing of the same units on day and gave no signal. from the platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to days. bacterial contamination testing on day and a storage time of maximum days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from - were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of platelet products were tested during the year period. thirty ( . %) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in ( . %) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with - units of concentrated red cells, depending on their problem. a total units were delivered from the beginning of their problem until december . the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: patients anti-hbc (+) and anti-hbs (+). patients anti-hbc (-) and Ánti-hbs (-) patients anti-hbc (-) and anti-hbs (+) patient anti-hbc (+) and anti-hbs (-) patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is % safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables and ) . for hiv and hcv the specificity was . %. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in % plasma and % ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: - hour after donation, about ml of pc were sampled into the ebds collection pouch and then incubated at °c under continuous agitation (incubator helmer ) for hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso ). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency and run control panels. ( . %) out of samples were positive for anti-hbc. ( . %) out of anti-hbc positive samples were hbsab positive, and ( . %) were hbsab negative. all of samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from . ng/ml bbi to . - . ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than . and . ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in patients referred during the aug. to dec. . all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. ( . %) were hbv dna positive and ( . %) were negative. ( . %) out of patients had evidence of delta infection and ( . %) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in out of hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in out of hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that . % of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker ( . %) than those with hbv dna (pcr) positivity ( . %). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested sera samples from the general population and 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv- , hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b (pb ) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb and hav in portuguese blood donors. we gather, during four months, plasma donations and joined them into pools, with no more than donations each. [ ] [ ] [ ] [ ] [ ] in voluntary donors the anti-hcv prevalence ranged from . % to . %, in family replacement donors from . % to . %, autologous donors from % to . %. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is . % and women . % (p = . ). over the year periods the prevalence in men has a decline tendency ( . % to . %; p = . ) and increasing tendency in women ( . % to . %; p = . ). according to age group the anti-hcv is . % in - age group, . % in - age group, . % in - age group, . % in - age group (p = . ). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [ ] [ ] [ ] [ ] [ ] . a total ftd and ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the -year overall hbsag prevalence among first time blood donors was . %. and ad . %. among autologous blood donors was observed a decreasing hbv prevalence from . % to . % in . according to age the prevalence was higher in - year group . %, while according to sex was higher in man ( . %) than female . % (p < . ). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from - hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. ( : ) . the hbv dna-emia in hbsag negative samples was . ¥ - . ¥ copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high ( . %) therefore the decision to introduce routine hbv nat screening is justified. ( / ) with stored apheresis and whole blood derived platelet concentrates. of these failed results there were confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing / . the bacteria detected were staphylococcus or streptococcus sp. of the fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing . % or / , and were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing / . there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of / , and with a low false positive rate (< . %). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only . % of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot . (genelab diagnostics) results: results from a total of screened donors were analysed. hepatitis b surface antigen rates was . %; anti-hcv seropositivity was . %; anti-hiv seropositivity was . % and tpha seropositivity was . %. one study calculated this risk to be one in for hbv, one in for hiv and one in for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in - was . %. but it had decreased to approximately . % in . anti-hcv positivity has been reported to be . % between and . but it was approximately . % in . rpr positivity in blood donors in turkey was reported to be < . % in and . % in . in , the rpr rates was . %. in our study these rates are . %, . %, . % and . % respectively. anti-hiv seropositivity was found around . introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn , tpn and tpn ) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be %, after testing specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over titer by tppa). the specificity generated from testing hospitalised patients previously screened as tppa negative, was . %. testing a mixture of sera and plasma from random donor specimens, generated donor specificity figures of . %. the precision (cv%) with a positive control was . % ( % confidence interval: . - . %) by the standard -day nccls analysis (ep a ). in a study conducted at asahikawa medical college hospital, in which, positive and negative specimens were tested, concordance with fujirebio tppa was determined to be %. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of - days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns , ns ) and a sandwich test for core antigen detection. results are available within . hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of days earlier than the monolisa hcv plus v test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around . days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products ( ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools ( bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was . ¥ e /pool with mean leucocyte count . ¥ e /pool. ebds installation and training occurred over a day period. platelet pools were tested for bacterial contamination over the subsequent weeks. ebds pouches were sterile connected onto platelet pools hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for hours on a shaking agitator at °c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, technician was able to make oxygen measurements in less than minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. ( ) was . the percentage collected from volunteer blood donors was % (n = ) and the rest % (n = ) was given from patient-related donors. the age of donors ranged from to years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of tested units, of them were found to be positive to at least one marker of hbv infection, that means the . % of the health adult population was infected in the past by the hbv. the . % (n = ) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and . % (n = ) were chronic carriers of the hbv with hbsag(+). the . % (n = ) of the positive donors were patient related donors and . % (n = ) were volunteer donors. in other words, of the not volunteers ( . %) and of the volunteers ( . %) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions ( . %) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, . % compared with the . % of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type , hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv- , hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv- (n = ), hcv (n = ), and hbv (n = ) seroconversion panels were tested neat and diluted ( : and : ) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv- , hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p- effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv- /hcv and hiv- /hcv/hbv (procleix ultrio) assays for the detection of hiv- rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, out of ( % of the positive, . % of total) were reactive for hcv rna only and out of ( % of the positive, . % of total) were reactive for hiv- rna only. none were positive for both hiv- and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv- /hcv assay. of the samples tested by the ultrio assay, ( . %) tested reactive for hiv- /hcv/hbv. in discriminatory assay testing, out of ( . % of the positive, . % of total) was reactive for hiv- rna, out of ( % of the positive, . % of total) were reactive for hcv rna, and out of ( . % of the positive, . % of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than virus. three out of positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in ), and hcv testing (introduced in ). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of patients tested, cases ( %) were hbsag positive, cases ( %) were anti-hcv positive, and cases ( %) were anti-hdv positive. co-infection of hbsag and hcv was found in cases ( %), whereas co-infection of hcv, hdv, and hbv was found in persons ( %). the highest rates of infections and coinfections were found in patients above years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: / ( . %) samples from rbd were anti hiv + nonreactive and rr for p ag both being nonreactive in the neutralization test, they were interpreted as false positives. / ( . %) sample from fbd was rr for p ag/anti hiv + nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion days later: p ag nonreactive, anti hiv + reactive and western blot positive. the only bd p ag positive/anti hiv + nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~ days), this suggests that some - infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in , the general incidence in rbd has dropped from . ‰ to . ‰, suggestive of a : escape rate. the prevalence in ftbd has stabilized at ± ‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that potentially infectious donation in rbd escapes the screening (= to a total of aprox. , annually). a limited investigation using hcv-antigen eia evidenced a ‰ escape rate in ftbds (= to a total of aprox. , annually table and are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than hours. background: the hbsag, anti-hcv, anti-hiv / , p antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: . hcv rna pcr testing in anti-hcv negative blood donations; . correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of samples. pools of ml were tested using the cobas ampliscreen hcv test v. . (roche). results: pools of samples from a-hcv serologically negative donations were tested from october to july . no one pool was initially reactive. invalid tests: ( . %) run failures were observed, due to: invalid internal controls ( . %) and invalid positive controls ( . %). invalid tests were repeated. in none of pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b , such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b by nat since april . methods: over the last years . million donations were screened for b by nat. samples with a virus load over iu/ml were defined as positive, whereas samples with a virus load between the detection limit ( iu/ml) and iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of b positive donors (case group) were determined over a time period of one year. virus load and b antibody status was compared with b negative donors (randomised control group). b antibodies (igg vp , igm vp , ns ) were analysed by two commercial antibody tests. results: overall b nat-positive donors were identified with a virus load over iu/ml out of . million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp igg was detected in . % and % of the case and control group, respectively (p = . ). these data demonstrated statistically significance (p = . ). all donor samples which were b nat positive for more than three months developed neutralizing vp antibodies. in contrast, ns antibodies were observed in % of the case group and in % of the control group (p < . ). ns antibodies were detected more frequently in samples, which were b nat positive for more than six months. conclusion: b nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below iu/ml. these data support our testing algorithm all components of high positive donations (virus load over iu/ml) were discarded. donors with ns antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: . ( . %)cases turned out negative with both technologies; . ( . %) cases were positive in both methods; . cases were positive only using tmpa recombinant [of which ( . %) were confirmed positive by tpha/fta abs. as seen we found samples ( . %) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the ¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between : to : per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least ¥ cfu. the positive control has to be stored at room temperature and is stable for years. after dilution in pbs, the preparation has to be used within hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was . in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per million transfusions, the risk for bacterial infections is estimated to be : to : . especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of cfu/ml to cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of cfu/ml and cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at °c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between cfu/ml for e. coli and cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation ( - h) at °c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within minutes when the sample was taken hours after spiking. background: since year our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from ml plasma by using either the qiagen biorobot and qiamp virus biorobot kit or the manual extraction with cobas ampliscreen hcv pcr kit v . . results: between march and december a total of seronegative donations ( pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs samples in hours where the manual method needs hours for samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than samples/day while preserving the availability of blood products. anti-hcv similarly was high till ( . - . %), but in trend to decrease afterwards ( . %). anti-hiv reflected the low endemicity of the disease in public setting and was % through the mentioned years. rpr test for syphilis was around . %. directed donors were % of all and volunteer donors consisted nearly %. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since . from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was of (% ) were female and (% ) male in age ranged from to years. results: sera tested, ( %) were found to be positive for toxoplasma igg antibodies and ( . %) were igm antibodies positive and of them ( . %) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group to years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are patients suffering from thalassemia major who are aged between and , and who have been receiving erythrocytic transfusion - times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia . ). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least transfusions a year. the tests in all patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years ( ) ( ) ( ) ( ) ( ) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv / was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv / positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: ( ) we observe that there is a decrease in all four infections. ( ) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches % in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least out of the estimate of first-time donors, involving blood transfusion centres from of the italian regions. each centre was required to enrol all first-time donors born before december st, , and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first months of the study the sites saw almost first-time donors, of whom . % belonged to the required age groups. among eligible donors, . % were both hbsag and anti-hbc positive, and . % were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, % were positive for anti-hbe, % had raised alt and % were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, % were negative and % were positive for anti-hbs. among anti-hbs positives, % showed values < miu/ml and % > miu/ml. the avidity index results suggested that approximately % of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately % of the italian first-time donors are older than years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that . % of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly % of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv / and tp ab. to c + ns + ns , ( . %) to c + ns , ( . %) to c + ns + ns , ( %) to ns + ns + ns , ( %) to c + ns + ns , ( %) to ns + ns . the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients ( . %) had anti-hcv ab for or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv & , anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october and january . the multi-marker qc sample 'pelispy s type ' (s ), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > ) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s lots were exported from edcnet and analysed. results: nearly results were submitted: all results were reactive (s/co > ). fifty of these results ( . %) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = ) or due to unacceptable results (n = )]. a further results were excluded because data provided by laboratories were inconsistent or incorrect. a total of results, reported using different prism reagent lots ( for anti-hiv, for anti-hcv, for anti-htlv and for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of < % for all s lots. results from prism anti-hcv and anti-htlv produced cvs between . % and . % for all s lots. data reported for s lot ps (n = , range for anti-htlv to for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with < % bias for the prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a + % bias. prism anti-hcv showed the greatest variability within reagent lot with results from of reagent lots showing a cv between % and %. conclusion: in results in a qc sample distributed to laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in , the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv- rna screening of all blood donors started late . aim of the study: to implement hiv- rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv- rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer ( ml/ml of each). from the final volume of the nucleic acid eluate ( ml) ml was used for the detection of hiv- rna (roche cobas ampliscreen) and ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv- rna ( iu/ml) and hcv rna ( iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv- rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = ) and minipool samples (n = ). results: detection limits of the hiv- and hcv assays ( % hit rate) were calculated to be . iu/ml and . iu/ml respectively. specificity for both assays was % and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of was below the recommendations by the council of europe for blood donor screening (for hiv- rna iu/ml and for hcv iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv- nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of donations, ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • out of donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in to the relevant institutions of the eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the - age limits for blood donation ( - for a first donation) were the rule in countries. in other countries the age limit could be brought forward to and extended to years old. the time interval between donations was identical for men and women in countries, and varied from to weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the / /ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be % correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from to months. previous transfusion history could not be a ci or could be one varying from months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the /ec/ directive on these practices in the eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average whole-blood donations are performed per inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection . % -bruise assessed using post-donation interview . %, sore arm - . %, nerve irritation . %, arterial puncture/pseudoaneurysm/arteriovenous fistula . - . % etc) are most commonly reported, while negative systemic reactions (vasovagal reactions . - . %, syncope . - . %) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol ; : - .) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare ( . %). fatigue ( . %- . %) and diminished physical working capacity ( . %) are reported to occur during days after the donation. a recent study of a consecutive sample of swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - .) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common ( % of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; % of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - ). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa- establishing a national adverse event reporting system for blood donors -a prospective study of . there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: . to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; . to define and categorise adverse events; . to record data systematically and prospectively using the existing computerised donor database. methods: from summer , a small project team of senior clinical and operational staff took months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over staff working on mobile collection teams, static sites and blood centres. daer was fully implemented by january . adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first months . million donors attended sessions throughout england and north wales. results were very consistent month on month. donors ( : ) had vasovagal symptoms but only % of these suffered syncope. % of all vasovagal reactions occurred in women. % occurred in donors aged - and a further % in donors aged - . (donors aged - represent only % of the total donor base.) donors ( : ) reported a delayed reaction, % of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in donors ( : ) and, more rarely, arterial puncture was diagnosed in donors ( : ). bruising was reported after the session by : donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about % of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only % of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around % of the global blood population has access to only % of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population ( participants). the survey was performed in november . results: five percent reported being active donors (had donated during the last months), % were passive donors (had not donated during the last months), % were non-donors with a positive attitude towards becoming donors, and % non-donors with no intentions ever to donate blood. in the youngest age group (age - ), % reported being active donors and % were passive donors. however, % of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and % reported they did not care about blood donation. in comparison only % of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and % reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion ( %) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: . a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. . specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. . consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to first-time and repeat blood donors, in order to be used as an indicant of donor's satisfaction. . first-time donors increased . % in the months of application. among them, . % were donors 'for relatives or friends' turned into volunteer donors and . % were first-time volunteer donors. the questionnaire analysis further revealed: • % were motivated by the use of magnetic card. • % appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • % were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • % considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. . the turnover of repeat donors also increased . % after replacing their plain old paper cards with new ones. further analysis revealed that: • % appreciated the quick cross-checking of donor's identity. • % were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in , greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa- emerging technologies in transfusion. dna based assays until the late s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short ( - nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing donors were genotyped for hpa- through - and ; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and , all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested ( epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons , , , , and . in all known weak d types, all six rhd specific exons are amplified (except for weak d type which lacks rhd exons and ), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between and , samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types ( t > g), ( g > c), ( c > g) and ( c > a). weak d type was identified by the combination of serology and absence of exons and by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all samples were subjected to the rhd mpx pcr and sample showed absence of rhd exons and , indicative of a weak d type when combined with serology. the remaining samples were analyzed by the weak d pcr-ssps, resulting in weak d type samples, weak d type samples, weak d type samples and weak d type sample. two samples remained undetermined and were sequenced for all rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau partial d phenotype ( g>a, g>a and c>t). the other sample had only one, not previously known mutation ( a>t), which is located intracellularly at the coohtail. extensive serology using the epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type . conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types , and should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, out of patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a . % incidence of anti-kel is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel acquired by transfusion/previous pregnancies: st case: in july , a -year-old woman (gravida , para ), rhdnegative, kel -negative was referred at weeks gestation. the father's phenotype was rhd-positive, kel -positive. a maternal antibody screen revealed d and kel alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron and exon of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel homozygosity; nd case: in august , a -year-old woman (gravida , para ) was referred at weeks gestation. she had a history of transfusion with rbcs units in -one of the donors was kel positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel -negative. the maternal antibody screen revealed anti-kel . doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron , exons , , and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel /kel by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel /kel result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of . g/dl was found to be favourable to one of g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger < . mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < . mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of months (hb transfusion trigger of hb < . mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then months. hrqol is measured after inclusion, after randomisation, weeks, , , , and months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july till june patients were included ( ra, rars, rcmd, raeb, cmmol) in general hospitals and university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and patients for the liberal transfusion policy. the mean follow up period in the liberal group was . months (inclusive run in period) and . months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group patients finished the study, received growth factors and patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about % reduction in amount of transfused red cells was found ( . units per pt per month in the liberal group vs . in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa- table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < . ). t-pa- thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches % at age . this rate is even better in well-treated patients, almost % of whom survive at age . regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the -year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from % to . % per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of adult patients with b-thalassaemia major transfused with a total of . rbc units from january to december . all units were fresh, stored less than days. . units were lr-rbc, . units were ld-rbc and . were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was . % and . %/per unit respectively, in those receiving ld-rbc was . % and . %/per unit respectively, while in those receiving washed lr-rbcs was . % and . %/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients . times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa- viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b ). pasteurisation (liquid heat treatment at °c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than nm pore size was introduced in the early s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately ml of plasma, whereas the improved set accommodates up to ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo ( ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below - °c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from % to %. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts- ), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained % to %. plasminogen and alpha -antiplasmin were retained % and %, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive / /ec require that fresh plasma prior to freezing contains < residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in fresh plasmas divided into groups: group : plasmas from leucoreduced whole blood, group : plasmas from packed cells after removal of buffy coat and specific filtration, group : : apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is . ¥ residual rbcs/l; linearity study with concentrations of . , . , . , . , . , . , and . ¥ rbcs/l showed excellent correlation between observed and expected values (r > . ); reproducibility study showed c.v of respectively . % and %. for values > ¥ rbcs/l, it appeared necessary to introduce a correction factor of . for the anti gpa pharmingen. quantification of rbcs in plasma: group (plasma from leucoreduced whole blood): . ± . rbcs/l; group (plasma from packed cells after removal of buffy coat and filtration): < . °¥ rbcs/l in all the cases; group (apheresis plasma): < . °¥ rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. . improving basic transfusion knowledge amongst health workers; . improving pre-operative preparation for surgery . strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao ) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o (hyperoxic ventilation) can be employed to rapidly raise cao by increasing the amount of physically dissolved o in plasma (hyperoxia). however, molecular o causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received homologous units in addition. the patients undergoing tkr and the patients undergoing removal of implants predeposited and units respectively, but finally and of them have been used. according to our patients data, the . % of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate autologous units. all of them underwent thr except one woman who also had a tkr months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of . homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran % is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f + . in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes / . subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo units three times per week (group b). rhuepo was given for a minimum of weeks or until icu discharge or death. the maximum duration of therapy was weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is > % chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa bb. ten percent of these women make anti-hpa a antibodies in their first hpa a incompatible pregnancy. in - new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of pregnancies show that only babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count < ¥ e /l. in previous studies, it is reported the ich appears in - % of the pregnancies where antibodies are present and that % of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets ( ¥ e mean) from donors with same blood group were pooled and divided equally into two bags, po- and control (pl , baxter), which have and ml/m *day*atm of oxygen permeability, respectively. on days , , , , , and of storage, swirling, mean platelet volume, po , pco , ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to days in po- ( / ) than in control ( / ) bags. dropped ph less than . on day was observed / in po- whereas / in the control. aggressive drop of glucose ( mmol/l) with prominent lactate accumulation ( mg/l) was also observed on day in of control bags. the po level in the control dropped more significantly by % ( . mmhg) on day than in po- ( . mmhg) compared with the initial level ( . mmhg) (p < . ). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po- bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day , and became a detrimental defect in of control bags on day . p-selectin expression was higher in control bags than in po- on days and with no statistical difference. in two control bags p-selectin expression reached > % and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for - days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa- background: maintenance of a neutral ph in the range of . - . is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of . - . cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph . ) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of bcs obtained from standard day -old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from - %. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields ( . ± . vs. . ± . °¥ e ) were similar (pags vs t-sol). statistically significant (p < . with paired ttest) improved platelet quality at days , , and of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at and days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = . for esc, and r = . for hsr at days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from to days provided the quality has been validated and bacterial screening is performed. irradiation up to gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to days is not known. method: two wbc reduced pcs, each made from buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at - °c. swirl, ph and cd p expression were determined on day , and . twelve experiments were performed and compared with a paired t-test, p < . was considered significant. results: see table (day values; mean ± sd; n = ). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day , the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day was < . in / pcs, versus / in group 'b' (not significant). the cd p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to days after blood collection. - b, - a, - b, pra %, donors. in patient -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient and were refractory to two and one hpa compatible platelet units respectively. in patient and compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: . the frequency of occurrence of anti-hpa antibodies in transfused patients was: - b, - b, - b, - a, - a; in three patients they were monospecific, in four polyspecific. . in patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. .the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa- the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b ). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen- or s- . the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about / of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of reactions in years with reactions more common in platelet pools ( : transfusions) than with single donor platelets ( : transfusions). these reactions caused fatalities in of cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the / of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march , the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of : true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: / repeats of x dilution and / repeats of x dilution were hcv rna negative, whereas / repeat of x dilution and / repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype a. the low hcv viremia (below iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a . % take-up rate ( . % where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd -enriched cord blood cells maintained at °c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at °c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd cells were cultured continuously at °c or °c for days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to °c, the cultures maintained at °c produced significantly more total cells ( . fold) and total mks ( fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets ( . fold). accordingly, the cells cultured at °c contained an increased frequency of cfc-mk ( fold) at day . cultures done at °c and °c were also more efficient than at °c but less than at °c. platelets produced in °c cultures could be normally activated by thrombin. as expected, the cells cultured at °c contained an increased amount of the heat shock protein hsp . control experiments showed that the culture of several cell lines was inhibited or unaffected by the °c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of > °c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at °c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than °c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only - % of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a . -year period. requests were received for deliveries from mothers and of these, collections were successful including pairs of twins. failed collection was most often due to a damaged cord at delivery. collections were made for siblings possibly requiring transplant (median age ) with for leukaemia, for erythroid disorders, for immune deficiency, for enzyme deficiency and others. the remaining collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was ml (range - , % exceeded mls) and mean tnc count was . ¥ (range . - . , % exceeded . ¥ ^ ). the mean cd +ve count was . ¥ (range . - . ). all collections were cryopreserved within hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was % (range - %) and the mean cd +ve viability post freezing was % (range - %). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v. . in an abi (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than %. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day . both test and control samples (n = ) were tested on day for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd +, cd +cd +, cd +cd +, cd + and cd +cd + human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd , was observed in . % (sd = . %) of control t cells upon activation with pma, while only a . % (sd = . %) of the test t cells increased cd expression. proliferation assays showed that h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il- , il- , il- b and il- cytokines after -h incubation in culture media increased significantly to pg/ml (sd = ), > pg/ml, pg/ml (sd = ) and > pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of . pg/ml (sd = . ) for il- , . pg/ml (sd = . ) for il- , pg/ml (sd = ) for il- b and pg/ml (sd = ) for il- . addition of lps further stimulated the release of tnf-a, il- , il- , il- b and il- in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k cells (k -rna). methods: dcs were derived from human pbmncs, which were incubated for days in the presence of gm-csf and il- , and then were transfected with k -rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k -rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared hours later, while the cells were expressing p bcr-abl protein and expressing increased cd , cd , cd , hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k cells. conclusion: human dendritic cells transfected with total rna of k cells in vitro could induce effective p bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl - donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in - % of relapsed patients. the success rate is % or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (< %) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately % of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately % to % of patients, is generally mild, and transient, but in < % of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to . j/ml uv light in the presence of um riboflavin, followed by storage under blood bank conditions with various concentrations of -deoxyglucose from to mm for days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without -dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days , , and . results: lactate production and glucose consumption increased from . mmol/ cells/h (sd = . ) and . mmol/ cells/h (sd = . ) for control samples to . (sd = . ) and . (sd = . ) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from . (sd = . ) for controls to . (sd = . ) for treated platelets at day , hsr from % (sd = . ) to % (sd = . ), esc from . % (sd = . ) to . % (sd = . ), swirl from . (sd = . ) to . (sd = . ), and increased p-selectin expression from . % (sd = . ) to . % (sd = . ). addition of -dog up to mm significantly reduced lactate production rate to . mmol/ cells/h (sd = . ) and glucose consumption rate to . mmol/ cells/h (sd = . ), and maintained ph above . (sd = . ) for days of storage. the effect of -dog exhibited a dose-dependent response. however, the addition of -dog had no effects on hsr ( . + . % at day ), esc ( . + . % at day ), swirl ( . + . at day ) and p-selectin expression ( . + . % at day ) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above % of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of -dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv- ( / ), international standards for hcv ( / ) and hbv ( / ) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv- subtypes other than hiv- b, for hiv- and for hepatitis b and c genotypes as well as specificity was evaluated with > specimens. results: results from this study indicate that high analytical sensitivities ( iu/ml hiv- m, cp/ml hiv- o and . cp/ml hiv- , iu/ml hcv and iu/ml hbv) and a specificity of > . % are accomplishable for the mpx test. the % detection rate for hiv- m subtype isolates (a through h) was between to iu/ml, for hcv genotype isolates ( a through ) between to iu/ml and for hbv genotype isolates (a through g and precore mutant) between to iu/ml. investigating seroconversion panels, hiv- rna was detected an average of and days earlier than hiv- antigen with abbott hivag- monoclonal and coulter p antigen tests, respectively, hcv rna an average of or days earlier than hcv antibody with the abbott hcv eia . or ortho eia . tests, hbv dna an average of days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october and september , apheresis ip units have been transfused in sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated ( . %). the population of patients receiving at least one transfusion (n = ) included . % of males, . % of females, the median age was (range - ). the most frequent broad diagnostic categories were hematology-oncology ( . %) and cardiovascular surgery ( . %). the patients received their transfusions either in regular hospital wards ( . %), intensive care units ( . %) or as outpatients ( . %). the number of transfusions by patient ranged from to (mean . ± . , median ). half of the patients ( . %) had previous transfusion experience and . % had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in . % (n = ) of the transfusions ( % ci . - . ), and . % of patients. only ( . %) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, . % ( % ci . - . ) of the transfusions were confirmed as having caused reactions in . % of patients, none of them serious. the most often reported symptoms were chills ( . %) and fever ( . %). itching, skin rash or urticaria was observed in . % of transfusions. of the serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio . , p = . ). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n mÜller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using single donor plasma units. for the evaluation of the plasma factors ml were drawn at different stages (before treatment, after plasma filtration with plas , after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha -antitrypsin complement: ch activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen ( . %), factor viii ( . %), and factor x ( . %). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days , and . the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was . ¥ ( . - . ¥ ; sd- . ). platelet pool volume was . ml (sd- . ). plasma % was within . % and . %. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had . ¥ (± . ). the average platelet loss was . ¥ . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for days. patients were randomized to receive one -day ip transfusion and one -day cp transfusion in random order. after each study transfusion, the hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, -hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included patients, randomized to the ip-cp sequence and to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence ( % vs %; p = . ). mean platelet dose (¥ e ) was . for ip and . for cp (p = . ). there was a significant period by treatment interaction (p = . ) at the . significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) -hour cci (¥ e ) was . ± . for ip vs . ± . for cp. the mean paired difference for both sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). for the first period only, mean -hour cci (¥ e ) was . ± . for ip vs . ± . for cp) the mean paired difference for the first period sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). the non-inferiority margin for the study was . ¥ e for mean treatment difference in cci (cp-ip). median time to next transfusion was h for ip vs h for cp following the first transfusion (p = . log-rank test; data censored at days after transfusion) and h ip vs h cp after the second transfusion (p = . ). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding -hour cci was not met. however, transfusion with -day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower -hour cci compared to -day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood ( ml/kg), across a major antigen (hgd) mismatch. high anti-s- ab titers (≥ : ) were induced after repeated immunization (days , , , , ) with klh-(s- ) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc ( ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken , , , , and days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = , g ), or o-srbc (n = , g ). no ab against o-srbc developed after biweekly transfusions over weeks in g rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = ; g ). transfused rabbits (g & g ) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s- abs were then induced by klh-hapten immunization in rabbits from g (n = ) and g (n = ), and in a group (n = , g ) of naïve rabbits. non-immunized rabbits (g , n = ), and (g , n = ) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g rabbits, but not any of the g rabbits. g rabbits exposed to o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g and g were transfused with m-srbc. viability of m-srbc in all g rabbits (hyper-immune or not) and the hyperimmune g rabbits was equivalent to crbc circulation in g rabbits. in pre-immunized rabbits with high titer anti-s- ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s- ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s- rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed + reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; + reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year are kept in computerized system that has patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr , rzr , r¢r¢, r¢r≤, r≤r≤, ge- , , and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge , anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally . examinees were typified. with o blood group were . ( . %); with blood group a were . ( . %); b blood group . ( . %) and ab blood group were . ( . %) examinees. totally . ( . %) were d positive and . ( . %) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system . ( . %) and d blood group in rh system with . ( . ) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence . % of anti-d in females versus . % in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than . %. however, the rare phenotype jk(a-b-) is more common in polynesians ( . %). individuals with jk(a-b-) phenotypes typically form anti-jk with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was . g/dl. she was given units of crossmatchcompatible rbc. on day her haemoglobin was . g/dl, with a total bilirubin of . mg/dl and lactate dehydrogenase of u/l. on th day an unexpected fall in hb ( . g/dl) occurred with an increase of bilirubin to . mg/dl and of lactate dehydrogenase to u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka: / and jkb: / ). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was g/dl. two months later her haemoglobin was . g/dl and anti-jk was present in the same titer. a year later her blood cell count was normal and the anti-jk was detected in a lessened titer ( / ). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at °c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer ( / ) were revealed. the rare anti-jk alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case . a years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer / ) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated . % red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated . % red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared . % enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc month to months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d( ), c( ), e( ), k( ), cw( )] and were tested with enzyme treated red cells in use and rbc to months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells to months post-expiration, except anti-k antibodies (negative with k positive cells - months post expiration). conclusion: most rbc antigens studied were detectable months after rbcs expiration date. tests with . % cells were valid in gel test (nacl/enzyme) for at least months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated . % red cells. background: wr(a) is a low-incidence blood group antigen ( : ) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: -year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in blood centres using different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard ng/ml anti rh (provided by cnrgs), a positive control anti kel and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions ( tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a + intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving blood donors from the university hospital of coimbra during the year . the incidence of weak d and rh (d) phenotype was determined in individuals who were rh (d) negative. the ab and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented . % of the total sample. we also describe ab , rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in s and s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from to times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about / of tested samples the number of igg molecules per rbc was small (< ) and the laboratory signs of haemolysis were present in . % of them as well as in . % samples with moderately coated red cells ( - igg/rbc). the large number of igg molecules per rbc (> ) was significantly associated with high frequency ( . %) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c d. in % of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in % of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting + with o rbcs at all phases and + with a rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c b,-c d (table ) . prewarming of test system did not change the reactivity ( + at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from + to + when tested with o red cells, as well as a significant reduction in antibody titer from : to : tested at immediate spin (table ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied . women, - years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in cases; moreover a rbc autoimmunization was found in women. anti-d ( cases), e ( ), c ( ), k ( ), c ( ), s ( ), d + jka ( ), d + s + e ( ), d + c + k ( ), m ( ), c + e ( ), d + c + g ( ) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in . % of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department ( . %) and ( %) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types , and were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum- , th- , ms- (bioscot/serologicals) and d e / - (immucor). results: out of samples molecular typing results were as follows: in samples dw were not determined, in samples, weak d type ; weak d type ; weak d type , weak d type ; weak d type and weak d type were determined. by all monoclonal reagents % weak d type , % weak d type , % weak d type and % weak d type negative results were given. by all monoclonal reagents weak d type and weak d type positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type , type and type . such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in > % of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in . according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting + - + s at c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs ( . % ficin) and no reactivity using . m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity > grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment ( . m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in samples from patients; ( . %) patients had > episode. these findings comprised . % of unexpected results found at pt and bg. incidences were . % at routine and . % at urgent bg ( and bgs, respectively), and . % both at routine and urgent pt ( and pts, respectively). . % patients were female, . % over , but . % < years, coming mostly from surgery ( . %), internal medicine ( . %), hematology ( . %), ginecology ( . %), cardiac diseases ( . %) and cardiac surgery ( . % patients). frequent diagnosis were solid tumors ( . %), cardiac diseases ( . %), hematologic malignancies ( . %), uraemia ( . %), orthopedic surgery ( . %) and hepatic diseases ( . % patients). . % patients were previously transfused, with only . % patients proved as not transfused or pregnant. subsequently positive antibody test during the study had . % tested patients. at pt positive crossmatch was found in . %, antibody screening in . % and both tests in . % cases. majority of reactions were ' +' ( % at pt and . % at bg); reactions ' +' or ' +' were found in only . % cases at pt, compared to % at bg. one crossmatch only was positive in . % positive crossmatches, with / patients having > crossmatched unit. ahg identification was positive in . % tested patients; in % of them papainized panel was also positive. lymphocytotoxic antibodies were found in . % tested patients; . % ( %- %) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in . %, hla lymphocytotoxic antibodies in . %, 'igg antibodies of unknown specificity' in . % (hla noncytotoxic antibodies in / elisa tested samples!), contaminated sample in . %, anti-bga in . %, non-specific cold antibodies in . %, subsequently recognized specific antibodies in . % ( lua, m, kpa, yka), non-specific autoantibodies in . %, carry-over of dat-positive cells and antibody to reagent in . % cases each, while in . % cases antibody screening and in . % cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < . ) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt ( . % vs . %), positive lct ( . % vs . %), lymphocytotoxic hla antibodies (lytxab) ( . % vs . %) and reactivity 'positive screening, negative panels' ( . % vs . %); in males non-specific cold antibodies ( . % vs . %) and antibodies to reagents ( . % vs ) were noted. age. in patients > vs < reactivity was often found at pt ( . % vs . %), caused by lytxab ( . % vs . %), anti-bga ( . % vs . %) or 'positive crossmatch, negative panels' ( . % vs ), but rarely by laboratory mistake ( . % vs . %) or later recognized antibody ( of patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt ( . % vs . % at bg), as positive antibody screening ( . % vs . % if positive crossmatch), with positive panels ( . % vs . % if negative). subsequent tests were positive in only . % patients with lytxab, % with anti-bga, of with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in % and . % tested samples found at urgent and routine pt (diamed), vs at bg. all cases due to lytxab, of anti-bga and of 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) . % cold antibodies and . % laboratory mistakes were found. positive antibody test: identification was negative in . % screening-only cases; % of them were caused by laboratory mistake. lytxab were found in . % crossmatch-only cases; . % reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in % cases with lytxab (in with papainized panel) and often due to 'igg antibodies of unknown specificity' ( . %), anti-bga ( . %), contaminated sample ( . %), but also to later recognized specific antibody ( . % cases). strength of reaction: laboratory mistake was noted in . % of 'w', . % of ' +', . % of ' +' and . % of ' + and +' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt ( % and . % of patients, respectively), due to positive crossmatch ( % and %; and . % patients with liver and cardiac diseases) and caused by lytxab ( . % and . %) or 'crossmatch/screening positive, panels negative' ( . % patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg ( . % and . % of patients), due to laboratory mistake ( % and %) or cold antibodies ( . % and %), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of patients ( male and female) were evaluated. the mean age of the patients was years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal ( / ) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in patients with the incidence of . %. antibodies were registered more frequently in females ( / , . %) than in males ( / , . %). patients ( . %) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table ). in patients ( . %) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. patients ( . %) were dat positive. autoantibodies were found in patients ( . %), all of which had specificity to rhesus system. cold agglutinins were positive in patients ( . %). no specificity could be assigned in patients ( . %), while in patients ( . %) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was . %. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about % of women form red cell allo-antibodies in pregnancy and . % of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that % of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and % of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case : a -year-old asian woman had three previous uneventful pregnancies. in her th pregnancy she presented with miscarriage at weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique ( . iu/ml by quantification using r r cells). in a th pregnancy, no antibodies were detected until weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case : a -year-old asian woman had a positive antibody screen post caesarean section in jan . no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at and weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was . iu/ml using rr cells and the anti-d level was < . iu/ml using r r cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of / . in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and ( ) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > % being s+. conclusion: there is a unique distribution of red cell antigen groups in the races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table . conjointly, in order to obtain the frequency of mi.v phenotype, we screened samples among with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype ( . %), there were also mi.i/ii phenotype ( . %), mi.v phenotype ( . %), mi.vi phenotype ( . %), mi.x phenotype ( . %), and most interestingly two miltenberger related not yet classified variants ( %). related variant a ( . %) was phenotypes as mia+, anek+ and hil+. related variant b ( . %) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore . % (table ). the discovery of unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period patient samples were screened, of the positive results were evaluated. blood samples with positive screen tests by the ortho auto vue tm method (av) performed with % cell suspension, and patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into categories according to antibody specificity; . rh system antibodies (n = ), . clinically significant non rh system antibodies (n = ), . clinically non significant antibodies (n = ), . auto antibodies (n = ), . not identified (ni) antibodies (n = ), . negative screening results by the diamed technique (n = ). the categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of samples were compared, . % of them gave stronger reactions by av, . % gave equal reaction strength and . % of them gave weaker reactions by av. positive screen tests by av only were detected in samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in samples, were rh system related, kell system related and not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to . % suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh and rh rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh or -rh antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague , † blood bank, faculty hospital m, prague , czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab grouping was performed with registered immucor reagents (immuclone: anti-a birma , anti-b lb ) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre . titration of reagents /+ aliquot of serum added/: anti-a: titre (both patient's serum and control); anti-b + patient's serum: titre , anti-b + control: titre . conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed % agreement between all three methods. samples were tested per min with the autovue innova, compared to min required for the same number of tests performed with manual testing and min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcÍa borrÁs, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples ( - years old, n = and > years old, n = ) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for h at °c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . , nrbc: . + . ; srbc: . + . . the values of am obtained with young individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . . nrbc: . + . ; srbc: . + . . conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < . ) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < . ) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < . ) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < . ). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification ¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by d-fluorescence microscopy after lipid extraction with triton x- in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa- tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at nm the flim-fret from alexa tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from to %, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in transfused sickle cell patients. methods: all the patients included in the study had received at least unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was - units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c c, c d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the patients. the dat was positive in ( %) of the patients. in cases ( %), the dat was igg type, in case ( %) igg + c d and in case ( %) igg + c c + c d. the acid elution was performed in cases. the eluate was positive in cases ( %). in ( %), of the cases, autoantibodies were pointed out whose specificity were specificity public, anti-e (rh ), anti-c (rh ), anti-ce (rh ). in cases ( %) we found alloantibodies whose specificity were anti-e(rh ), anti-k(k ), anti-c (rh ) and anti-jka (jk ). in the cases ( %) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in ( . %) of the patients. of these, ( %) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of patients was %. seventeen patients ( %) of the patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang , (suppl. ), ). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having distinct incubation wells, application zones and detection areas. microliters of % suspensions of reagent red cells for reverse grouping (reverse-cyte a , a , b, , medion diagnostics, switzerland) are mixed in each incubation well with microliters of plasma. the resulting suspensions are incubated for min, followed by the transfer of microliters each to the application zones, where migration starts immediately. results can be read after min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho screening rbc since the year . aim: (i) to summarize the performance of the autovuetm system for rbc abs, during years ( ) ( ) ( ) ( ) ( ) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in . % of , blood donations that were screened, during - , using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in . % ( . % of the donations). an increased percentage of confirmed tests is noted, since , probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, samples with low positive results (agglutination degree of . ) were retested twice on the autovuetm. / ( %) gave negative results in both repeats. only / was confirmed positive and anti-m was identified by diamed gel test. the remaining samples had at least one positive repeat. / ( . %) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and blood donors of azeri nationality were examined. patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee ( . %) •• ccdee ( . %) phenotypes were twice as much higher than the occurrence of those in the donors ( % and . % respectively). phenotype ccdee occurs more frequently in the donors ( %), whereas it is significantly rare in the patients ( . %). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors ( . % and . %) than in thalassemic patients ( . % and . %). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c c and c d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c c or anti-c d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of heterozygous sickle patients and homozygous patients were analysed. of the homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. ( ) and are present in more than % of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a years old swedish woman, a rh negative, at th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre ( : ) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from . /ul to . /ul) was observed. this pattern showed no variation until the end of the pregnancy. at th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to . /ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p- rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of alloantibodies on blood samples over . these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia ( . %), e ( %), le a ( . %), le b ( . %), p ( . %), m ( . %), d ( . %), c ( . %), jka ( . %) and c ( . %). within the chinese community, the most frequent alloantibodies were similar: mia ( . %), e ( . %), le a ( %), le b ( . %) and p ( . %). in the malays, the most frequent alloantibodies were le a ( %), le b ( . %), mia ( . %), e( . %) and p ( . %); while in the indians, these were mia ( . %), le b ( . %), le a ( . %), d ( . %), and e ( . %), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians ( %) compared to chinese ( . %). anti jka tended to occur mainly in the malay race and anti-c was rare in all (< %) reflecting the high prevalence of c in the singapore population (r r phenotype). the ratio of alloimmunised male to female (m : f) is : . most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at : . . alloimmunisation increased with age for mia, e, k, p , jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies ( or more) within the alloimmunised subjects is . %. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a years period, from / / - / / , we studied newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in ( %) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in ( . %) (newborns of group a with mothers of group o), anti-b antibody in ( %) (newborns of group b with mothers of group o), anti-d antibody in ( . %) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in newborns and in the rest , no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d ( ), anti-e ( ), anti-k ( ), anti-fya ( ), anti-s ( ), anti-jkb ( ), anti-n ( ), anti-kpb ( ). in two patients antibodies were identified, while in / ( . %) no antibody was identified (unspecific). it is remarkable that only in out of patients with both dat and iat positive, an irregular antibody was identified, while the rest patients had unspecific antibodies. in patients with only iat positive, had an irregular antibody and had unspecific antibodies. in out of patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is . % and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. . % of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than % in china) were reported from % to %. moreover all del individuals in this population were found mainly carrying a same allele, rhd a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in rh-positive donors with cde/cde and cde/cde genotypes, respectively, and del phenotype individuals carrying rhd a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and isoforms were detected in rh-positive and del, respectively. among them, isoforms have identical sequences, which are transcripts with exon , exons and , exons and , and exons to spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon or exons - were spliced out, but both possess a bp segment of sequence from intron of rhd. through additional reversedtrancriptase pcrs, which amplified exon to ¢-region and exon to ¢-region, the results showed that exon did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd a allele since the exon was always spliced out in all isoforms. all transcripts in del maintain a normal open reading frame and encode proteins with different numbers of amino acid residues and different c-terminals (genbank ay , ay , ay , ay , ay , ay ). among them, the sequence of del (isoform with exon spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in . it encodes amino acid residues and has amino acids more than normal rhd. it is different from rhd after codon . in normal rhd protein, the amino acids after ( residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del that was found in del. (iii) there is only one polymorphism in the region of bp segment between rhd intron and of the del transcripts, which indicated that other polymorphisms may exist in intron of rhd a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh ( pooled we reported earlier that metabolic arrest followed by incubation at °c reduces the platelet storage lesion (badlou et al. transfusion ) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium ( min, °c) followed by storage ( h, °c) and recovery with glucose ( h, °c). controls were (i) platelets in glucose-rich medium stored for h at °c and recovery with glucose (c ) and (ii) platelets stored for h at °c (c ) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. binding of msp, c , c was ± , ± , ± % of total platelets. phagocytosis of msp, c and c was ± , ± , ± % of total macrophages (means ± sem, n = ). before recovery of msp, binding/phagocytosis was % higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in patients with leukemia and lymphoma, males and females with mean age ± years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of compatibility tests were performed, of which were compatible. twenty five compatible platelet concentrates out of were clinically evaluated. twenty from compatible crossmatches ( %) were resulted in successful transfusion while only from ( %) in unsuccessful. the incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~ . ). additionally patients out of ( . %) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june and is still in course: in january , blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the blood samples analysed, were reactive and the specificity of the antibodies were: anti hpa a: , anti hpa b: , anti hpa a + hla: , anti hpa b. , anti hla: , auto hpa- b: . specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa- , , , , , ) using pcr-ssp or pcr-rflp. the infants with hpa immunization suffered from severe (plt count - /ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa- and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa- a is the most frequently implicated antigen. other antigens such as hpa- a, or hpa- a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa- bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa- bw (maxa+) mismatch as the sole antigenic incompatibility in out of families. in the last one, incompatibility was found for hpa- without anti hpa- b maternal alloantibody. as the father was found to be hpa- bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa- bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa- bw (maxa+) fmait is not uncommon and was found to be around % of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa- a antibodies after non-myeloablative allogeneic hsct. a year-old male patient was diagnosed with multiple myeloma in / . treatment consisted of cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received cycles of bortezomib, and after complete remission a stem cell allograft ( . ¥ /kgbw cd + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine ( ¥ mg/m ) and alkeran ( mg/m ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d but was accompanied with a continuous decrease of plt counts. between d and d the patient received seven plt transfusions, containing a median of . ¥ plt/unit (range , - , ¥ plt/unit) from random donors. the corrected plt count increments at to h after these transfusions were < /ml. therefore, and because of even a further decline of platelet counts to /ml on d we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak ®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa- a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa- , - , - and - of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d after hsct reacted strongly with the donor's plt due to anti-hpa- a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa- bb, - aa, - ab, and - aa; the donor was hpa- ab, - aa, - aa, and - aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d , d and d after hsct contained antibodies against hla class i antigens but hpa- a antibodies were not anymore detectable. no hla antibodies were detectable on d after hsct. the severe thrombocytopenia was caused by hostderived hpa- a antibodies. fortunately, plt counts started to increase on d spontaneously and the patient could be discharged at d (plt . /ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa- b antibodies: case report p moncharmont, m vignal, y mÉrieux and d rigal efs rhone alpes site de lyon, lyon cedex , france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa- b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a -year old mother, gestation /partum , gave birth to a male neonate by caesarean section at weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted . g, was . cm tall and had a head circumference of . cm. the apgar score was , , , at , , and min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during h. the plt count which was . , . and . giga/l at day (d) , d and d respectively dropped dramatically at . giga/l at d . simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa- a/a and anti-hpa- b ab were detected in her serum. the baby was heterozygous, hpa- a/b. plt were transfused to the baby and the plt count rose to . , . and . giga/l at d , and respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa- b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor antibodies. hit type ii occurs in up to % of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf antibody test (diamed id-card). results: from the studied patients, ( . %) developed thrombocytopenia (determined by a decrease in the platelet count below %, after the introduction of heparin therapy); ( . %) did not show decrease in the platelet count. six ( . %) out of thrombocytopenic patients were positive for anti-heparin/pf antibody. three ( . %) out of non thrombocytopenic patients were positive for anti-heparin/pf antibody. the results demonstrate that ( . %) patients were positive for anti-heparin/pf antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects . - in live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to % of reported cases). the commonest antibodies are anti-hpa- a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week of gestation to a healthy mother after first uneventful pregnancy; birth weight g, apgar score . immediately after birth, severe thrombocytopenia ( ¥ /l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa- a/a) were given. twenty-four hours later platelet count rose to ¥ /l and no new petechiae were observed. on third day of life the blood platelet count was ¥ /l and the newborn received ivig g/kg and corticosteroides. twenty-four hours later the platelet count rose to ¥ /l and further clinical course was uneventful. natp due to hpa- a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa- a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of . % to % in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: samples were referred after being characterized as weak d. in cases we obtained a positive result, with a preponderance of weak d type ( . %) over type ( . %), ( . %) and ( . %). two samples were not categorized. the high incidence of weak d type in our population is in marked contrast to studies performed in other european populations where weak d type was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a -bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to ¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the ¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a -bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = ). results: ( . %) rhd-positive and ( . %) rhd-negative samples were phenotyped. of the rhd-positive donors, ( . %) were rhd homozygous and ( . %) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee ( . % vs . %) and dccee ( . % vs . %) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in . % of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v. . in an abi (applied biosystems). results: see table . the substitutions of rhce-specific nucleotides in exons , and with their rhd-specific counterparts lead to different new rhc-antigens with weakened expression. since one amino acid change in allele lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a -year old mother (w.o.g. ) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron , exons and were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron , exon and exon while sample of fetus ii lacked rhd-specific intron . therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons - , & ; polymorphisms for dweak type - , d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi . rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons , , , and were possible and failed only in exon - . determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron pcr. results: weak d type was not identified in our research population. weak d type was identified in cn, weak d type was identified in cn and weak d type was found in cn, bsa, be, bc and saa. conclusions: although weak d types to represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type in all populations. however, regarding rhd phylogeny, the weak d type mutations ( c > g and t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from a, b, ab subgroups, and provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon and , and allele separation were performed on these samples. results: abo*a allele was observed in an aint subgroup. two new a alleles that showed g > a base change and c > t of intron and a polymorphism of c > t in a(pro) intron were discovered. o and o alleles were also observed. in b subgroups, a silent substitution c > t (leu leu) was observed as a new b allele. another new b allele which showed g > a was also found in b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > . ) verifying that the monoclonal anti-ab associa- study aim: to prevent the sensitization to rh (d), to a d-patient who was transfused with d+ blood. material and method: on september , ( / / ), we admitted to our hospital through air-carriage, a female of years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: %) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally units ( ml) of d+ condensed red cells, out of that were initially asked. before the h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh (d). the indicted dose was - iu/ ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = ) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table ) . for the long-term follow-up samples, additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with copies of negative dna in a single amplification tube for all primer pairs. we were also able to calculated the clinical sensitivity that using donor-recipient pairs. . % of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with . % of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to -chloro- - dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag- /common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with million viral particles of murine cmv (mcmv). at different post-transfusion time-points ( h, weeks, weeks, weeks, weeks), different female recipients plus non-transfused female knock-out controls were challenged with million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp ( mg sc once or q weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day and day after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ % during blood donations and blood donation was not performed if ht < %. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was . € in the aranesp group and . € in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from operations in patients with anti-erythroid antibodies. materials: three patients ( male and females), aged between - years old, had to undergo selective operations, total hip replacement surgery and aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of consecutive patients (group ) who donated two red cell units (rbc) of autologous blood and weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ ) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ . ; postop-hb = hb ( - h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as g/l) < (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next patients who were also assigned for pabd (group ). pabd was refused if the probability of rbc requirement (prr) was < %. between - % one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded % two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group ( vs and vs g/l). % of autologous blood of group were discarded, / patients needed additional allogeneic rbc. hb-loss caused by rp was ± g. mean prr in group was . %. / patients donated one rbc, which was later discarded, and no patients donated two rbc. / of group needed allogeneic rbc. mean prr of these patients was % (range . - . ). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other ( . %) patients who required autologous and homologous blood, had average predonation hb level of (sd ± . ) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = . ), predonation htc values (p = . ), weight (p = . ) and gender (p = . ): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = . ). ( . %) patients with ptka were transfused with autologous blood only, and had average predonation hb level of (sd ± . ) g/l. other ( . %) patients transfused with autologous and homologous blood had average predonation hb level of (sd ± . ) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = . ): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = . ) predonation htc values (p = . ), gender (p = . ) and age (p = . ) of patients and the need for postoperative transfusion. conclusions: our results show that over % of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight - g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, cm proximal and cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, patients, including males and females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about - percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at - day intervals. the blood volume taken from patients in every collection differed from - ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, cc to cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately % of the total blood volume withdrawn] had platelet counts of - /mm . the procedures of agp labelling were performed in laminar flow chamber. to seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed h after application of labelled agp (early scan) and at , , , h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at , h and at lapse time = h for days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every - weeks. by the th week of gestation the patient had donated four units of blood, her hematocrit was %, anti-ku titre was / and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were and at and min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day . the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above °c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+ °c, - °c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature + °c, + °c and + °c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+ °c) or freezer (- °c). the temperature was observed in min intervals for three hours, first measurement was min after putting into freezer or termostat. results: (see table ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since , we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between - %, the mean volume about ml and the mean platelet-content is . ¥ per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from to days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: twins made out of pools from buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at °c, and sampled at day , day and day . ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day with cb was . , with nfb . ; glucose with cb . mmol/l, with nfb . mmol/l; lactate with cb . mmol/l, and with nfb . mmol/l, hsr with cb %, with nfb %; p-selectin with cb % and with nfb %. the new platelet storage bag showed better results of in vitro quality markers, especially after day of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at °c or rt simplifies the preparation process. filtration at + °c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, % of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by . mm. a decrease of the process time is expected. methods: units of whole blood were collected with the new t 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within min in all cases. after donation whole blood units were stored at room temperature for h. subsequently, whole blood filtration was performed by gravity at a standard height of cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at g ¥ min by an heraeus cryofuge i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx -abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table . results are given as mean and standard deviation. whole blood filtration was completed within min in all cases. red cell units were transfused after a mean of days to patients affected from transfusion dependent ( %), post-surgery ( %), and post chemotherapy anemias ( %). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day , , and , and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day , and , and cd expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years ( , .) aberrations from these principles have taken place (self-sufficiency). nbti collected x = ( ) blood units into blood bags. in serbia x = ( ) blood units. retrospective analysis: ldpc-bc was administered x = % with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted ( %). increase of the use of leukocyte poor red blood cells was also registered ( % introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at °c with platelets stored at °c, and to determine the in vitro-effects of pre-incubation at °c for h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at - degree on a flat bed agitator; (ii) at - degree on a flat bed agitator and pre-incubated for h prior to analysis; (iii) at °c; and (iv) at °c and pre-incubated for h prior to analysis. this paired in vitro study (n = ) over days include volume, platelet counts, mpv, volume, ph, po , pco , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf ). results: platelet count (day and ; p < . ), mpv (day ; p < . ), ph (day and ; p < . ), pco (day and ; p < . ), bicarbonate (day ; p < . ), glucose (day , , , and ; p < . ), atp (day and ; p < . ) was significantly higher in platelets stored at °c and platelets stored at °c with preincubation. ldh (day ; p < . ), bicarbonate (day and ; p < . ), lactate (day , , , and ; p < . ), ph (day and ; p < . ), esc (day , , , and ; p < . ), hsr (day , and ; p < . ) was significantly lower in platelets stored at °c and platelets stored at °c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf was significantly higher in platelets stored at °c than in platelets stored at °c (day , , , and ; p < . ). hsr (day and ; p < . ) and esc (day , , and ; p < . ) was significantly higher in preincubated platelets stored at °c compared with platelets stored at °c. conclusion: platelets stored at °c maintain metabolic and cellular characteristics to a great extent during days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at °c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: whole blood donations were collected in a whole blood filtration set with cpda- and stored at room temperature for h before filtration at room temperature. the leucodepleted whole blood was stored for days. following parameters were analysed on day , , : free haemoglobin in%, k. in addition leucocyte count was performed on day and a blood culture on day (see table) . blood cultures on day remained negative and all counts of residual leucocytes were below ¥ (exponential) /unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day . however the results were within the required specifications from the european and swiss guidelines up to day . we conclude that autologous leucodepleted whole blood can be stored in cpda- -for days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a -month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: ( ) blood of female donors was not used for ffp production, and plasma was only used for fractionation ( ) plasma of female donors was not used for kt-bc pools ( ) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: %*; %** ffp produced by plasmapheresis: %*; . %** female donor units on cell separator: . %*; . %** ffp from total plasma units: %*; %** plasma units used for bc-pc pools: %*; %** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~ %), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of days. extended storage to days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd p and cd b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = ) using apheresis technique. the pc from each donor was divided in units, one served as control and the other was gamma irradiated with gy. the pcs were stored on a flatbed agitator at °c for days. samples were taken on day (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day , and were also analysed for hrs, cd p (p-selectin) and cd b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day , and . the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd b expressing cells were well maintained for days of storage. glucose decreased and lactate increased significantly during the storage period, from . mmol/l to . mmol/l for lactate and from . mmol/l to . mmol/l for glucose. the percent cd p expressing cells increased significantly during storage from % on day to % on day . po was well maintained but ph increased and pco decreased significantly between day and whereafter ph decreased and pco continued to decrease. the for parameters g'max and time to g'max increased significantly between day and and the time to g'max continued to increase significantly between day and . the results indicate a well preserved platelet quality after storage for days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b (transforming growth factor b ) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from buffy coats. following overnight storage at - °c, buffy coats were pooled with ml t-sol (baxter). forty-two pcs were produced either manually (n = ) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = ) using the orbisac validation bc set (gambro) equipped with the lrp leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl series analyser (radiometer). samples for testing of cytokines were centrifuged for min at g, °c; supernatants were harvested and frozen at - °c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days , and . results: platelet concentrate volume (mean): m-pcs: ml, a-pcs: ml. platelet yield was found to be . ¥ for m-pcs and . ¥ for a-pcs (p < . ). in all pcs ph levels were between . - . . glucose consumption and lactate production from days - and days - did not differ significantly. rantes levels (pg pr plts) were significantly higher in a-pcs than in m-pcs (p = . , repeated measures analysis of variance), but no significant difference was found in tgf-b levels (pg pr plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/ - . pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd + cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in °c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd + cell count were evaluated by flow cytometry with a no-wash, single-platform technique and aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h , stemcell technologies) and colonies were counted after days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables , and . post-thawing tnc and mnc, as well as cd + cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd + cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd + cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: cord blood units ( from males and from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb . blood units were also randomly typed for hla-dqa and dqb at high resolution. results: mean birth weight was g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was . g. babies were < th centile ( g) and were > th centile ( g). comparing the hla allele distribution in these extreme bands we found that hla drb * was significantly associated with high relative birth weight: . % in th centile vs . % in th centile, p = . . on the contrary, hla-drb * and dqb * were associated with low relative birth weight: . % and . % respectively in th centile vs . % and . % in th centile p = . and p = . . all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb * and higher relative birth weight (mean . vs . ; p = . ) as well as the association of hla-drb * with lower relative birth weight (mean . vs . , p = . ). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb * and hla-drb * genes encode for different amino acid sequences in the pocket of the molecule (aa , , ). this implies distinct functional restriction patterns. the sequence motif of hla-dr is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retarda- which provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a %- % dextran solution in . % saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation °c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was % to % when stained with gimsa or wright staining method. the viability of isolated pmnls was also % too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during to . at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged g to g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: exchange transfusions were carried out. four out of et due to abo incompatibility mother-newborn. five out of due to rhesus incompatibility mother-newborn and eight out of due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at °c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: samples were taken from newborns at first h of life for abo blood group typing during - . simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in out of newborns were found detected antibodies anti-a, anti-b. in out of newborns no antibodies were found. in out of were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from + . to + . conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab incompatibilities, we have discovered alloimmunized women with the following specificities: anti-d, anti-c, anti-c, anti-e, anti-jka, anti-d + anti-jka, anti-d + anti-s, anti-d + anti-c, anti-d + anti-c + anti-k, anti-s, anti-k, anti-m, anti-c + anti-e and anti-d + anti-c + anti-g. the most severe hdn were the d + c + g, the c + e and out of c newborns, with mean hemoglobin between and g/l, bilirubin = . g/l, reticulocyte count = %. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, years of age, in th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the th gestation week covered with mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < % or hemoglobin (hb) < g/dl and reticulocytes < %; b) hct < % or hb < g/dl in these conditions: o required < %, recurrent apnoea and bradicardia, cardiac rate > bpm and respiratory rate > bpm for more h; c) hct < % or hb < g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of % after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume ( ml per kg if premature or ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of - weeks) show a range of weight from to . g, while at term newborns from . to . g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always - ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of neonates admitted during one year, ( male, female) received blood components. fifty four percent received one, % two and % received three types of blood components. the frequency of transfusions were times. the most common used blood products were fresh frozen plasma ( %), red blood cell ( %), whole blood ( %) and platelets ( %). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were %, %, % and % respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january and december we studied d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of d-negative studied samples, ( . %) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that patients ( . %) had in their serum other anti-d associated alloantibodies: anti-k ( . %), anti-c ( . %), anti-e ( . %) and anti-c + e ( . %). during the immunohematologic follow up, it was determined that in / patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (- °c and - °c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at ¥ g for min within h of collection. plasma was removed. four units of plasma were pooled into a ml bag, mixed, divided into parts (aprox. . ml) and immediately frozen. two of these units were stored at - °c and units of ffp at - °c. after one month the plasma was thawed at °c during - h. the fibrinogen concentrates ( - ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at - °c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored - °c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center ( donations per year and utilization of approximately of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in the national center for the production of blood products was established, under the direction of elias politis and years later in begun the production of dried plasma from greek donors. by the year the center started the production of fibrinogen and by the year the production of antihaemophilc factor. in all the activities of the center settled down due to administrative aspects. at the beginning of s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early s, when techniques for the isolation and concentration of clotting factors were improved. in , matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl , is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation ( ¥ g for min) of whole blood. four units of plasma were pooled into a ml bag, mixed, divided into parts and immediately frozen. one of them was stored at - °c and after one month the plasma was thawed at °c during - h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well- (light cycler, roche diagnostic systems, nj) was used for the identification of the c y, h d and the s c point mutations of the hemochromatosis gene, and were based on protocols developed, for c y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c y allele was obtained ( . %) in individuals with genotype , however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than . patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c y, h d and s c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from % to - %, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of samples ( mothers and newborns), from / / and / / , were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the cases studied it was found cases without incompatibility ( %). from the incompatibilities, . % were abo, . % were rhd, . % were other rh incompatibilities, and . % were due to auto-antibodies. % of the mothers were rhd+ and % rhd-. conclusion: of the pregnant women studied, only were rhd-. from this group ( %) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the pregnant women rhd-, % had incompatibility abo, which decreases to near % the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a haplotype expression has been found about - % in caucasian and in greek population particularly, . %. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a expression. in of these donors the expression of cd , cd , cd , cd , cd , cd , hla dr, cd +cd +, cd +cd +, cd -cd +, cd +cd -cd +, cd -cd -cd +, cd -cd -cd + was examined. meanwhile, patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a expression, while in of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a expression in healthy donors was . % and in cancer patients % (p > . ). in table the mean, standard error, t-test and p of the two groups are included. (see table ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd expression, which was higher in cancer patients. summary and conclusion: the expression of hla a in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in ), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in , dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march . after keeping it for days at storage, blood was forwarded on a two-day journey to a small town, kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are - nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deter- objective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd ) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = ) and umbilical cordon (n = ) were used. several dilutions oh hailuronic acid sodium salt solution % (sigma l- h ) were confronted with % erythrocyte suspension in phosphate saline buffer (pbs) ph . . the rbcs were previously treated with an enzymatic solution of % bromeline in pbs ph . (sigma l h ). agglutination readings' were been by slow sharking after of h incubation at °c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à si. di- . - (i = , , . . .) where si represent the score and di- is dilution inverse. the adult' rbcs showed a = ± , while en the newborn the parameter was a = ± . our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd ) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during min at °c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p -dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig , is induced by p through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele ( tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (< %) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele ( repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a +/ + reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a + reaction in a test with monoclonal anti-d and a +/ + reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against anti-d reagents in the id-partial d typing system. one of these (number ) gave negative reactions and the remaining five gave positive reactions (ranging from +/ + to +), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a +/ + reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk applied conventional and no conventional (hemolytic medium time) techniques in sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in ae from b patients and in ae from ab patients; a epithopes in ae from ab patient and in ae from a patients; p and p epithopes in ae and only p epithopes in ae. these patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in sera and anti a immune antibodies in sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c a and c a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during - period all anemic patients on hemodialysis were divided in groups. the first group pts with febrile non-hemolitic transfusion reaction. the second group- pts immunized toward leukocyte and hla antigen. the third group young candidates for kidney transplantation for prevention of hla immunization. the fourth group pts with sle (for immune-complexes and autoantibodies). total patients ( males and females) received units of rbc with residual number of leukocytes. commercial filters of baxterÔ (lekostop lds) and terumoÔ (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with - microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until days of collection. result: aabb permits maximum < ¥ wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of ¥ the le reduction of - . %. the number of rbc after filtration is minimum % - g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g a, mthfr c t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients ( . % heterozygous, . % homozygous) compared to controls ( . % heterozygous), p < . . the or for heterozygous carriers was . ( % ci . - . ), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients ( . %) and controls ( . %), or . ( % ci . - . ), p = . . although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c t mthfr than the control group ( . % vs . %), or was not significant ( . , % ci . - . ), p = . . analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c t mthfr gene (tt and ct) (or . , % ci . - . ), p < . . conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to ¥ /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease ( % atipical plasma cells). to treat heavy anaemia (hgb g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol mg per day). coagulation parameters were tested: pt . inr, aptt s, fibrinogen . g/l, trb ¥ /l, d-dimer mg/l, atiii %. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with dose of cryoprecipitate, dose of fresh frozen plasma, iu atiii, doses of red blood cells and vitamin k mg. two days after the substitution therapy we saw pt . inr, aptt s, fibrinogen < . g/l, trb ¥ /l, atiii %. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till th day of therapy, fibrinogen was below . g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with mg/kg/dose of rfviia (*) for days, after which there was clinical and endoscopic recovery, and an inh decrease to . ub/ml (fviii dosage %) . he began to take meprednisone ( mg/kg/day) for days, after which the inh titre was . ub/ml. (table a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of . ub/ml and was treated with mg/kg/dose of rfviia (*) for days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to . ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was . ub/ml. (table a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a -year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio ( : ). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, to november, , we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than °b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo < ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of patients (ra, rars and raeb < % blasts) receiving epo ± gcsf between and and having serum epo < ui/l, the response rate at week (iwg criteria) was %. six variables were associated with response to epo ± g-csf for mds: age > years (p = . ), number of prior red blood cell transfusions < packs/months (p = . ), serum epo level < ui/l (p = . ), percentage of blasts < % (p = . ), percentage of erythroblasts > % (p = . ) and low ipss score (p = . ). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p- pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on february , during the course of the first general meeting of the pbsa, which comprised of founding patient organizations. the objectives of the pbsa are: . to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. . to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl - gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl - gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to - u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than patients with haemophilia have now been treated in phase gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, < u/dl rise, in / patients enrolled were observed, although less factor ix concentrate was needed in / subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (> % of normal) for > year in mice following a single treatment. in / patients treated repeated factor viii rises ( . - . u/dl) were seen, but no improvements lasted beyond months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. / patients enrolled sustained levels of factor viii > u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~ u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. con- reference: petz ld, garratty g. immune hemolytic anemias. nd ed. philadelphia: churchill livingstone, , pp - . w-pa- autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [ ] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [ ] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [ ]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (< h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [ ] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about % of cases. gift detects igg antibodies and is positive in about % of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa- practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab ( + = adsorption; + = adsorptions; + = adsorptions; + = or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time ( - h) does not allow for adsorptions, one can dilute the patient's serum (e.g. in , or in ) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that - % of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [ ] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [ ] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [ ] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [ ] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb (cd ) and gp - . the respective antigens, frequencies and alleles are illustrated in table . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [ ] . references are available from the author upon request. w-pa- a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a -yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c -independent binding of c b complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia ( and neutrophils/ml). the diagnosis of annanti hna- a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day and , respectively. omphalitis was successfully treated with -day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for days without success. this was followed by an attempt at neutrophil count increase with -week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at -week intervals. in spite of prolonged neutropenia ( and months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: ( ) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; ( ) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna- a immunization, and ( ) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors ( . %), aged between and years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during , % of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the % of them were successfully processed to ffp. during , % of the donations were fulfilling the specifications and . % of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc / ). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about % during - , suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc / hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa- transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september , , with almost deaths and more than injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the -year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than units of blood used in the first to h. in in new york, the five hospitals closest to the disaster site admitted only disaster victims. the new york blood center, which supplies percent of blood for the city's hospitals, added units to routine inventory at hospitals. the center received telephone calls and collected more than units of blood in the first h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september , america's blood centers collected more units of blood, and the american national red cross collected more units than in the same period the previous year. more than units were collected for the disaster victims, but only units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach %s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june , , a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august , , as shown in the following sections: . the development of a voluntary blood donor system. . removal of the upper age limit for blood donors. . funding for blood donations. from january , , each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa- can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from to %. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around . % of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv , hhv ), parvovirus b , hepatitis g ( . %) and tt (transfusion transmitted) virus ( . %). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around : to : depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values < . g/dl in the first and third trimesters, and < . g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa- impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris , bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach / above years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than % bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts > %) evolving in a short timespan with severe cytopenias, and to aml in approximately % of cases. at diagnosis, % of mds patients are anemic, with an hemoglobin level less than g/l, and % of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of - units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) ( - u/kg tiw, or a - u as single weekly dose) are typically used in low-risk mds. the response rate to epo is - %, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than g/l). absent or low rbc transfusion needs and a serum epo level less than u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable ( - months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to - %, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in , compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin or ug/week) with response rates varying from % to % in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over years and backed by postmarketing experience in countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa- iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss > l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (< weeks) of the anemia is desirable in cases with transferrin saturation < % and ferritin levels < mg/l. with this regime it is possible to collect up to autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed red cell apheresis in patients with diagnosis of hereditary hemochromatosis ( ¥ c y homozygotes, ¥ c y + h d heterozygote) using haemonetics mcs p cell separator (protocol tae) in which red cells are removed from patients in - cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december , and february , have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter and days after drbc procedure. donors suitable to drbc apheresis had to have: age between and years, weight > kg, hb > . g/dl and serum ferritin between and ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of donors who donated drbc during the study period, only males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb . ± . g/dl, ferritin ± ng/ml, serum iron ± microg/dl. on day mean hb was . ± . g/dl (p < . ). on day mean hb was . ± . g/dl (p < . ), ferritin ± ng/ml (p < . ), serum iron ± (p ns). only out of donors ( %) had a ferritin value > ng/ml. in the studied donors the collection of units of rbcs induced an expected reduction of about grams of hb, however only % of this reduction was recovered after days (p < . ). similarly, also iron stores have not been restored after months from donation, as shown by a % reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of units of rbcs (approximately - mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively % of subjects at first blood donation and, in these subjects, hfe mutations prevalence was . for c y and . for h d (velati et al., ) . aims: the role of the c y mutation is well known in the patho-genesis of iron overload, whereas the role of the h d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h d mutation in comparison to a population of blood donors wt/wt for the h d mutation. methods: a total of repeat blood donors were examined in italian transfusion centers ( in northern italy and in southern) for c y and h d mutations. out of those, blood donors heterozygous for the h d mutation and wt/wt for the same hfe mutation, both groups wt/wt for the c y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table summarizes the allelic frequencies in the blood donors. table reports the haematological evaluation in the subjects heterozigous for h d mutation and the wt/wt for the same mutation. conclusions: these data suggest that subjects with h d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h d heteroxygotes or even homozygotes when available, in order to get further insights on the h d role in iron metabolism. background: cd is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and females aged to (median ). twenty of them had never been transfused (group a) and were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd b, anti-cd , anti-cd , and anti-cd . leukocytes were gated according to cd . we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < . ) and controls (p = . ). this downregulation concerned the proportion of cd + cells, that was lower in the transfused patients than the non-transfused (p < . ) and controls (p = . ), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < . ) and group c (p = . ). negative correlation was observed between the cd expression and cd b (p = . ) and cd (p = . ). cd b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = . and . respectively) while the percentage of cd b cells did not differ significantly between the various groups. increased expression of cd was also found in the group a compared to group b (p < . ) and c (p = . ). the proportion of cd + cells did not differ between the various groups. the levels of immuno-reactive svcam- as determined by elisa were found . + . in group a, . + in group b and . + . in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in centers and the gfm has the intention to extend the study to a larger population in at least centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after blood donations or plasma donations. before , each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g % for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g % for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g % for intravenous use (ivig) has been used in the study. the preparation is liquid, % stabilised with glucose of a ph value of . ± . . it is used in those cases where sepsis developed after surgery. both an ivig group (n = ) and a control group (n = ) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = ), and the control group (n = ); pancreatitis (n = ) control group (n = ); intestinal obstruction (n = ) control group (n = ); abdominal organ perforation (n = ) control group (n = ); abdominal perforate injuries (n = ) control group (n = ); serious abdominal interventions (n = ) control group (n = ). the period of hospitalisation of the patients in the ivig group was ± days while the period of hospitalisation in the control group was ± days. the mortality rate in the ivig group was % counter . % in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g % for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: pc were produced by this device and some parameters were studied. for the preparation of pc, bc were pooled using the orbisac set, with an integrated filter (pall lrp ). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol / . the pc was stored in a gambro elp bag. results: the average platelet count per unit was . ¥ e . the platelet recovery from pooled bc was . % (range . %- . %). all products of the tested pc containing < ¥ e wbc (by flow cytometry). the values of ph on day and of storage were . and . . the swirling phenomenon was good until day °. the average loss of haemoglobin per bc was . g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood ( ml) was collected in top-and-bottom bags, and randomly selected to be processed by either ( ) current routine or ( ) atreus c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc d (atreus). lr-rccs were stored at °c and sampled until day . various in vitro measurements were performed (n = per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was % lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained ± ¥ platelets in ± ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. . ± . . ± . < < . aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day . on day , either , , , or pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days , and for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein b (gp b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of pools were studied, each of , , and pc's. the mean platelet yield was . ¥ e with a range of . - . ¥ e . the five pc's had a mean yield of . ¥ e and all maintained a ph > . on day . all products had less than ¥ e residual wbc. platelet quality data is presented in the table. data are the mean ± sd, n = . conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may . the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were blood units (ek) issued for the mentioned period to pediatric clinic for pediatric patients (~ , % units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to - bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was - ml er (depends on age and body weigh). ek was issued as washed concentrates, ek were filtered and ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than times than obstetric ones ( vs ) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk is an immunohaematological analyser for detection of red blood cells antigens of ab , rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: blood samples of donors were tested between july and january . all samples were analysed for ab blood group. samples were tested for rhd antigen and ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab , rh (d, c, c, e, e) and k were investigated truly by first testing at . % samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about . % samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify bp in sregion of hbv genome in the first pcr and bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to . iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of % (hc-ii; %) and a specificity of % (hc-ii; %) (p < . ). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p- blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in . since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from . january to . december . results: in whole blood participated in the consumption with . %, packed red blood cells with (rbc) only . %, washed rbc were used in . % of the cases. in whole blood participated in the consumption with . %, packed rbc with . %, washed rbc with . % and rbc in additive solution with . %. as far as plasma preparations are concerned, there has been, since , a great consumption of plasma -witch was separated from whole blood in period up to five day in . % cases, and small consumption of fresh frozen plasma (ffp) only . %. since , there has completely been cancelled the production of five day old plasma, only ffp is being used. from to for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from units to units per year. in , after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from units in to units in . conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we total febrile non haemolitic male transfusion reaction f e m a l e alloimunisation on le/hla male antigens f e m a l e kandidates of renal male transplantation female lupus nephritis male f e m a l e total female male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, cgy of gamma radiation reduce lymphocyte response to mitogens by %.the aim of the study: . to estimate potassium level increment in stored irradiation blood units. . to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. . to evaluate the expiratory date of blood units post irradiation. the study included units of blood collected in cpd-adsol (as- ). in twenty units the blood collection bag was with inline leucodepletion, while the other units were non leucodepleted. all the units were irradiated using caesium as a source of irradiation, with a dose of - cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from st day post irradiation and continues to day post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day , p value of more than . . . gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. . there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within h post irradiation. . there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. . an out date of days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within - h post irradiation. . the percentage of irradiated blood units requested by our physicians ( . %) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from % ( ) to . % ( ) and increasing trend of using packed red cells component therapy from . % ( ) to . % ( ) . there is also increasing trend of using fresh frozen plasma (ffp) from . % ( ) to . % ( ) . comparing and year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during year period, a total of units of blood and units of f.f.p were used. more specifically, the results can be shown in the following table . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a -year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt s, pt %, fibrinogen . g/l, fv % (other factors were in normal range), platelet count ¥ /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about . bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of mg/kg (~ ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding ( %), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted ( %) along with the presence of factor v inhibitor at a level of . bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of : respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr t/t cpd/sagm (macopharma). whole blood ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. ± °c for to h. after component separation (beckman coulter j mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. ± °c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit . wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr and abc leucoflex lcr ( . and . ) is highly efficient. the use of abc leucoflex lcr showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = . < . ). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = . < . ). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable : , rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst system and leucoflex lst system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed donations with the lst system and donations with the abc lst system. all the donors were random male volunteers and they were meant to donate mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows: . no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst system compared to the abc lst system. . the duration of filtration has been found without statistically significant differences between the two systems. . the loss of volume in the filter of lst is higher than in the abc lst (p = . < . , which is statistically significant). . there was very good leucodepletion with the lst systems (median reduction of the wb cells . log) but there was a superiority of the abc lst over the lst concerning the leucodepletion per litter and per unit (p: . and p: . respectively). . the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst . evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged - days, filtered and consecutively washed with ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were: . the duration of priming and filtering the rccs. . the loss of volume in the filter. . the efficiency of the filtration. . the acceptance of the personnel of a new (to them) filtration system.our results have as follows: . we counted the duration of priming and filtration. median time of priming was s (range - ) and of filtration was min (range - ). . the median volume lost in the filter (as calculated) was . ml (ranging from . to . ). this narrow range is apparently due to the non-flexible cell of the filter. . the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was . ¥ (range . - . ¥ ) and per unit was . ¥ (range . - . ¥ ). the median reduction of the wbc count was . log (range . - . ). . the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly blood banks and blood stations in whole country. the overall blood collected at those centers is about . . units. nearly one in third is being collected at government hospitals ( . %), nearly the same is collected at university hospital blood banks ( . %). the third major group is the red crescent society blood centers-rcsbc ( . %), followed by the social security hospitals ( . %). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) > . . . - . . . - . . . - . . < . . conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among topics in five broad categories. the top two topics were selected for the forum from the completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord- -r datur authors: berger, mitchell; shankar, vidya; vafai, abbas title: therapeutic applications of monoclonal antibodies date: - - journal: the american journal of the medical sciences doi: . / - - sha: doc_id: cord_uid: r datur abstract researchers have sought therapeutic applications for monoclonal antibodies since their development in . however, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases. i n , kohler and milstein revolutionized the field of immunology by developing monoclonal antibodies (mabs). since that time, many mabs have been developed for use in diagnostic procedures and in immunotherapy. ever since it was observed that the therapeutic use of heterologous mabs elicited immunogenic responses in humans, significant research efforts have been devoted toward creating chimeric and humanized antibodies for use in human patients. major achievements have been in the production of mabs in transgenic plants and animals. the use of phage display libraries has created customized antibodies with defined affinity and specificity. this review describes how rodent, chimeric, and humanized antibodies have each been used, with varying degrees of success to treat cancer, septicemia, autoimmune disorders, and infectious diseases. we also describe here recent applications of antibody engineering, such as the use of bispecific antibodies and antibody fragments in immunotherapy. von behring and kitasato discovered in that the serum of vaccinated persons contained certain substances, which they termed "antibodies." in , they treated diphtheria with an antiserum raised against the toxin. on the basis of research on tetanus toxin and trypanosome parasites, ehrlich proposed in the "side-chain theory" of antibody formation, which hypothesized that physiologically active substances, including toxins, attach to cell surface receptors that are produced in response to toxin-cell interactions and then ejected from the cells into the bloodstream, leading to circulating antibodies. , not until the s, however, did scientists' understanding of antibodies become sufficient to lay the foundation for the development of mabs. jerne postulated in a theory of natural selection for antibody formation. animals vaccinated with an antigen were expected to produce several distinct antibodies against several epitopes of the antigen. frank macfarlane burnet subsequently refined and expanded jerne's theory. burnet's "clonal selection theory," as it is generally known, postulates that cells specific for synthesizing type of antibody are spontaneously generated due to random somatic mutations during the maturation of the immune system and that these cells proliferate when exposed to an antigen. at about the same time, porter isolated fragment antigen binding (fab) and fragment crystalline (fc) from proteolytically cleaved rabbit ␥-globulin. until the s, antibody-producing cells were difficult to maintain in culture, because they died after a few days. in addition, only polyclonal antibodies could be obtained. in , littlefield developed a way to isolate hybrid cells from parent cell lines using the hypoxanthine-aminopterin-thymi-dine (hat) selection media. in , khler and milstein provided the most outstanding proof of the clonal selection theory from results of heterokaryons-cell hybrids formed by the fusion of normal and malignant cells. twenty-five years after kohler and milstein produced the first monoclonal antibodies, dramatic progress has been made in using antibodies for diagnostic purposes, but the uses of mabs to treat disease have-until recently-remained somewhat limited. , however, as this review indicates, the potential of mabs to aid in the treatment of a wide range of diseases is now beginning to be realized. antibodies are y-shaped proteins composed of peptides called heavy and light chains, the ends of which vary from antibody to antibody. each combination of heavy and light chains binds to a particular antigenic site. these glycoprotein chains are folded into domains of about amino acids that become twisted into the immunoglobulin (ig) fold and are stabilized by disulfide bonds. structurally, each ig molecule consists of -kd heavy chains and -kd light chains, linked by disulfide bonds. human immunoglobulins are divided into classes or isotypes based on the amino acid composition of their heavy chains: ␣, ␦, ⑀, ␥, and , denoted iga, igd, ige, igg, and igm, respectively. there are kinds of light chains, (k) and (l), which are common to all classes. four subclasses of igg (igg , igg , igg , and igg ) and subclasses of iga (iga and iga ) exist, each with a distinct function. secretory iga exists in dimeric form held together by a j chain and is associated with a secretory component that helps it pass through the cell membrane. , each chain has a constant domain to bind host effector molecule and a variable domain to bind to the target antigen. light chains have variable (v l ) and constant domain (c l ), whereas heavy chains have variable (v h ) and either (␣, ␦, and ␥ chains) or constant domains (⑀ and heavy chains) depending upon the isotype class. each variable domain contains regions known as "hypervariable loops," also known as complementarity-determining regions (cdrs), that identify the antigen. the other amino acids in the variable (fv) domain are known as framework residues and act as a scaffold to support the loops. the v l and the v h , the c h and the c l , and the c h domains are paired; the c h domains have carbohydrate side chains attached to them and are not paired. the folded constant domains may be homologous among different species, allowing hybrid domains (eg, mouse-human) to be produced. the variable domain confers specificity and affinity. the cdr amino acid sequences are extremely variable and play a large role in interac-tion with the targeted antigen. the chain type, region, and distance from the amino terminus characterize the domains. thus, c h domain refers to the second constant domain of the heavy chain. upon digestion with papain, the antibody molecule is cleaved on the amino-terminal side of the disulfide bridges into identical fabs and an fc fragment, whereas pepsin cleaves the antibody on the carboxy-terminal side of the disulfide bridges into f(ab') fragment containing both the arms of the antibody, and many small pieces of the fc fragment. currently, the following are available: whole antibodies, enzymatically produced -kd fab fragments, engineered -kd single-chain fv (scfv) antibodies consisting of the v h and v l connected by a flexible peptide linker, diabodies (noncovalent dimers of scfv), minibodies (scfv-c h dimers), and heavy chain iggs found in species of the camelidae family, which are devoid of light chains and are referred to as v hh . the first mab described by kohler and milstein was created by the fusion of murine myeloma cells with murine-antibody-secreting lymphocytes. , myeloma cells are immortalized b lymphocytes capable of secreting homogeneous antibodies. the immortal myeloma cell lacks the enzyme hypoxanthine guanosine phosphoribosyl transferase (hgprt) and is sensitive to the hat media. however, a hybrid cell known as a hybridoma, generated by the fusion of myeloma cell and an antibody-producing b cell, can survive in the hat media. the spleen b lymphocytes contribute the hgprt gene to the hybrid cell and, hence, unfused myeloma cells and spleen cells die in the hat media. the conventional method of generating mabs is the hybridoma technology in which spleen cells from immunized mice are fused with murine myeloma cells. whereas the myeloma cell imparts immortality to the hybridoma allowing cells to be cultivated indefinitely, the immune spleen b-cell confers antigen specificity. because each hybridoma is derived from a single cell, the cells within a hybridoma cell line are identical and make the same antibody molecule with same antigen-binding site and isotype, hence it is called a mab. among several excellent reviews that detail mab production with hybridomas is a recent review by dean and shepherd. initial attempts to bypass the mouse to make human mabs involved fusion of human immune spleen lymphocytes with nonsecreting human myeloma partners to obtain hybrid cells that continually secrete a specific antibody. however, poor fusion of human myelomas, unsatisfactory performance of the hybrid cells, and the difficulty in accessing immune lymphocytes have prevented success. although heteromyelomas-which are fusions of hu-man and mouse myelomas-work better, these hybrids are usually unstable. attempts to use mouse myeloma cells to create hybrids and derive human mabs led to the loss of human chromosomes and the inability to make human igs. unfortunately, in vitro immunization is limited by its inability to produce a secondary response and by the absence of the affinity maturation process that occurs in vivo. affinity maturation process is a complex phenomenon, a consequence of intense bcell proliferation, somatic hypermutation of ig variable domain genes, and selection for b cells with high-affinity antigen binding, all occurring in the specialized microenvironment of the germinal center within the lymphoid tissue. thus, the search for an ideal fusion partner for generating human mabs has been difficult, which is why the epstein-barr virus (ebv) technique for immortalization of b lymphocytes is preferred. but immortalized b cells do not always replicate exactly the in vivo antibody response, because the tissues from which these cells were selected and the manipulations to which they are subjected to in the laboratory may alter the antibody specificity. , protocols for the preparation of ebv virus, b cells, and cell fusion have been described elsewhere. , , methods used for large-scale production of mabs may include the generation of ascites tumors in mice or in vitro mammalian cell culture fermentation by using bioreactors and continuous perfusion culture systems. , the key issues in scale-up productions are the growth media, fermenter size, fermentation time, and purification procedures. purification or downstream processing is accomplished by chromatography, fragmentation, conjugation with chelating agents, ultrafiltration, and controlled precipitation. a more recent technique for producing antibodylike molecules uses what is known as the phage display library. it involves the construction of v h and v l gene libraries and their expression on the surface of a filamentous bacteriophage. developed in the s, the phage display method requires repeated "panning" or screening of different antibodies based on their affinity for a specific antigen. antibody genes are linked to bacteriophage coat protein genes and the bacteriophages with the fusion genes are used to infect bacteria to create the phage display library. the resulting bacteriophages express the fusion proteins and display them on their surface, and the phage display library comprises recombinant phages, each displaying a different antigenbinding site on its surface. the phage expressing an antigen-binding domain specific for a particular antigen can be detected and isolated by binding to the surface coated with that antigen. libraries of v h and v l genes may be generated from nonimmunized donors, immunized donors who have an immune response against a particular antigen or from a synthetic library consisting of antibody fragments. , one promising way to increase antibody yield or develop new antibodies may be by using genetically altered animals and plants. abgenix, a company in fremont, ca, has developed the transgenic "xeno-mouse," in which the mouse antibody-producing genes have been inactivated and functionally replaced by approximately % of the human ig gene loci in germline configuration, coding for the heavy and light chains. , upon immunization with any specific human or nonhuman antigens, the "xeno-mouse" generates mabs, which are fully human igs, with high affinities and antigen-binding specificities. "xenomouse" strains producing specifically igg , igg , or igg isotypes have also been created to generate panels of diverse and highly specific mabs. kirin brewery company, japan, has developed another transgenic mouse known as the "trans-chromo" mouse. the endogenous igh and igg loci of the "trans-chromo" mouse were inactivated, but it harbors individual human chromosome fragments, derived from human chromosomes and , that contain whole human ig light-and heavy-chain loci, respectively. these mice are capable of producing every subtype of fully human ig, including iga and igm. in these transgenic mouse models, human antibodies with high affinity to an immunized antigen are naturally selected by the murine immune system via an affinity maturation process, and thereby show increased diversity of the mabs. transgenic mice may be a suitable alternative to chimeric or humanized antibody production or the use of phage display systems to create less immunogenic or novel antibodies. for instance, transgenic mice that express mabs to coronavirus in their milk have been developed. , because ruminant animals, such as cows, goats, and sheep, produce relatively large amounts of milk, genetically-modified members of these species could also be used to produce large quantities of therapeutic proteins, including mabs. plants may be a potential source of recombinant proteins, including mabs. - plant virus vectors, such as the tobacco mosaic virus, may be used to make mabs. transgenic tobacco plants may also be used for large-scale production of recombinant iga, which is used in passive mucosal immunotherapy. , this mab could be added to toothpaste to effectively protect against bacteria that cause tooth decay. , recombinant antigens obtained from plants may also have therapeutic applications. for instance, attempts to immunize mice with escherichia coli heat labile enterotoxin b produced in transgenic tobacco and potato plants have proved promising. hepatitis b viral surface protein produced in transgenic tobacco plants has been shown to be immunogenic in mice. the genes coding for murine malignant b-cell specific markers are inserted into tobacco mosaic virus to cultivate an immunogenic protein in tobacco plants that may eventually be used in developing a vaccine against non-hodgkin lymphoma. , other immunotherapeutic proteins under development include norwalk virus capsid proteins produced in tobacco and potatoes, cholera toxin and ct-b produced in potatoes, hepatitis b antigen produced in tobacco, anti-human igg used to detect nonagglutinating antibodies produced in alfalfa, and humanized anti-herpes simplex virus (hsv)- grown in soybeans. - the food and drug administration (fda) considers antibodies to be "biopharmaceuticals"; as such, mab applications are regulated by the agency's center for biologics evaluation and research (cber) and the center for drug evaluation and research. , the fda has created a "points to consider" document advising manufacturers of factors to consider in the production and testing of mabs intended for human use and identified information that should appear in investigational new drug or biologics license applications. the "points to consider" document serves to "indicate the agency's current thinking on mab products for human use." the agency's recommendations are aimed at protecting human health because viruses and cellular dna from antibody-producing cells with malignant phenotypes may be integrated into host cells after transformation. accordingly, among the points to consider are several steps-such as taking care to ensure purity of immunoconjugates and demonstrating the ability of any purification scheme to remove adventitious agents-designed to prevent contamination of the final product by human pathogens. manufacturers must also adhere to animal care standards and detail steps to prevent contamination of cell culture. the points to consider document includes a list of normal human tissues used in cross-reactivity testing, tests for murine viruses, and organs to be considered in dosimetry estimates. the fda recognizes that because of species differences, animal models expressing the antigen of interest or cross-reactive epitopes are not always available. the agency has also published a guidance entitled s preclinical safety evaluation of biotechnology derived pharmaceuticals based on international conference of harmonization technical requirements. before beginning phase clinical studiesconducted to assess the safety of the drug and its mechanism of action-the fda recommends that researchers conduct in vivo and in vitro testing of the mab to assess cross-reactivity with human tissues or non-target-tissue binding. preclinical safety testing aims to evaluate the immunogenicity, crossreactivity, stability and effector functions of mabs. the metabolism, carcinogenicity, and genotoxicity of the product should also be evaluated. guidance has also been published for mabs used as reagents in drug manufacturing. the guidance emphasizes biological safety, performance characteristics of the reagent, and potential presence of residual amounts of the reagent in the final product. the fda general principles for testing and manufacturing are broadly applicable to many classes of antibodies, including those produced by phage display systems or transgenic plants and animals. depending on the expression system being used, other fda guidance documents, such as cber's points to produce biologicals should also be referenced. [ ] [ ] [ ] humanizing monoclonal antibodies rodent mabs with excellent affinities and specificities have been generated using conventional hybridoma technology, but their use in clinical medicine is limited due to the immune responses they elicit in humans. for instance, the human antimouse antibody (hama) response can compromise the clinical effectiveness of murine mabs. although hama responses are directed against the murine constant regions, which represent the major antigenic features of the mouse ig, significant responses are also directed toward the murine variable regions. as a result, patients may mount an immune response against the injected murine antibodies, leading to allergic or immune complex hypersensitivities, rapid clearance of the antibody, and reduced clinical efficacy. , - initially, morrison and colleagues , introduced chimeric mabs in , which showed several advantages over unmodified rodent antibodies. generally, chimeras combine the human constant regions with the intact rodent variable regions, replicating the rodent antibody variable regions by pcr and then cloning them into eukaryotic expression vectors containing human constant regions. ideally, this allows better interaction with human effector cells and the complement system. because the fc region has little influence on the structure of the fv region, the chimeric constructs' affinity and specificity are virtually unchanged, and both rodent and chimeric antibodies cause apoptosis at a similar rate and intensity against target cells in vitro. , , although chimeric antibodies have helped solve some of the problems associated with the use of rodent mabs, they still show significant immunogenicity in humans; because of their approximately % mouse sequence, they cause an human-antichimeric antibody response. humanized antibodies containing only the cdrs of the rodent variable region grafted onto the human variable region framework have been introduced to overcome these deficiencies. the early work on recombinant, chimeric, and rodent/human antibodies happened during the mid s and, by the late s, greg winters and his colleagues demonstrated that a functional human-like antibody could be created by grafting the antigen-binding cdrs from variable domains of rodent antibodies onto human variable domains. numerous humanized antibodies have now been designed and constructed, and many are currently being evaluated in clinical trials. , , , , efficient procedures for constructing humanized antibodies have been developed. , , , the first step is to clone and sequence the complementary dnas (cdnas), coding for the variable domains of the mouse antibody to be humanized. the mouse hybridoma cell line is grown in an appropriate culture medium, and cells are harvested for rna isolation. polymerase chain reaction (pcr) primers that hybridize to the ' ends of the mouse leader sequences and to the ' ends of the mouse constant regions are designed for cloning light chain variable regions and heavy chain variable regions. cdna is synthesized from total rna, followed by pcr amplification with light and heavy chain specific primers. positive bacterial colonies containing mouse variable regions are then screened. construction of a chimeric antibody involves modifying the cloned mouse leader-variable regions at the '-and '-ends, using pcr primers to create restriction enzyme sites for convenient insertion into expression vectors, to incorporate sequences for efficient eukaryotic translation, and to incorporate splice-donor sites for rna splicing of the variable and constant regions. the adapted mouse light and heavy chain leader-variable regions are inserted into vectors containing, for example, human cytomegalovirus enhancer and promoter for transcription, a human light or heavy chain constant region, a neomycin gene for selection of transformed cells, and the simian virus origin of replication in cos cells. these vectors are designed to express chimeric or reshaped human light and heavy chains in mammalian cells. the design and construction of an engineered human antibody require an analysis of the primary amino acid sequences of the mouse variable regions to identify the residues most critical in forming the antigen-binding site. a structural model of the mouse variable region is built on the basis of homology to known antibody variable regions. the framework regions (frs) of the new variable regions are modeled on frs from structurally similar immunoglobulin variable regions. the design process involves selecting human light and heavy chain variable regions that will serve as templates for the construction of a reshaped human antibody. the mouse cdrs are then joined to the frs from selected human variable regions. the primary amino acid sequences are then carefully analyzed to ascertain whether they would recreate an antigen-binding site that mimics the original mouse antibody. within the frs, the amino acid differences between the mouse and the human sequences are examined, and the relative importance of each amino acid in the formation of antigen-binding site is evaluated. minimum changes in the frs are desirable and should closely match the sequences from natural human antibodies. any potential glycosylation site in the frs of either mouse or human sequence needs to be identified and its influence on antigen binding considered. the dna sequences coding for the reshaped human variable regions, either made synthetically or based on an existing sequence that is very similar to the newly designed reshaped human variable region, are modified by pcr with specially designed oligonucleotide primers. the human variable regions together with their leader sequences are then cloned into a mammalian expression vector that already contains human constant regions. each human variable region is linked to the desired constant region via an intron. preliminary expression and analysis of the reshaped human antibodies are done by cotransfection of mammalian cell-expression vectors, coding for human light chain and coding for human heavy chain. the vectors will replicate in the cos cells and transiently express and secrete reshaped human antibodies. the concentration of the antibody produced can be analyzed by using an enzyme-linked immunosorbent assay. specific changes in the amino acids of the framework region may also be required to preserve the orientation and structure of the rodent cdr required for binding. computer modeling using databases containing human variable genes will identify sequences homologous to the rodent v regions. , a computer model of the rodent fv can identify the non-cdr residues that interact with the cdr sequences, and choices can be made regarding which residues need to be included in the variable region. antibodies humanized in this way may have binding affinities up to one-third greater than the corresponding murine antibodies. allergenicity is also significantly reduced. about to % of patients exhibit a hama reaction to murine antibodies, whereas only about % have a similar reaction to humanized antibodies. , , - humanization of mabs still has several practical difficulties. first, a detailed knowledge of the antibody structure and function is required. second, methods for efficient construction of humanized mabs are limited. third, unpredictable immunogenicity may result when a new amino acid sequence is introduced to balance affinity retention. fourth, the antibody repertoire is limited to the animal in which the progenitor mab originated. fifth, the rodent mab producing hybridoma must be isolated and thoroughly characterized. however, obstacles to humanization are gradually being surmounted. karpas et al reported creating a hat-sensitive and ouabain-resistant human myeloma cell line that can fuse with human lymphoblast cells. a hat-sensitive subline of the myeloma cells that secreted only light chains was fused with ebv-transformed white blood cells that produced igg mabs to hiv- gp . eventually, a clone was isolated that was polyethylene glycol-resistant and would not revert when placed in hat medium. standard polyethylene glycol fusion protocol was followed to fuse the myeloma cells with both ebvtransformed white cells and fresh white cells obtained from the peripheral blood of adults and tonsil cells from children. the authors reported promising rates of hybridoma formation, stable ig production, and high yield of secreted antibodies compared with antibody-producing cell lines from mouse myeloma cells. the authors have now developed hybridomas that have been secreting cells for more than months. a human myeloma cell line may eventually prove useful in creating mabs against certain autoimmune diseases and cancers. this technique may also make it easier for other researchers to generate human mabs for use in therapy. antibodies may act directly when binding to a target molecule by inducing apoptosis, inhibiting cell growth, mimicking or blocking a ligand, or interfering with a key function. , in addition, antibodies may modulate or potentiate drugs or other therapies. the antibody may itself act as an effector-as in antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent complement-mediated cascade-or it may involve effector elements such as cytotoxins, enzymes, radioactive isotopes, signals for other parts of the immune system, and/or cytotoxic drugs. , , - adcc occurs if fc regions on the antibodies are recognized by receptors present on cytotoxic cells, such as natural killer cells, macrophages, granulocytes, and monocytes. complement-mediated cytotoxicity ensues if the antibody binding prompts a complement cascade to occur. active immunotherapy can be accomplished if the antigen can provoke a long-lasting t-cell response, which may be achieved by administering whole cancer cell extracts or by using small antigenic peptides isolated from tumors in experimental patients or animals. for instance, mabs mimicking breast cancer-specific antigens elicit anti-idiotype antibodies; this is another way of creating active immunity, which can lead to a humoral auto antibody-like immune response. the use of bispecific molecules that recognize antigens on both the target cell and effector cell can increase adcc. the most effective mechanisms are blockade of a crucial ligand or growth factor or adcc in which tumor cells are killed by fc receptorbearing cytotoxic effectors. cell killing by adcc is proportional to the amount of antibody bound to a cell, whereas the blockade of an essential growth factor may not show effects until most of its receptor is saturated. a higher antigen expression by the target cell will increase antibody binding and subsequent adcc. but high receptor expression will also make it difficult to prevent the binding of a cytokine or ligand at minimum threshold. , , with a bispecific antibody, the specificity of mab is combined with the cytotoxicity of immune effector cells, for instance, to neutralize a tumor. , , , bispecific antibodies link the tumor cell directly to the killer cell via cytotoxic trigger molecules, such as t-cell receptors or fc receptors, leading to lysis and/or phagocytosis by the effector cells. although bispecific antibodies can enrich effectors at the tumor site and activate tumor bound effector cells by enabling cross-linking between effector and target cells, they can cause system-wide immune activation because of t-cell receptor cross-linking. bispecific antibodies can also mediate cellular cytotoxicity via various effector cells, including phagocytes, natural killer cells, and t-lymphocytes. another way to use the binding properties of antibodies is by conjugating antibodies to cytotoxic drugs, radioisotopes, or toxins. , , , , techniques for conjugation have been described in several recent reviews and articles. , , , mabs can be conjugated to chemotherapeutic drugs such as doxorubicin, mitomycin, and methotrexate. chemotherapeutic agents constitute cytotoxic or cytostatic drugs that can be conjugated to antibodies; thus far, however, these drugs have shown poor specificity for target cells and frequently lead to toxicity. , , often, antibodies may lose reactivity upon conjugation with such agents. immunotoxins used in cancer therapy are conjugated antibodies that combine plant (ricin, gelonin, saponin, abrin, pokeweed antiviral protein) or bacterial (diphtheria toxin and pseudomonas toxin a) toxins with antibody specificity. , , , , , , , toxins may inhibit protein synthesis even at picomolar concentrations. natural toxins, such as diphtheria toxin, can act at low concentrations, enter cells, and disrupt key cellular processes. enzymes may also be conjugated to antibodies in antibody-directed enzyme-prodrug therapy. , , , , an enzyme that can convert a prodrug to an active form may be conjugated to an antibody and targeted to a desired location using the antibody-binding region. they may more effectively localize tumors than chemical conjugates, and a prodrug is converted to an active drug by the enzyme at the target cells. another therapeutic technique relies on using antibody fragments, which may be produced by the traditional method (proteolytic digestion with papain) or with newer methods that may reduce damage to the binding sites. , some newer methods attempt to construct mimetics that combine multiple cdrs from several antibodies into single molecules with molecular weights substantially lower than single chain fragments. because of their smaller size, antibody fragments may be able to penetrate tissues and tumors more readily and be less immunogenic than whole mabs. however, although antibody fragments may have better penetration than whole antibodies, their shorter half-lives may compromise clinical usefulness. heteropolymerized antibodies have recently been developed by chemically linking a mouse igg mab, specific to a complement receptor site (cr ) on the human or nonhuman primate erythrocyte, to a second mouse igg mab that is specific to the targeted antigen. the heteropolymer technique is based on immune adherence, in which antibody-antigen immune complexes bind to a complement receptor on the red blood cell, facilitating the phagocytosis of these complexes. once introduced into the patient, the heteropolymers will bind to both the red blood cells and the targeted antigen. the antigen-antibody heteropolymer complexes are then transported to the liver and spleen, where the complexes are destroyed by macrophages, whereas the red blood cells return to the circulation unharmed. [ ] [ ] [ ] [ ] a wide variety of conditions, of both autoimmune and foreign origin, may potentially be treated with this method, including hiv infection, systemic lupus erythematosus, marburg virus infection, and myasthenia gravis. - although mouse antibodies are currently being used with this technique, chimeric mousehuman antibodies could also be constructed. it may also be possible to use the heteropolymers as "sentinels" by injecting them before antigen exposure. in recent study, multiple infusions of heteropolymers provided nonimmunized monkeys with protection against an antigen (⌽ ) for as long as weeks. when milstein and kohler announced their isolation of a mab in , many thought mabs would provide an effective cancer treatment. however, early clinical trials were disappointing. - producing mabs to tumor antigens is a complex process. first, proteins peculiar to human cancer cells are identified; then, mice are injected with human tumor cells (antigen) to stimulate an immune response. after about days, the mouse lymphocytes are removed and fused with myeloma cells to isolate and reproduce hybrid cells specific to a predetermined antigen. rodent antibodies alone have been used in several trials, but the clinical effects are often minimal because of poor activation of human effector or complement cells by the fc region of the murine antibody and the accompanying hama responses. , substituting the rodent fc region with a human fc fragment may help overcome these effects. with the discovery of proto-oncogenes, secondgeneration trials in the s turned to more specific tumor antigens that could be potential targets. in , mab - a, an antibody to epithelial cell surface antigen expressed on human colorectal carcinomas, was approved for the identification of adenocarcinomas; mab - a may reduce the mortality and occurrence rate of colorectal cancer. , in , rituximab, a mouse-human chimeric anti-cd antibody, was approved for the treatment of non-hodgkin b cell lymphoma. , rituximab binds to cd antigen on b cells and b cell tumors and then elicits a natural immune response that can kill malignant cells. recent animal trials have shown the potential of mab therapy in cancer. increases in epidermal growth factor (egf) receptor expression have been found on many human cancers, and a fully human anti-egf receptor igg mab has been shown to inhibit human cancer growth in vitro and in vivo. other efforts are focusing on antibody to her antigen in breast cancer. patients with cancer often mount an immunologic response to tumor-associated antigens with increases in cytotoxic lymphocytes and antibodies. several tumor-reactive antibodies have been cloned against melanomas, colon carcinomas, ovarian, breast, and lung tumors. often, these antibodies cross-react with other malignant tissues or cell lines. recently, trastuzumab (herceptin), a humanized mab that binds directly to the c-erb protein (her ), has been approved by the fda for the treatment of breast cancer. , - herceptin can be used to treat metastatic breast cancer in patients whose tumors overexpress the her protein (about % of breast cancer patients) and may be used in conjunction with other therapies such as paclitaxel (taxol). antibody was developed by humanizing murine mab d by inserting antigen-binding regions of mab d into the framework of a consensus human igg , resulting in rhumab-her or trastuzumab. cancer cells have antigens that are specific to the tumor (tumor-specific antigens) or are present in greater concentration than normal (tumor-associated antigens). antibodies may eliminate target cells by complement action or through adcc. the variable region of the antibody recognizes and attaches to a specific antigen and the constant region, then joins with an effector cell capable of killing the targeted cancer cell. when the antigen and antibody bind, precipitation and agglutination may isolate the complex. anti-idiotype mabs that mimic-tumor associated antigens also may be used in cancer therapy. the idiotype network hypothesis, formulated by lindemann and jerne, suggests that because each mature b cell secretes an antibody with unique antigenbinding specificity in the variable domain (referred to as the idiotype), anti-idiotype mabs can be generated and used as surrogate antigens or vaccines for immunization against the tumor. , a murine anti-idiotype mab, aca , which mimics the tumor-associated antigen ca , was recently found to induce a specific anti-anti-idiotypic immune response in of patients with platinum-pretreated recurrent ovarian cancer enrolled in a phase i/ii clinical trial. , a positive immune response was associated with statistically significant (p Ͻ . ) prolonged survival times ( . Ϯ . months in patients shown to have an anti-antiidiotypic response compared with . Ϯ . months in patients who were anti-anti-idiotypic negative). , in addition, peripheral blood lymphocyte mediated lysis of ca- expressing tumor cells increased in of patients after vaccination with the mab. despite the use of a murine antibody, both this study and a previous phase i study involving patients with ovarian cancer found minimal side effects. , promising early results have also been demonstrated in patients with advanced colorectal carcinoma who received a murine anti-idiotype mab that mimics an epitope of carcinoembryonic antigen (ceavac) , and in patients with malignant melanoma who received an anti-idiotype mab (trigem) that mimics disialoganglioside gd despite promising developments, however, there are still several obstacles to effective cancer therapy with mabs. problems with the tumor, the mab, or conjugate characteristics all continue to challenge researchers. , , thus far, chemotherapeutic mab therapies have faced obstacles due to the poor ability of agents to affect tumor cells preferentially over healthy cells, the intrinsic insensitivity of many tumors to these drugs, and the rapid development of resistance in tumor cells. , , , mabs often decrease the size of tumors, but rarely lead to complete remission of solid tumors. at the cellular level, mabs at low doses ideally should bind with excellent affinities. because no antigens have been identified that are expressed exclusively on tumor cells, cross-reactivity of mabs must be examined with histochemical tests on tissue sections or in animal models. careful testing must also be done to ensure that the effector molecules such as drugs, toxins, or isotopes in the conjugates do not inadvertently target healthy cells. although humanized antibodies have greater affinity than murine antibodies, this potential has yet to be translated into improved clinical outcomes against cancer. however, recent success using the xenomouse technology to develop a fully human igg mab specific to epidermal growth factor receptor indicates the potential for future advances in this area. recombinant immunotoxins, fusion proteins containing the fv of a mab and a bacterial toxin, are under development for cancer therapy. because immunotoxins exploit only the variable region-binding function of the mab, in theory just a fragment of the binding region, such as the bivalent f(ab') fragment, monovalent fab, scfv, or disulfide-stabilized fv fragments may be used as opposed to the larger and probably more immunogenic whole antibody. disulfide-stabilized fragments may be more stable or have higher affinity for the antigen than scfv, in which the linker may interfere with binding or fail to stabilize the fragment. recombinant immunotoxins derived from pseudomonas enterotoxin have been shown to be active against lymphomas, solid tumors, and leukemias. because binding specificity and affinity are the key elements, it is not known which form of the immunoconjugate is usually more effective in treating human tumors; some animal tests suggest that the whole antibody, with its longer half-life and bivalent binding, is more effective than the antibody fragments with decreased binding affinity. bispecific antibodies and single chain fvs are being developed that may be more effective because they clear faster from nontumor tissues and more deeply penetrate tumors than whole antibodies. , , bispecific antibodies may be used to target tumor vascular endothelial cells, thereby limiting the tumor's blood supply and possibly inhibiting its spread. advances in radiolabeling have allowed immunoconjugates to be delivered to cells and showed promise in clinical trials. , , , , radioimmunotherapy uses a radiolabeled mab to deliver radioactive isotopes to targeted cells. radioisotopes such as iodine- and yttrium- , which are ␤ emitters, can cause damage not only to the bound cell but also to cells adjacent to tumor cells that antibodies may not be able to reach within the tumors. lack of knowledge about the appropriate dose, biodistribution, and shedding of antigen hinders use of radioisotopes. radiolabeled mabs may also affect normal cells, depending on the extent to which reticuloendothelial cells expressing fc receptors bind to the constant regions of intact antibody molecules. using antibody fragments or constructs may modify this nonspecific uptake. , , , radiotherapy exerts most of its effect by emitting a low dose, which exponentially decreases continuous radiation, but the antibody per se may have a cytotoxic effect. the duration of radiotherapy is determined by the half-lives of the antibody and the isotope used. the success of radioimmunotherapy is affected by the specificity, affinity, dose, and immunoreactivity of the antibody, heterogeneity of antigen expression, diffusion rate, tumor volume, blood supply and tumor location, dose rate effects, and variability in dose deposition. the choice of radionuclide, selection of the chelate used to link the mab to the radionuclide, and the mab selected are critical in development of radiolabeled mabs. suitable radio nuclides include yttrium- , iodine- , and copper- . iodine- was the first radioisotope to be used in treatment of hodgkin disease and other lymphomas. yttrium- is potentially useful for lymphoma therapy because it decays with ␤, but not ␥, emissions that may kill other tumor cells in a cross-fire effect. the energy released by yttrium is times higher than that of iodine- , which degrades rapidly after uptake into a tumor cell, causing toxicity. in addition to their uses in radiotherapy, radiolabeled mabs can also be used to diagnose cancer. , , , , radioactive isotopes linked with mabs may help localize tumors in a form of diagnostic imaging called immunoscintigraphy. for instance, oncoscint, a mab coupled to indium- , may be used to detect an antigen (tag- ) found on colorectal adenocarcinomas. mabs may potentially be used to suppress the immune system after transplant or to induce tolerance to transplanted organs or tissues. thus far, however, only antibodies to cd and cd are licensed for clinical use. okt , a murine igg a antibody to human cd , and antibodies to cd (il- receptor) have been used to reduce allograft rejection. graft-versus-host disease (gvhd) is a complication of allogeneic stem cell transplant that occurs despite histocompatibility testing and use of cyclosporine and its analogs. gvhd is a frequent cause of illness or death in allogeneic transplant patients and occurs when alloselective donor t cells recognize and interact with major and minor histocompatibility antiigens in the host, leading to cytokine release. mab therapy against gvhd is most effective when it is administered after a bone marrow transplant but before gvhd development by targeting t-cells before their activation. for instance, target is the interleukin- receptor ␣-chain (il- r␣ tac protein or cd ), whose expression is a crucial step in the activation of alloreactive t cells. , recently, it was reported that daclizumab, a humanized anti-il- a antibody, was an effective complement to dual immunosuppression therapy in renal transplant patients. because only activated cells express il- , antibodies to this cytokine might inhibit t cells during allograft rejection and prevent generation of cytotoxic t cells. the development of hama response in patients and the decreased effectiveness of murine mab relative to human mab have thus far compromised the effectiveness of this approach. however, humanized anti-tac mabs, with better human effector functions, may survive longer in vivo and may prove less immunogenic than its murine counterpart. short-term cd antibody therapy may also contribute to long-term acceptance of skin and islet allografts, even after gaining immunocompetence. more generally, the ability of mabs to induce tolerance to transplanted tissues and self-antigens may hold great therapeutic potential. , mabs to cd and cd have been studied, although thus far these have not resulted in clinical success. depletion or blockade of t cells by antibodies may facilitate tolerance by preventing t cells from attacking the graft, and recent t cell depletion studies in primates have proven promising. mab treatments for other complications after transplants are also under study, including potential treatment of posttransplant lymphoproliferative disorder with the anti-cd mab rituximab and the use of anti-lfa- mab (odulimomab) to protect against ischemia-reperfusion injury after kidney transplants. [ ] [ ] [ ] daclizumab, a humanized antibody that targets the anti il- receptor, may reduce the risk of acute rejection of a renal transplant and also lower cytomegalovirus infection rates among transplant recipients. the inflammatory bowel disorder known as crohn disease has also been treated with mab therapy. a chimeric igg k antibody, infliximab (remicade), acts by binding to soluble and transmembrane tumor necrosis factor ␣ (tnf␣), preventing it from binding to its receptors on activated macrophages. the anti-tnf␣ antibody may provide relief to patients with moderately to severely active crohn's disease. in addition, the fda recently approved infliximab for the treatment of rheumatoid arthritis in combination with methotrexate. - high levels of ige may cause bronchial hyperresponsiveness, a risk factor for asthma. [ ] [ ] [ ] im-therapeutic applications of monoclonal antibodies mune responses mediated by ige are important in the pathogenesis of allergic asthma. in recent study with subjects reporting moderate to severe allergic asthma, twice-weekly injections of recombinant humanized anti-ige antibody (rhumab-e ), which forms complexes with free ige and blocks its interaction with mast cells and basophils led to a fall in serum ige levels and slightly decreased asthma symptom scores relative to the placebo group. patients receiving anti-ige were able to reduce reliance on corticosteroids. although the study must be interpreted cautiously, it is nonetheless a promising step in finding more effective asthma therapies. , , approximately , cases of sepsis in the us and about , cases in the uk are reported each year. , the mortality rate could be as high as to %, depending on the population studied. , mabs have been targeted to tnf␣ and tnf␣ receptors, which are key elements of the inflammatory response. unfortunately, this may inhibit many cytokines, which can impair the patients' ability to fight infection and increase the risk of secondary sepsis; also inhibiting a few cytokines may not be sufficient. although mabs targeted toward components of the host immune system, such as individual receptors or mediator cells, help in the treatment of septic shock, targeting the bacterial endotoxin or lipid a of gram-negative bacteria with mabs may be more efficacious. lipopolysaccharide endotoxin, a component of the outer cell membrane of gramnegative bacteria, consisting of a highly variable o-linked polysaccharide chain, an r core region, and lipid a, which in turn is composed of a glucosamine disaccharide backbone substituted with amide-and ester-linked long-chain fatty acids, is believed to be important in many cases of septic shock and septicemia, and can trigger an inflammatory cascade that can cause serious injury and even death. , lipid a is linked by the core region to the o-linked side chain, and variations in the o-linked side chains result in an enormous diversity among gram-negative bacteria, making adoptive immunotherapy against the o-linked antigenic determinants difficult. directing antibodies to the core region or lipid a, which tend to be more conserved, would allow for therapy against a diverse array of gram-negative bacteria. favorable results with polyclonal j antibody have spurred attempts to develop a mab. , a murine igm mab, e (xoma, berkeley, ca) binds to an epitope on lipid a of e coli j . a human igm, ha- a (centoxin) is derived from a heterohybridoma fused from spleen cells of a patient vaccinated with e coli j before splenectomy. , , , both the antibodies have thus far shown mixed success in patients with sepsis and neither is currently cleared by the fda for therapeutic use. because patients with bacteremia often have endotoxemia, ha- a, an igm antibody to endotoxin may be effective against endotoxin in the bloodstream as well. unfortunately, the high cost of antibody ($ /dose in europe) combined with the uncertainty that the sepsis is caused by gram-negative bacteria has hindered the widespread use of ha- a. , , although it is less efficacious, the only other option may be to target tnf␣, but recent study reported a high ( . %) rate of adverse reactions in patients receiving an anti-tnf␣ antibody. the antibody was no more effective than the placebo given to control subjects. however, research on a mab for the treatment of septicemia continues. cytomegalovirus (cmv) causes serious illness affecting the immunocompromised, such as patients with aids and those undergoing organ transplants. infection rates may be up to % in those negative for cmv who receive kidneys from seropositive patients. cmv infection can result in retinitis and gastroenteritis in hiv-infected patients and may also cause chronic intrauterine infection. , up to , cases of congenital cmv infection are reported each year; mental retardation and hearing loss may occur in about % of these cases. currently, there is no vaccine against cmv. ganciclovir, foscarnet, and (s)- -[ -hydroxy-( phosphonylmethoxy)propyl]cytosine are some of the potential treatments for cmv infection. another mode of treatment is via administration of anti-cmv hyperimmunoglobulin, derived from pooled sera of cmvseropositive persons. passive immunization has been shown to reduce the severity of cmv and prevent mother-to-infant transmissions. moreover, antibodies may be able to clear the virus from infected tissues, a function previously thought to be exclusive to cytotoxic t lymphocytes. many physicians use a combination of antiviral agents and immunoglobulins in patients at risk for cmv infection. mabs may also decrease the amount of antiviral agents required for treatment. mabs against murine cmv polypeptides have been shown to be protective in animal models. although mabs may act synergistically with foscarnet or ganciclovir in vitro, it is unclear whether this advantage could be extrapolated to in vivo conditions. experiments in which murine cmv is used as a model system suggest that high concentrations of antibody, along with ganciclovir or (s)- -[ -hydroxy-( phosphonylmethoxy)propyl]cytosine, synergistically inhibit the growth of murine cmv in cell culture, whereas lower antibody concentrations produce only an additive effect. however, antibody ad-ministered along with ganciclovir in severe combined immunodeficient mice also produced only an additive effect. the extent to which a mab can protect the host from cmv infection cannot reliably be predicted from its immunoreactivity, neutralizing titers in vitro, or from its antigen-binding data, because the mechanism of action of a mab in vitro and in vivo may be entirely different. if an antibody is used along with drugs, not only is the treatment efficacy improved but also the side effects of a high drug concentration may be avoided. for instance, mg of ganciclovir/kg of body weight, along with an antibody, was almost as effective as mg/kg ganciclovir administered alone in cmv-infected severe combined immunodeficient mice. the msl antibody, a human igg directed against human cmv glycoprotein, is marginally synergistic in inhibiting cmv in vitro when conjugated to ganciclovir or foscarnet. in a recent trial involving aids patients with active cmv retinitis, the rate of retinitis progression was similar in the group receiving the antibody ( mg, intravenous) for weeks and the placebo control group. surprisingly, mortality increased in the msl- group, and the poor performance of msl- in this trial may have been due to the inability of the antibody to neutralize the established cmv infection in aids patients or its failure to cross the blood-ocular barrier in sufficient quantities. liver transplant patients are also at risk of cmv infection and may require treatment with the murine mab okt , an anti-t cell antibody that is directed to cd antigen, along with ganciclovir to mitigate transplant rejection. human mabs neutralizing cmv were tested for their safety and pharmacokinetics over a period of to days in bone marrow transplant recipients and were found to be safe and efficacious, as evidenced by a steady increase in the neutralizing activity. also, a human anti-cmv mab designated c that was purified from hybridomas generated by the fusion of human lymphocytes with mouse myeloma cells has virus neutralization titers about -fold higher than those produced after conventional human ␥ globulin used in humans. a humanized mab has been developed that binds to the -kd glycoprotein, gpul (gh), of cmv, recognizes a variety of virus strains, and neutralizes clinical isolates of cmv; it could be used as a potential agent for the prevention or treatment of cmv infections in humans. respiratory syncytial virus (rsv) causes serious lower respiratory tract disease requiring considerable supportive care, administration of humidified oxygen, and respiratory assistance. the fda approved the use of a humanized mab against rsv, which afflicts mostly infants and children younger than months of age. , known as palivizumab, the antibody (produced by synagis and medimmune, inc.) treatment showed a % reduction in rsv infection in hospitalized patients. , the american academy of pediatrics' committee on infectious diseases has published recommendations on the use of this antibody in children at risk for rsv infection. , mabs have also been used in the therapy of hsv infections. the incidence of neonatal herpes is to % in babies born vaginally to mothers with primary infection but only to % in babies born to mothers with recurrent infections. this difference is attributed to the passive transfer of protective antibodies to the fetus. antibodies to hsv- may also confer partial protection against type strains. based on this observation, the potential of mab therapy against herpes has been studied for more than a decade. experiments in mice, for instance, indicate that microgram quantities of antibodies may promote healing of corneal opacity and blepharitis caused by herpes simplex. however, attempts to confer immunity in humans and other higher animals by passive immunization alone have been disappointing, because antibodies to hsv are restricted to only a few specific epitopes. among the glycoproteins expressed by the herpes virus on its surface, glycoproteins (gb, gd, gh, gk, and gl) are thought to be crucial to infection. the initial step in hsv infection seems to be the binding of gc to heparan sulfate proteoglycans followed by the binding of gb, and the binding of gb and gc is stabilized by gd. gh is involved in initiation of viral fusion and other glycoproteins may help in expanding the fusion process. evidence that neutralizing antibodies to gh, gd, and gb prevent membrane fusion but not viral attachment is consistent with this model. most human antibodies are directed against gd and gb epitopes. mabs can target at least different antigenic sites on hsv glycoprotein d in the murine model. , glycoprotein d is necessary for virus replication in tissue culture. yamamoto et al reported the antibody-dependent cellular cytotoxicity effect of hs , a neutralizing mab to glycoprotein b of hsv, in athymic nude mice inoculated with hsv intracutaneously. when hs is administered, the skin lesions healed in % of the mice given the antibody. also, latent infection in the ganglia was prevented in mice that survived, as evidenced by the failure to detect hsv upon co-cultivation with vero cells. administration of hs after development of zosteriform lesions ( to days after infection) reduced the virus in the ganglia and prolonged survival time; however, the disease was not totally avoided and eventually the mice succumbed to the disease. the antibody therapy could prevent or decrease the severity of herpetic keratitis, iritis, and blepharitis after corneal infection by hsv. , thus, mabs could potentially be used to prevent congenital herpes and sexual transmission of the herpes virus. , , , currently, no treatments exist for the hemorrhagic fever caused by the filovirus ebola. recently, a team of researchers at the us army's medical research institute identified protective antibodies directed against epitopes on ebola virus membraneanchored glycoprotein. some of the antibodies protected mice as long as days after exposure; doses were equivalent to acceptable ( to mg/kg) human levels. antibody specificity is important, because some mabs studied bound to ebola zaire but not to the ivory coast or sudan serotypes. much research remains to be done before the potential of antibodies in the treatment of ebola will become realized. toxins are poisonous proteinaceous substances. antibodies were first clinically used in the s for protection against the toxin digitalis. , today, antidigitalis immunotoxicotherapy is a standard therapy. digoxin is produced from sheep immunized with digoxin-serum albumin conjugate. igg antibody specific for digoxin is purified and cleaved into fabs, because the smaller molecular mass of the fragments allows faster renal clearance and more rapid action than the whole antibody. the fab fragments bind to circulating digitalis molecules and generate a complex that is unable to bind to receptors. adverse effects are rare, and the immunotherapy is generally effective against digoxin and digitoxin within hour. , however, because polyclonal antibodies may be difficult to produce and may cause hypersensitive reactions, researchers have attempted to develop humanized mabs against digoxin and other toxins. for example, group has used a transgenic mouse to produce hybridomasecreted human mabs against digoxin. more recently, the effect of goat anti-colchicine igg antibodies has been studied in mice exposed to a lethal intraperitoneal dose of anti-inflammatory colchicines, and preliminary studies in a -yearold woman with colchicine poisoning showed promise. , attempts have also been made to use drug-specific goat antibody fragments to treat anti-tricyclic antidepressant (tca) overdose. , antidepressant overdose is the most common cause of intentional drug overdose in the us. however, lethal doses of tricyclics are -to -fold higher than those of colchicine, digoxin, and snake venom, which require higher antibody doses (up to several g/kg) to reverse the toxicity. , high-affinity tricyclic antidepressant-specific mabs have been shown to reverse the cardiovascular toxicity of antidepressant desipramine in rats and prolong their survival, and tricyclic antidepressant-specific fab fragments, along with sodium bicarbonate, a standard treatment for tricyclic overdose, also minimizes the required dose of antibody. the use of smaller antibody fragments, such as single chain fv fragments, half the size of a fab, may also be a promising approach because the single chain fragment retains affinity and has shorter half-life in the body. monoclonal antibodies may also help to alleviate poisoning due to environmental contamination. hexachlorobiphenyl, paraquat, atrazine, , , -trichloro- -pyridinol, the chief degradation byproduct of the insecticides chloropyrifos, triclopyr, and chloroprifosmethyl, and other chemicals may soon be detected and remediated with the help of antibodies. , [ ] [ ] [ ] antibodies may also be used in the treatment of poisoning due to paraquat, hexachlorobiphenyl, domoic acid [amnesic shellfish poisoning], and other contaminants that are otherwise difficult to remove from the body or surrounding environment. [ ] [ ] [ ] [ ] the primary target of many abused drugs is the central nervous system (cns), and immunotherapy against such chemicals must be able to penetrate the cns. pcp or phencyclidine (angel dust) is a type of arylcyclohexamine that affects multiple sites in the brain. , it is linked to violent psychotic episodes that are similar to schizophrenia. treatment is difficult because there is no known antagonist identified, pcp has a high volume of distribution, and about % of it is cleared after being metabolized. recently, mabs have been described that could bind to cocaine or pcp and act like sponges in the bloodstream to prevent them from reaching the brain. it has been shown that a single dose of antibody reduced pcp effects for up to weeks in animals, which might be equivalent to a several months in humans. high-affinity antibody b fragments against pcp function better in that the fragments with bound pcp are more quickly cleared from the body than the whole antibodybound pcp. the crystal structure of the antibody fragment complexed with pcp has been studied in detail. antibody fragments are also preferable to whole antibodies in the treatment of pcp addiction because of their lower antigenicity and improved pharmacokinetics. , anti-idiotype antibodies could potentially be produced against the drug-mab binding site and mimic drug structural features, although these antibodies do not cross the bloodbrain barrier. by increasing protein binding in the vascular compartment and lowering the drug's volume of distribution, the antibody acts like a pharmacokinetic antagonist. evidence that the antibody can also reverse effects of other potent arylcyclohexylamine drugs suggests that antibody medications can be used to treat different classes of drugs. , an anti-pcp igg has been produced from a hybridoma cell line using both ascites and bioreactor methods, and the pharmacological and immune specificity of the antibody has been confirmed by administering the anti-pcp fragment to rats in three different studies without producing observable effects in behavior. active immunotherapy against methamphetamine addiction is also being contemplated. antibody therapy for cocaine addiction is also a possibility, especially with the development of catalytic antibodies having enzyme-like characteristics that can be used to inactivate drugs. , [ ] [ ] [ ] antibodies with esterase activity have been successfully mimicked, and because cocaine is believed to be metabolized by in vivo esterases, such antibodies would be useful in the treatment of cocaine addiction. catalytic antibodies probably would not be as useful as high-affinity anti-cocaine antibodies, but could be used in medical emergencies or as part of a withdrawal therapy. potential antibody applications against cocaine addiction have been tested in rats with a vaccine approach by ( ) attaching cocaine molecules to a carrier protein for immunizing rats to produce antibodies against cocaine and ( ) humanizing anticocaine antibodies derived from rats and producing them in bacteria. , a catalytic mab ( a ) has recently been reported to attenuate cocaine's cardiovascular effects in mice. , conclusion about years have elapsed since edward jenner vaccinated a young child against smallpox. since that time, the field of immunology has evolved at a rapid pace and has yielded many critical developments. although vaccination has thus far proven to be the most cost-effective method of preventing diseases worldwide, the development of mabs that use the specificity of immunological responses is one of the most successful applications of immunology to date. chimeric and humanized antibodies have reduced the risk of allergenicity from exposure to nonself antibodies and heightened the clinical effectiveness of mab treatments. developments in radiology and pharmacology have allowed radiolabeled and immunoconjugated antibodies to be produced. antibody fragments, heteropolymers, and bispecific antibodies are now available in addition to whole mabs. these promising developments may soon allow mabs to be used to treat afflictions as varied as substance abuse, cancer, asthma, viral infection, septicemia, and poisoning. as heddy zola observed in , "the therapeutic application of mabs, whilst still limited in scope, promises to break its substantial shackles and realize the potential forecast by its proponents." on immunity: with special reference to cell life ehrlich's passion: the origins of his receptor immunology the natural selection theory of antibody formation a modification of jerne's theory of antibody production using the concept of clonal selection the hydrolysis of rabbit gamma globulin and antibodies with crystalline papain selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants continuous cultures of fused cells secreting antibody of predefined specificity with the benefit of hindsight antibodies in diagnostics-from immunoassays to 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antigen produced in transgenic plants tobacco can be good for you. the new york times rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants production of antibodies in transgenic plants a humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes production of a diagnostic monoclonal antibody in perennial alfalfa plants using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases points to consider in the manufacture and testing of monoclonal antibody products for human use guidance for industry. s preclinical safety evaluation of biotechnology-derived pharmaceuticals center for biologics evaluation and research and center for drug evaluation and research, us food & drug administration points to consider in the production and testing of therapeutic products for human use derived from transgenic animals points to consider in the production and testing of new drugs and biologicals produced by recombinant dna technology points to consider in the characterization of cell lines used to produce biologicals. center for biologics evaluation and research, us food & drug administration monoclonal antibodies: the second generation human antibodies by design genetically engineered antibody molecules engineering antibodies for therapy recombinant antibodies: alternative strategies for developing and manipulating murine-derived monoclonal antibodies monoclonal antibody-based therapy for solid tumors: an overview monoclonal antibodies: innovations in diagnosis and therapy unconjugated monoclonal antibody therapy of lymphoma monoclonal antibodies: the second generation. herndon (va): bios scientific preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by cdr grafting the promise and pitfalls of monoclonal antibody therapeutics radiolabeled monoclonal antibodies monoclonal 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to the c b receptor of human erythrocytes: a potential therapeutic treatment bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor facilitate virus clearance in a monkey model bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model the primate erythrocyte complement receptor (cr ) as a privileged site: binding of immunoglobulin g to erythrocyte cr does not target erythrocytes for phagocytosis antigenbased heteropolymers facilitate, via primate erythrocyte complement receptor type , rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys quantitative studies of heteropolymer-mediated binding of inactivated marburg virus to the complement receptor on primate erythrocytes antigen-based heteropolymers, a potential therapy for binding and clearing autoantibodies via erythrocyte cr infusion of bispecific monoclonal 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selfadministration model we thank carolyn m. black and the national center for infectious diseases editorial office for the critical review of this manuscript. key: cord- -f ciho o authors: nan title: tuesday plenary session tuesday: posters date: - - journal: vox sang doi: . /j. - . . .x sha: doc_id: cord_uid: f ciho o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases ed- - standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be g (fda criteria). however, actual measurements have found a mean hemoglobin content of . ± . g per unit, with variability between manufacturers. percent of units may contain less than . g of hemoglobin while percent may contain more than . g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be . to percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of . g/dl. for pc transfusion, about percent of transfusions achieve the targeted dose of - ¥ platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of g/dl rather than g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of % yielded the same outcomes as one of %. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below % were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of g/dl and unlikely to be necessary at a hb above g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: ( ) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; ( ) the quality of retrieved studies is assessed systematically; ( ) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and ( ) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. ed- - biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf- /cxcr pathway has been emphasized, either by down regulation of cxcr , changes in sdf- gradients, or by disruption of sdf- /cxcr signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf- /cxcr , disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla /vcam- pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt- l, il- ) or chemokines (i.e. il- , gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf- molecules (i.e. ctce ), or cxcr antagonists (i.e. amd ) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. ed- - the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by - fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by %. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. ( ) ann med : - ). determination of the coding sequence of all the genes giving rise to antigens within the blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl ( ) human blood groups, nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. ( ) transfusion : - ), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. ( ) transfusion : - ). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. ed- - the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd + t cells and class ii molecules present exogenous antigens to cd + t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 's and 's -protect oncology patients from clinically relevant bleeding in more than % of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below per microliter (or in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol ; : - . additional data on lumbar puncture are reported in ann hematol ; : - . another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about % to about % of patients. ed- - ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over % in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: . studies of interventions comparing ffp with no ffp; . studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; . studies of interventions comparing ffp with a different blood product; . studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range - patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication ) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in . first in the commission agenda was a recommendation on donor selection criteria, given in . then came the european blood directive / /ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over donations per inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of nd august which came into force as of january st . this act was then updated in november according to eu directive / /ec and came into force as of january th . this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of nd august , in force since january st , has been supplemented by decrees: . procedures for external bts audits; . requirements for donor selection; . requirements and procedures for organization and safe management of blood transfusion in hospitals; . requirements for implementing of national and regional donor registers; . employment criteria for bts personnel; . training requirements for hospital personnel involved in blood and blood product administration; . national, uniform price list for blood and blood products; . organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age years, range - ; median weight kg, range . - . kg) with solid tumors at onset or on relapse underwent collections with the p y kit of the fresenius hemocare com.tec blood cell separator. our cd + cells target was ¥ e /kg. collections were started if a peak of at least . e /l cd + cells ( per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio : - : was combined with heparin u/kg. in children with < kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in % albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of ml (range . - . ml) was processed in a separation time of min (range - min). the median product weight was g (range - g) and yield of cd + cells was . ¥ e /kg body weight (range . - . ¥ e /kg body weight). three poor mobilizing patients (peripheral blood cd + peak of - cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose ( . and ). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under kg. m-pa- alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in , aubuchon and birkmeyer published a paper (jama ; ( ) : - ) where they concluded that the cost was usd per qaly, which is far above the 'acceptable limit' of usd . this estimate was adjusted to usd . mill. per qaly in a letter to jama (jackson, jama ). in , riedler et al. published (vox sang : - ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp for neonates and gbp for patients aged . the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = ) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas ) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination ( . mmol/l), maximum storage time of whole blood before separation ( °c, h), maximum storage time of mb plasma before freezing ( h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+ . %), fibrinogen (clauss) (- . %), factor v (- . %), factor viii (- . %), factor xi (- . %) and protein c (- . %). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha- -antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment + , thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at °c for months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of < . mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within hours (usually - hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but : plasma-containing transfusions is the most commonly cited figure. in % of patients, recovery is well underway within hours, and leads to complete resolution. death occurs in - %. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: ( ) antibody-mediated and ( ) -hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than % of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in % these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since . since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past years, cases of trali and cases of possible trali have been confirmed by critical review of each questionnaire. of cases of definite trali, donor specimens were obtained in cases. of cases, anti-leukocyte antibodies were detected in cases ( %) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (< %). of cases of antibody positive donors, anti hla antibodies were detected in cases, anti hna antibodies were detected in cases, and both were detected in cases. of cases of positive anti hla antibodies, class i antibodies were detected in cases, class ii antibodies were detected in cases, and both were detected in cases. on the other hand, the anti-leukocyte antibodies were detected in % of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion ( %). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of adverse events analysed by the serious hazards of transfusion (shot) scheme ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , ( %) were haemolytic transfusion reactions (htrs). were due to incorrect blood component transfused (ibct): / were abo incompatible and / caused by other red cell antibodies. a further cases were reported as acute htrs (ahtrs; i.e. occurring within hours of transfusion) whilst were recognised more than hours after transfusion and reported as delayed htrs (dhtr). / ( %) patients died and suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. / ahtrs occurred in group a ( / ) or group b ( / ) patients given group o platelets. of the ahtrs related to red cell transfusion, were due to errors, were in patients with auto-antibodies, in only of whom alloantibodies had been adequately excluded/identified. at least / dhtrs were potentially avoidable; in cases the antibody was detectable retrospectively in the pre-transfusion sample; in cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. % htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in of patients ( %) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations - allogeneic from matched sibling healthy donors and autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf ( - g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was - ¥ e /l (autologous setting) or on the th day - hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was . - . l, and . l for one pbsc transplantation in average. results: using a minimal target dose of cd + cell count ( ¥ e /kgbm), performing one mnc-apheresis procedure for % recipients sufficient number of pbscs were obtained. the mnc yield was . ¥ e /kgbm in allogeneic and . ¥ e /kgbm in autologous setting in average. the mean cd + yields for allogeneic and autologous transplantations were . ¥ e /kgbm and . ¥ e /kgbm, respectively. hematopoietic reconstitution was achieved on the . th day for leukocytes and the . th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd + cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late s but it was only in the s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive / /ec off. j. european union. . . :l / -l / ) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa- years of shot data - : a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to deaths and cases of major morbidity in the uk between and . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that % of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for % of all pre-transfusion testing but % of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. % of hospitals reported that they participated in shot in , but only % of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa- passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b ) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds ( depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa- evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for days. red cell quality was determined by measuring the in vivo red blood cell recovery hours after re-infusion of the -day stored red cells. storage hemolysis and atp were also determined. methods: units of blood ( ml) were collected from normal volunteers into the leukotrap wb system containing cp d/as- anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within hours at room temperature (rt units), or after hours at - °c (cold units). the prion filter set was sterilely connected to the units on day , and the units were filtered and stored for days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with -cr radioisotope, re-infusion, and determination of the -hour in vivo rbc recovery. a donor sample was also labeled with m-tc to allow for red cell mass determination. thus, both single-and double-label -hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on units. the mean single-label -hour recoveries were . % and . % for the rt and cold units, respectively. the mean double-label recoveries were . % and . % for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the -hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than % of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp d/as- (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~ ¥ e platelets in ml of % plasma and % intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria ( , , cfu) were added to the double units. each double unit was divided into two identical products containing , , or cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process ( mm amotosalen + j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days , and of storage. samples ( ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at and cfu per product, strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and strains (e. coli, c. perfringens) tested negative after days of platelet storage, respectively. k. pneumoniae tested positive after - hours of culture when sampled on day of platelet storage for both and cfu per product. at cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after days of platelet storage. the anaerobic cultures of p. acnes became positive after hours of culture when sampled on day of platelet storage. of the strains studied, only s. aureus consistently tested positive after - hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire -day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv- and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from to showed a significant decrease from / and / before nat implementation to / and / after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is / , five times higher than hiv one and times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples / , / , and / , in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of days, ultrio assay is projected to close the wp by an average of days on undiluted samples, days in minipools of samples, days in minipools of samples and only days in minipools of samples. the projected yield calculated on the basis of . million donations collected per year in france, would be . unit per year for minipool-nat and to units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: of clinical samples ( . %) typed identically with this system and cat, and of the discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average genome equivalents(geq)/ml early in pregnancy to about geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t / being only minutes. in we have shown that this cell free fetal dna could be used for rhd genotyping. in the last years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over %, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only - % of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. ( ) transfusion ( ), ]. this system utilized low-speed centrifugation of -well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for . % of samples, compared to . % for cat (p > . ). when samples were tested for unexpected alloantibodies, fc determined the correct result for . % of samples, as compared to . % for cat (p > . ). this novel method was better than cat at detecting weak anti-a (p < . ) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (< bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = . ) but a better correlation was observed with the langmuir equation (mean r † = . ). in maternal sera, the mean ka of anti-d was . ¥ to the m- (from . to ¥ to the m- ). in the sera from immunized donors, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ) and in lots of anti-d ig, the mean ka was . ¥ to the m- (from . to . ¥ to the m- ). the comparison of anti-d affinity measured in cases of hdn in which infants presented a fetal anemia and in cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was . ¥ to the m- whereas in the cases of postnatal anemia the mean value of ka was . ¥ to the m- . conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa- alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa- alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, ; anti-hpa- a, ; anti-hpa- b, ; anti-hpa- a, and anti-hpa- b, ). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges . - . ). interestingly, we used lyophilized platelet with a high expression of cd bearing the hpa- system and we have detected anti hpa antibodies among sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa- system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between and g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between and g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for cases of cardiac bypass surgery. in addition, exclusive use of fuwb for burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa- transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention . the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from centres ( patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < . ) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < . ). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults ( ml/kg in premature; ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < ¥ cells/l if bleeding is observed; (b) a plt count < ¥ cells/l without bleeding; (c) a plt count < ¥ cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in children ( premature infants and at term newborns). the weight of premature infants ranged from - g and at term newborn from - g. gestation age of premature infants ranged from - weeks and of at term ones, of course, from - weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of - ml/kg, with a plt count < ¥ cells/l. results : in the study period, plt transfusions have been performed: children have been only transfused one time, while multiple plt transfusions (ranged - ) needed for children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome ( cases) , haemorrhagic syndrome without sepsis ( cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome ( cases). after hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all little patients. the highest increase in plt count was ¥ cells/l, while the lowest ¥ cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. % of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies fusion society of turkey (bbtst) in with contribution of blood transfusion centers. according to these figures % of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the blood banks performing apheresis, were university hospital blood banks. another blood banks were producing both productive and therapeutic, produce only productive and produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only centers reported to prepare random platelets as well. each center had apheresis machines between - . a total of centers was carrying out around < procedures, around - , at centers about - , at a further centers around - procedures a year (one center was excluded). of the responders to the survey , all procedure were done at blood banks, whereas at of them all were carried out by the hematology clinics. at other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: centers < , centers - , centers - and centers > units have donations a year. around % of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are % lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the hospital based transfusion services, which are financed within the budget of the whole hospital. the hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to % ( blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population ( in the north, in the central and in the south region). each sub region will have one standard mobile blood collection team to collect blood units daily, i.e. annually. the blood units per teams provide the blood units ( %) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p- years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in . since and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in , / . codex of voluntary blood donation and health care staff has also been established; blood donors donates blood annually. in the past years, there was over million blood donations, performed in accordance with who regulations. over transfusion medicine specialists and technicians specially trained for the work in blood transfusion service (n = ), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with . million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from . now, they have been regulated by tender. in , test to lues was introduced, to hbsag in , to a-hiv in and to a-hcv in . information system was introduced in . nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso implementation. in the current reform of transfusiology system we are aiming for percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde . the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in in stations of blood transfusion and in blood transfusion departments at big hospitals. amount of donors in was equal to , voluntary donors being . % of them. the average number of whole blood donations in relation to the general population is per inhabitants, and on average percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is . ml per inhabitant and ml per one bed. an average volume of one blood donation is ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about . % of collected blood is used for procurement of blood components and preparations, . % of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv- / , hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is . %. % of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: % albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first . cross-matches last year (in months) were found incompatible due to dat positivity of blood donors' red cells ( . %). dat positive [( +)-( +)] samples were only igg positive in cases, only c d positive in and igm positive and c d positive in case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c c, -c d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the donors that participated in the survey ( . %) answered the questionnaire, ( . %) men and ( . %) women. that the survey was useful thought % and % that it was not. opinions were elaborated by . %. that the questionnaire should be completed before each blood donation was the opinion of . %, % thought it should be filled out only the first time blood is donated and . % that the questionnaire should not be completed at all. the answers given were sincere in . % of blood donors, % were not and . % were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years ( ) ( ) ( ) ( ) ( ) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from . . till . . . statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total donors in nitm and ( . %) deferrals. . % of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in ( . %) blood donors, use of drugs - ( . %), low blood pressure - ( . %), high blood pressure - ( . %), infections - ( . %), cardiovascular diseases - ( . %) and others. relation voluntary/family donors is almost equal ( . : . ). in the last two years the number of voluntary blood donors is increasing ( : ), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~ %, which is result of insufficient data and it is increasing in the last two years (> %). reasons for deferral are predominantly from temporary character ( . %). permanent deferrals are only ( . %), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of % macedonians, % albanians and % others (serbs, gypsies, turks). over % of blood donors are voluntary non-remunerated and ~ % are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from till . results: there were blood donations for the mentioned period. predominant blood donors are employed and high school students in %. family blood donors are ~ %; between them % are from albanian population. the ratio between blood donors macedonians vs. albanians is : . woman blood donors are presented with %. first time blood donors are %, and regular donors arẽ %. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than %. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donat- conclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype b of hepatitis c virus is the most frequent in blood donors d, from a to f, from a to k, a and a. these are differently distributed in the world: types and are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from . to % of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may to december , blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: blood donors have resulted hcv-rna positive with identification of the following genotypes: a = cases ( . %); b = ( . %); a + b = ( . %); a/ c = ( . %); = ( . %); = ( . %); none was a or a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv- a has showed a double prevalence in men ( cases, . %) respect in women ( cases, . %), while genotype b is more frequent in women ( cases, . %) than in men ( cases, . %), moreover genotypes and do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt > iu, our results show that . : donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype b has resulted the most frequent in blood donors' population, followed by type , while the others have showed a very low prevalence. the high frequency of genotype in blood donors is explained by the observation that hcv is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype is principally linked to a not transfusion transmission way; genotype is linked to old age, to female sex and to post-transfusion transmission; genotypes and are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes and are still little known because extremely rare in europe. p- kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel . the kel (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family - ( . %) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger - ( . %). motives for their donation are the following: their wish to help the ones that need blood - ( . %), concern that some day everyone can be a potential recipient of blood - ( . %), because of offered benefits - ( . %), for a friend or relative - ( . %), care for their health - ( . %), because of citizen duty - ( . %), because the others donate - ( . %), curiosity - ( . %). they want to be invited every months - (%) students, every months - ( . %), every months - ( . %) and ( the mean age of case group was / ± / and the mean weight of them was / ± / , / % was male and the mean number of blood donation was / ± / . the mean age of control group was / ± / and the mean weight of them was / ± / . / % of them was male and the mean number of blood donation was / ± / . the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < . ). the rate of vasovegal rx was higher in blood donor (p < . ) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < . ) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than - and - whole blood units per year, respectively. we present studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. mg and mg daily combined with mg ascorbic acid over months (males) or months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (> mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period - . methods: the donation of whole blood and aphaeresis platelets from donors of age from to (max. years for each class) were count for the period of five years ( ) ( ) ( ) ( ) ( ) . the percentage of the man´s donation was calculated for each target class ( ) ( ) ( ) ( ) ( ) . results please see tables and . in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from to decreased during objected years. unfavourable state of total number of births in the czech republic ( birth in republic ( birth in , birth in ) and its decreasing tendency ( birth in !) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of women from the district of limassol (the second largest urban center of cyprus) between the ages of and . using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. % of women do not donate blood and attribute their reluctance to do so to health-related problems, while about % of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to % of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively and beat per minutes and blood pressures (bp) were both in the normal range ( / and / mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to / and / mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to / mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the - data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period ( ) ( ) , . percent of persons who presented for donation were deferred; . % were temporary deferrals ( % due to laboratory test results, among others low hemoglobin, . % due to risk of acquiring a transfusiontransmissible infection) and . % were permanent ( % due to the infectious diseases markers, . % due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption : percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure ( . % of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact - % of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia ( . % of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future ( - % of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn , abbott laboratories, usa), in a blood donor setting. methods: the nbm- device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of subjects ( females, males) aged - . subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm- device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device ( donors). hb levels were considered normal when readings were equal to or > . g/dl. results: venous hb measurements ranged from . - . g/dl. the mean nbm- hb level was . ± . g/dl, only . g/dl lower than the mean hb result obtained by venous sampling, which reached . ± . g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ± . g/dl. the mean absolute error (mae) of their difference was . g/dl. when checked against the cell-dyn in the usa center, where subjects had hb of . g/dl or lower, the nbm- and hematastat devices showed comparable sensitivity results. the nbm- using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the december to march were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: donors -control group: donors). computer records were evaluated during the next years ( march to march to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure ( %), deferred for medication ( %) and colds and/or sore throats ( . ). non deferred donors were a little more likely than donors with sttds to return over the next years ( . % vs. . % pv = . ) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers - ( %), while all other ethnic groups are present with only %. the most prevalent is the group of adults aged - ( - . %), with high school education - ( . %) and mostly those who donated blood - times ( - %). conclusion: having in mind that % of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with % in relation to the total number of blood donors surveyed in the period - . this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta -microglobulin (b -mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta -microglobulin (b -mg) is a low molecular weight protein ( daltons) and found in all biological fluids it is light chain of histocompatibility class -human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class -hla antigen plays a role in the elimination of the virus. method & samples: beta -micro globulin was measured in serum drawn from hbs ag positive blood donors include ( . %) female and ( . %) male in age between - years, and healthy ( %) female and ( %) male in the same age we detected serumic b -mg by enzyme immunoassay (ela). results: our studies showed b mg level increased in ( . %) hbs ag positive donor that was significant differences with healthy control (p = . ). conclusions: it seems that serum b mg is a good marker for hbs ag replication. the role of b mg in monitoring of response therapy needs to be more evaluated. and ( . %) were contributed by vd, rd and dd respectively. over the last / years, voluntary donations have shown a rising trend from . % to . %, where as rd ( . % to . %) and dd ( . % to . %) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd ( . % vs. . % vs. . %) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable ( . % vs. . % and . % vs. . %). hiv antibodies was found more frequently in vd as compared to dd [ . % vs. . % (p < . )] whereas, vdrl reactivity was lower in formal as compared to latter [ . % vs. . % (p < . )]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between and . results: during this period people volunteered to donate blood, out of which were allowed to donate blood, while were declined. out of the total number of blood donors were male and female donors. the reasons for declining potential donors were the following: . % had low levels of hb, . % were taking antibiotics, . % were ill, . % had low blood pressure, . % had high blood pressure, . % for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in and , but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. . the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. . the lack of motivation of the studied lot is another cause. the questionnaire was applied on a lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to , which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vÖrÖs*, c bercsÉnyi † , o petrÓ † , r jÁger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the . . in hungary. as the consequence of reorganization till . . , blood banks had been established instead of existing before, under direction of the hhnbts. the working profile of the regional blood centers and local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about inhabitants and hospital beds). local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually donors donate blood in this region. this donation activity covers about the % of all inhabitants. the acceptance ratio of the donors is good ( - % of the donors were deferred). there are hospitals in our region. the regional demand on rbcc is - . u/year, on ffp is . - . u/year and on pc is - . u/year. the poster shows the donor data and the details of blood application of this region since . p- implementation of rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pÉlissier and jl beaumont efs ile de france, paris, france background: single donor rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for rbc collection (weight ≥ kg, height ≥ cm, hb ≥ . g/dl, ferritin ≥ ng/ml for repeat rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the recruited donors, all donors were male, % were regular whole blood donors, % were regular whole blood or apheresis donors and % were new donors. the medical staff was trained on rbc collection with the sdr protocol and disposable set ln pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as to months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table . six percent of the donors had a ferritin level below ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: hematomas and citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of min). the results of the survey were very favorable as more than % of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: ( ) possible risk of infectious disease in seronegative study; ( ) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than % blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in (c = . ; p < . ) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs + apparatus were used for platelet apheresis. apheresis were performed between / / and / / . including criteria of donors are that estimated process time is smaller than minute and estimated postapheresis platelet count is higher than ¥ /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls ( procedures on patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count > ¥ e /l or blast count > ¥ e /l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was . %. tp-treatments ( procedures on patients with symptomatic thrombocythemia and/or platelet count higher than ¥ e /l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction ( . % in average) and with clearly noted clinical improvements, subsequently. tes ( procedures on patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from . % till . %. rbcx treatment ( procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with % albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of collections (range - ). the inlet blood flow ranged between . and . ml/min (median . ml/min). the median blood volume processed was ml (range - ). leukapheresis lasted a median of min (range - ). the median total nucleated cell yield was . ¥ e /kg (range . - . ), mononuclear cell (mnc) yield was . ¥ e /kg (range . - . ) and cd + cell yield was . ¥ e /kg (range . - . ). the median of mnc collection efficiencies was . % (range . - . ). in ( . %) patients, in only one apheresis procedure more than ¥ e cd + cell/kg were collected. during ( . %) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than ¥ e /l in cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately / pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a -year-old pregnant woman, alloimmunized to the hpa- a (p a , zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt . /mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was . ml in a short time procedure ( minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected ( . ¥ e ) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing . ¥ e each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from . /mmc to . /mmc and after a week the child had plt . /mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to - minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm ( - seconds), thrombin time ( - seconds), the dilute fibrin monomer complex ( - mg%), coagulation time ( - min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was - % of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of - procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: pts ( females, male), median age . years ( - ), affected with agvhd grade ii ( ) and extensive cgvhd ( ) gtx with median total granulocyte doses of ( - ) ¥ per gtx corresponding to . ¥ granulocytes/kg in children and . ¥ granulocytes/kg in adults. the wbc counts increased from baseline values of . ( - . ¥ ) g/l for both pediatric and adult patients to peak values of . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at one hour after gtx and to . ( . - . ) ¥ g/l (children) and . ( . - . ) ¥ g/l (adults) at hours after gtx. in out of patients ( %), the crp levels significantly declined ( ( - )%; p£ . ) during the granulocyte transfusion period; in almost all cases ( / ; %) after the initial or nd transfusion. thirty-eight patients ( %) were alive at day + after termination of neutropenia and gtx. patients without crp response to gtx ( / , %) and patients with severe viral infections / ( %) were not among the day + survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: of them with mcs p and with mcsplus (haemonetics), according to automatic standard protocols a p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was : ). most of the donors ( % men and % women) were patient´s relatives. half-hour before the initiation of the procedure, mg of calcium ( tablet cal-c-vita) were administered to each donor. the mean platelet yield was . e /unit. the overall rate of the donor related adverse events was . %. feeling faint was the most frequent event, which was occurred in . % of donations. hypotension and citrate related rates were . % and . %, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was . %. other adverse events were venipuncture related ( . %), machine related ( . %) and miscellaneous complications ( . %). ( ) plateletpheresis using the mcs p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. ( ) with the used acd-a-to-blood ratio ( : ) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. ( ) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis . from the donors immediately with the initiation of the procedure (citrated whole blood) and . from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap- c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs p device (p = . ). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a year old pregnant woman in the th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially mg/kg bw prednisolone for weeks and subsequently mg/kg bw for weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table ) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was . ml per apheresis. after day plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. again lead to a recovery of the patient which held on until day . conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: procedures were carried out with mnc program and procedures with autopbsc on patients with cgvhd. both hemoglobin increased from . ± . to ± mg/tu, k+ from . ± . to . ± . mmol/l, level of glucose decreased from . ± . to . ± . to . ± . mmol/l, ldh from . ± . to . ± . ukat/, lactate from . ± . to . ± . mmol/l, ph from . ± . to . . . the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was . ± . ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage and , in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/ - . background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the . to -fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in cases with bone marrow volume over ml, and cases with bone marrow volume under ml. fifteen of these cases were allogeneic, and were autologous procedures; a software uploaded with cobe pbsc coll vers . and (catalog no: - - ) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: - - ). results: the mean volume reduction was . % ( . - . ) for volumes over ml, and . % ( . - . ) for volumes less than ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd + cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of donors who donated platelets by apheresis procedure from january till april . procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than ¥ /l and the volume of the processed blood was not less than ml. according to histogram, the most frequent donors' initial platelet value ranged from ¥ /l to ¥ /l ( %). final concentration of the samples of tested donors ranged from . ¥ to . ¥ in the average volume of ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < . . leukocyte contamination of the final concentrate prepared without the filter ranged from . ¥ /l to . ¥ /l. presence of red blood cells in the final concentrate ranged from . ¥ /l to . ¥ /l. p- therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of diseases into categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in croatian hospitals in the period - , respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in patients suffering from various diseases. in ( %) patients ta was performed by membrane filtration, while in ( %) separation by centrifugation was used (table ) . according to the ctg, s of the aabb/asfa guidelines, ta was performed in ( %) diseases from ctg i, ( %) ctg ii, ( %) ctg iii, and ( %) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis ( %), collection of pbpcs ( %), sy. guillain-barré ( %), and plasmacytoma ( %). in ctg ii frequent indications were: poisonings ( %), systemic lupus erythematodes ( %), and rapidly progressing glomerulonephritis ( %), and in ctgs iii and iv: cytoreduction-polycythaemia ( %), thyroid storm ( %), gvhd ( %), and reumatoid arthritis ( %). ( ) the time spent for resolving h / , ( ) mtp / , ( ) discarded blood units / . iii group: wrong data input / , donor replacement / , marking errors / , error in determining blood group at the first blood taking / , errors in input medical consulting / and disregard of prohibitions / . the consequences are: ( ) the time spent for resolving h / , ( ) mtp . / . , ( ) discarded blood units / . conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso parameters (soon to be updated with iso ). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p- software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from to more than . indeed, bacterial detection is performed for pools of to platelet samples and to red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt . in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of different reports can be generated on the website as a pdf file. for this study, reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or , -dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) -phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis nd. edition. academic press, new york ; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from ml to a total volume of ml and tests were performed in microtiterplates. results: samples were analysed in hplc and modificated commercial testkits, another samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range - mm atp) and statistical analysis demonstrated a given linearity (r = . ). variability has been calculated as . % (intraassay; n = ) and . % (inter-assay; n = ). the hand-on-time calculated for samples has been decreased from . hours to minutes. at least the costs of atp-determination have been reduced from € . to € . per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: ( ) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. ( ) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about whole blood units were collected and processed by mda national blood services in [ ] [ ] [ ] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some different types of dm were defined. out of reports sent to the corresponding manufacturers for investigation, responses ( %) were received and analyzed. about % of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly % of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table analysis and characteristics of t ( ) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. ( ) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. ( ) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. ( ) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in / / and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso : standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of . million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of %. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of %. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of %, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min . %-max %). but because with vista any procedure brings between . and . products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between € and € (mean value €) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx- filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of units of red cells, measurements of volume, platelet concentration and platelet count/pool of ld platelet pools produced by a team of technologists and of ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by . % of participants. the remaining . % of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in . % and . % of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. % of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso -quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on / iso -quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into levels; -most severe, potentially harmful to patient. -severe, damage to process and result. -moderatly severe, damage to process only. -benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years - , events were detected and recorded in the blood bank, they comprised . % of all tests performed ( ). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level & , whereas technical events were mainly of severity levels & . analysis of event recurrence in the quality system revealed that % of events were resolved, whereas only %- % of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of . %. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: . administrative for defining the it security organization, the information security policy, and information security awareness and training. . physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. . technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: < . ¥ /l, leukocytes: < . ¥ /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv- group m arna, hiv- group o arna, hiv- arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv- group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing % of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives / /ec and / /ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since . blood samples are classified according to adr in division . under un as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. . of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso : standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso : standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and , dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on erythrocyte suspension (es) samples during days of storage at oc on days , , , and . for whole blood (wb) samples during days of storage, same parameters were measured on days , , and . the measurement of the complex impedance of blood samples were performed in the frequency range from khz to mhz. by using the a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and , dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and , dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and , dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that , dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of plateletpheresis procedures performed using two cell separators (cs baxter, usa, mcs p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs was . ± . ¥ and in mcs p, it was . ± . ¥ per unit, however, only - % of sdps showed wbc levels < ¥ . six of donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in , the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in , centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = . ), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured times in order to determine within-run imprecision. coefficient of variation (cv) calculated from consecutive measurements was . %. to investigate between-day imprecision of the hemocue method one sample was divided in aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was . %. during the period of validation measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were . % (low), . % (medium) and . % (high). in order to investigate possibility of batch testing, supernatants of different rbc products were divided in aliquots and measured periodically during -month period. cvs calculated from measurements of each sample were in range . - . %. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a . mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer ( wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the -year period ( - ) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w ' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in . purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is years, of which at least are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: . organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. . collection, processing, storage and transport of blood and blood components . laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) . clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is -year specialization in transfusion medicine, established years ago (over specialists finished this specialization till now). we have postgraduate studies in tm started years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of theoretical and practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory -month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished -mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at - % of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of - %). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than time in - years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost % from the general number of patients of hospitals with positive results at % from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to , whole blood was comprising the majority of transfused units (more then %) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized symposiums, national courses, national and international congresses since . due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to %. in most of the university hospitals and training hospitals this is around %. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for years. methods: this is a months programme which has basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost general practitioners are trained by this programme in the last years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was ± and postoperative ± mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived ( ± ) and survived patients ( ± ) was not significant (p = . ). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il- and anti-inflammatory cytokine il- in relation to clinical outcome and complications after cardiac surgery. methods: in participating patients blood samples were taken before and after surgery. using elisa il- and il- were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il- and il- were low. mean postoperative level for il- was ± and for il- ± . compared with patients without mods and without infections and survived patients only the il- was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il- were higher in patients receiving more then units blood transfusions compared to transfusions. conclusion: there is an association between mods and mortality and il- , not with il- . ld has no influence on mean levels of il- and il- . there is a correlation between transfusion of more then units and il- , not with il- . these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july to december . patients were enrolled within h of icu admission. follow-up time was days, hospital discharge or death, whichever occurred first. results: a total of patients ( male, female, mean age . ± . years, range - ) were included in the study. the mean hemoglobin (hb) level at baseline was . ± . , which level was descending during the study. overall . % ( / ) of the patients received one or more rbc units while in the icu stay (mean . ± . units per patients). the mean pretransfusion hb was . ± . g/dl and the mean time to first icu transfusion was . ± . days. more rbc transfusions were given in the first week of the icu stay ( units vs , , units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = . , p < . and r = . , p < . respectively). baseline hb level was significantly related to the number of rbc transfusion (r = . , p < . ), but was not an independent predictor risk factor of length of stay or mortality (r = . , p > . and r = . , p > . respectively). the mean baseline apache ii and saps scores were . ± . and . ± . respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of < g/dl ( . ± . vs . ± . and . ± . vs . ± . respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < . ). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population ( . mill) the trauma team performed initial assessment and resuscitation of patients, % males, during the first six months of . the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: units of erythrocytes (er), of thrombocytes (thr) (buffy coat preparations from donors) and of s/d-treated whole plasma (op) (octaplasÒ) were transfused in uuh during this period. . % of er were given to surgical, . % to medical and . % to gynaecological and obstetric patients. % of er were given to patients above years. er units were given to patients (mean . units/patient; range - ). mean age of trauma patients was ± , median , range - years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb < g/dl, irrespective of age and sex. eighty-eight patients ( . %) received er, of them as massive transfusion ( er units in < hours) ( . %), of these died ( %). altogether trauma patients received units of er ( . % of total uuh consumption), . thr ( . % of total) and units of op ( . % of total). massive transfusion consumed er ( %), . thr ( . %) and op ( . %) units. fourteen adult trauma patients ( . % of those transfused) received or er units only. lowest pre-transfusion hgb was . - . g/dl, median . , mean . ± ; highest post-transfusion hgb . - . g/dl, median . , mean . ± . five patients received er transfusion at higher pretransfusion hgb level than g/dl, but in er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for > days, while only % were issued before days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year . in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of - mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved health institutions across the country. as many as deliveries and abortions were reported in . the institutions reported the usage of doses of mg rhd gamma globulin or doses/ deliveries + abortions. we compared our data with those obtained from similar surveys conducted in , i.e. before the issuance of the guidelines. in that year deliveries and abortions were reported, and doses of rhd gamma globulin or doses/ deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of - beds, general hospitals with the capacity of - beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/ deliveries + abortions in the first category was - with the median being ; in the second category there were - doses with the median being , in the third category - doses with the median being , while in the fourth category the range was - doses with the median of doses. the number of pregnancies which should have been protected with rhd immunization in year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = . ¥ . = . . if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in , doses of rhd gamma globulin or mg/ deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in , the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in a study was carried out regarding the usage of plasma derivates. as many as health institutions took part having the capacity of acute beds out of a total of . the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of - beds and general hospitals with the capacity of - beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in . the consumption of albumin was . kg or kg/million inhabitants in and consumption per bed for the first category institutions was in range . - . kg with the median being . , for the second category the range was . - . kg with the median being . , while for the third category the range was . - . kg with the median being . . in in croatia the consumption of albumin was . kg or kg/million inhabitants. the consumption of i.v. gamma globulin was . kg or . kg/million inhabitants in and the consumption per bed for the first category institutions was in range . g- . g. with the median being . , for the second category was in range . - . g. with the median of . and for the third category was in range . - . with the median . g. in in croatia the consumption of i.v. gamma globulin was . kg or . /million inhabitants. the consumption of the concentrate of factor viii was . iu or . iu per inhabitant in . . iu, i.e. . %, was a recombinant factor viii. in the consumption of factor viii was . iu or . iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased . times and the use of the concentrate of factor viii increased . times if compared with the results in . self-sufficiency has not been reached in plasma derivates in croatia we needed l plasma for gamma globulin and l plasma for concentrate of factor viii for the level of usage of these plasma derivates in . significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than % following transfusion with rhd+ rbc's and as many as % in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital ( beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby . rhd-patients, from to , were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the . rhd-patients we found out identified anti-d, % ( ) detected before any transfusion in our hospital. of the left, nine were excluded because no information was available during some years. had history of pregnancy related to the immunization. patients were exposed to rhd+ blood components: were transfused only with rhd+ platelets (including two women of childbearing age); with rhd + rbc's. the remaining had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period ( ) ( ) ( ) ( ) ( ) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total hospitalized patients who have been analyzed, ( . %) received wb and bp, and there have been transfused units wb and bp. every patient, on an average, has been transfused with . units wb and bp. at the same time ( . %) units have been transfused as a wb, ( . %) have been transfused as red blood cells (rbcs) and ( . %) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during - and to compare them with other studies. one hundred sixty five ( ) unselected patients of whom ( %) were women and ( %) were men with a mean age of years (range - years) were studied retrospectively. seventy six ( %) patients had intracapsular fracture and ( %) intratrochanteric fracture. the mean hb concentration on admission was . g/dl (range . - g/dl) and the mean hb concentration at discharge was . g/dl (range . - . g/dl). a total of units of rbc were transfused (a mean of . units per patient). blood transfusion occurred in patients ( . %), ( - % in other studies) with a mean of . units per patient (range - units), ( . - . in other studies). patients with preoperative hb values < g/dl were transfused more often than those with hb values > g/dl ( % versus %) p: . . women were transfused more often men ( . % versus . %) p: . . patients aged > years were transfused more often than those aged < years ( % versus %) p: . . finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures ( . % versus %) p: . . conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb < g/dl, in patients older than years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of . we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine %, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) . % and trauma/surgery . %, cancer . %. summary/conclusions: the use of ffp corresponds to . % of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from to in our hospital. results: our data are presented in table . conclusion: after , wb usage rates systematically decreased from % to . %. however, in the period of - , the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about % of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since . background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® pre and post a platelet transfusion (n = ) and was compared with healthy controls (n = ). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = ). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < . ). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years ( ) ( ) ( ) , units of wb were collected, among them ( . %) units were collected for pre-storage filtration and in ( . %) of these qc was done. within hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at g for minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume ± . ml ( % corresponds with the guidelines of council of europe), hematocrit . ± . ( %), hemoglobin . ± . g/unit ( %), number of residual leukocytes . ± . ¥ ( %), hemolysis . ± . % ( %), the test of sterility was % negative. during filtration of wb, wbc were removed in approximately . %, the duration of filtration was ± minutes and the loss of hemoglobin was . ± . %. the pre-storage filtration of wb is highly efficient, . % of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from , when the cardiosurgery department was founded in our hospital, to . the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from to were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs - and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about - . % were produced with the use od these filters, and platelets from - % with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts ( ¥ cabg and/or valve; ¥ re-cabg and/or valve; ¥ valve with or without cabg). oncologic surgery patients were also included in three rcts ( ¥ colorectal cancer; ¥ gi-oncology or vascular surgery; ¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in or more of the rcts. multivariate analyses were performed on: in-hospital mortality, -day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from surgery patients were collected ( onco; cardiac; vascular; orthopedic, other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = . ) and at days post-surgery (p = . ). no association between randomization and stay in hospital (p = . ) or stay on icu (p = . ) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = . ), and 'hospital' to be far stronger associated (p < . ). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = . ) and the trial arm became more strongly associated with infections (p = . ). in the analyses of surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = ) and portugal (n = ). in november a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was % (uk) and % (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by % in wales (n = ) and % in england (n = ), but only % in scotland (n = ), % in northern ireland (n = ) and % in eire (n = ). in mainland europe, variation was also observed: % in denmark (n = ), % in portugal (n = ) and % in other countries (n = from countries). fewer laboratories selected c-blood for pmfs: % in england and northern ireland, % in scotland and eire, rising to % in wales. mainland europe showed similar variation: % in denmark, % in portugal (all ccee matched) and % in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged < years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a year old female (with pre-existing antibodies other than anti-k), from % ( ), % ( ) to % in . in this survey, k + blood was selected for a female aged (no antibodies) by %, whilst % selected a k + unit for a female aged (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the and year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data ( ) suggested that > % laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec- ) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c fresh showed ± and after hours storage ± % expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib- ), low (gpib- ) and in-between (gpib- ). the binding and phagocytosis of plt showed an increase of ± % which implicates an indirect and negative relation to gpib- , and direct relation to gpib- expression. anti-human pselectin (cd p) delayed ± % the binding and annexing v ± % the phagocytosis of stored plts, after -hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a -year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured g/l. he received ml . % nacl and ml ringer solutions. after that, he was transfused with units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for hours. two days after the patient was operated. after surgery he was transfused with units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for minutes at °c with fmlp, lps or pma. monoclonal antibodies against hna a and hla-i, or patient serum were incubated with whole blood for min. surface cd b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna a monoclonal antibodies induced neutrophil activation, which were also inhibited by p map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: out of transfusion reactions were febrile, were anaphylactic, were due to circulatory overload, out of transfusion reactions concerned the transfusion of incorrect blood component. out of transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)- beta, il- and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il- has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il- from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from blood donors and frozen at - °c. upon thawing, the level of the il- beta, il- and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il- beta was at the test detection limit in all donor plasma samples. in ( . %) bd, the level of il- was . pg/ml, exceeding the test sensitivity limit of . pg/ml. in ( . %) bd, the level of tnf-alpha was within the range of - pg/ml, with a test sensitivity limit of pg/ml. tnf-alpha levels > pg/ml were measured in plasma samples of ( . %) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il- and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao = mmhg, o saturation = %), hypotension (bp = / mmhg) and fever ( °c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp ( cm h o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for hours the patient improved significantly, was extubated and returned to the surgical unit on the th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a -year old female with a history of multiple abortions whereas the prc unit was from a -year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years - there were . blood components issued in slovenia and atrs were reported ( in blood components issued), in of them the severity grade was ( in . blood components issued). in the year there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from in to atr in blood components issued. . % of all atr were not classified. conclusions: although the number of reported atrs was increased by % and % a year in and respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, . years old, with average weight of kg were used. the animals were dewormed by albendazole ( my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. ( ) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed ( . %) out of ( . %) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm ( . %), igg ( . %) and iga ( . %). ( . %) out of patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa- a in a homozygous hpa- b patient. in a minority of cases the offending antibodies were directed against hpa- b, - a, - b, a, - a, - b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a -year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a -year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a -year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa- a, anti hpa- b and anti hpa- b, respectively. the platelets genotype of the first patient was hpa- b/ b, of the second hpa- a/ a and of the third patient, hpa- a/ a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in . the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: ( ) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. ( ) to use the information acquired to determine the morbidity of transfusion. ( ) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. ( ) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: ( ) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. ( ) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. ( ) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: ( ) the total number of reported cases has steadily increased since the introduction of the programme. ( ) the number of participating healthcare institutions has also increased to % (n = ). please refer to table entitled 'summary of the haemovigilance report - ' . ( ) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. ( ) there has been very good participation from the participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. ( ) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing ( %) were rated for seriousness. of these, ( . %) were rated as grade (moderate to serious morbidity) or worse. ( . %) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in ( . %) and as 'possible' in ( . %). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use ( . per the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested > months following the implicated unit. results: between between - suspected pth cases were reported: ( %) were pth-b, were pth-c ( %) and in patient both hbv and hcv infections were reported. investigation was completed in / ( %) cases, with % of the involved donors ( / ) being retested > months after the implicated donation. hbsag was not detected in any of the retested donors. anti-hbc was detected in donors involved in pth-b cases of which were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 'anti-hbc+ only' donors, and none was found positive. only in / donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in . in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in % ( / ) of the cases where the patient received up to blood components. conclusion: in the past years an average of cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in % of the reported cases. so far, there was no clear evidence of hbv transmission. in cases (out of . million blood units collected nationwide during - ) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. - . , p < . ). the seroprevalence for a-hiv in the bd population was . % (se: . ; ci: . - . ; p < . ) whereas in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-hiv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was . %. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was . , which indicates that the number of bd a-hiv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was . % (se: . ; ci: . - . , p < . ), in the a-hbc positive bd was . % (p < . ) and in the a-hbc negative bd was . %. from a-htlv positive donations, were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was . %. the rp for a-hbc as regards a-htlv was . , which indicates that the number of bd a-htlv positive is . times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. . despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. . our data show that the real anti-hcv prevalence of our donor population is very low ( . %). . the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. . given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. . donor re-entry in the pool of donors is an issue for further discussion. . the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: . to recognize the epidemiology of hepatitis c and how it differs geographically; . to investigate the risk factors for presence of anti-hcv antibody in blood donors; . to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january , using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was . % out of blood donations. the average prevalence of hcv infection by riba was . %, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was , with cases in males and cases in females. most infections were found among older persons ( % were aged - , and % aged - ). the seropositivity was higher in family/replacement donors ( %) than in volunteers ( %). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion %, hospitalization %, immigrants %, occupational %, sexual transmission %, injection drug use %, household contacts %, other %, tattooing %, unknown %. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested sexual partners and family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using % trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in ) the risk of bacterial contamination, ) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and ) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year . results: in the attached table the estimated risk reduction through % trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by %, the risk of known viral infections by %, and transfusion acquired bacterial infections by %. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly ( : ) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table shows the distribution of the blood donors in different professions. administrative clerks with ( . %) and the workers with ( . %) registered in the group of hbsag(+) blood donors, as well as workers with ( %) and administrative clerks with ( %) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = and p > . there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = . and p < . shows a significant difference, where the workers with ( . %) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table . in the group of hbsag(+) blood donors the number of those who work in production profession- ( . %), is dominant over the number of those that has non-production profession- ( . %). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c = . and p < . there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from confirmed hbsag(+) blood donors and confirmed hbsag(-) blood donors as control group. results: the table shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group ( %) donors that live in a flat dominate compared to ( %) of those that live in a house. having in consideration the presented distribution (table ) for c = . and p < . there is a significant difference, that comes from the bigger number of blood donors ( ) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table ) , we can show that ( . %) of the hbsag(+) donors have less than m living space, in compared to ( %) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between and m , and between and m of living space per person, obviously are not very big. for u = and p > . there is no significant difference in the number of the donors in the two groups, when the available living space in m is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in from cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table shows the distribution of blood donors concerning the use of condoms. table : in the group of hbsag(+) blood donors those who have not used condoms dominate with number of ( . %), in compared to those ( . %) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom- ( %), compared to ( %) who have not. the given distribution of blood donors concerning the use of condoms for c = . and p < . , shows significant difference, which is due to the prevailing of the number of blood donors ( ), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the th- st day and th- th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of - °c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, specimens ( . %) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were . %, . %, and %, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and of them were found positive for one of the three viruses (hiv- , hcv, hbv). with the elisa/axsym assay of the blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those blood units units were given for transfusion following our blood centre protocol and the remaining units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those that were found positive by the procleix ultrio assay was positive for anti-hcv and were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day or and, in case of negative result, prolongation of their storage time to days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to days at to °c with end-over-end agitation. other plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day , and . both aerobic and anaerobic culture bottles were inoculated with a -ml platelet sample. culture bottles were incubated at °c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of plt concentrates were cultured and bacterial contamination was assessed in each unit at day , and after collection. on of storage day two out of ( . %) plt units were found to be positive for bacterial growth. cases of unconfirmed positive results were noted at the beginning of the study. out of the other units which were negative on day and continued to be cultured for the next days, the assessment at day found no other positive. after further storage, at day , defined as the end of the prolonged incubation period, out of the plt concentrates ( . %) grew bacteria although testing of the same units on day and gave no signal. from the platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to days. bacterial contamination testing on day and a storage time of maximum days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from - were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of platelet products were tested during the year period. thirty ( . %) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in ( . %) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with - units of concentrated red cells, depending on their problem. a total units were delivered from the beginning of their problem until december . the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: patients anti-hbc (+) and anti-hbs (+). patients anti-hbc (-) and Ánti-hbs (-) patients anti-hbc (-) and anti-hbs (+) patient anti-hbc (+) and anti-hbs (-) patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is % safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables and ) . for hiv and hcv the specificity was . %. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in % plasma and % ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: - hour after donation, about ml of pc were sampled into the ebds collection pouch and then incubated at °c under continuous agitation (incubator helmer ) for hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso ). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency and run control panels. ( . %) out of samples were positive for anti-hbc. ( . %) out of anti-hbc positive samples were hbsab positive, and ( . %) were hbsab negative. all of samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from . ng/ml bbi to . - . ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than . and . ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in patients referred during the aug. to dec. . all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. ( . %) were hbv dna positive and ( . %) were negative. ( . %) out of patients had evidence of delta infection and ( . %) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in out of hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in out of hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that . % of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker ( . %) than those with hbv dna (pcr) positivity ( . %). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested sera samples from the general population and 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv- , hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b (pb ) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb and hav in portuguese blood donors. we gather, during four months, plasma donations and joined them into pools, with no more than donations each. [ ] [ ] [ ] [ ] [ ] in voluntary donors the anti-hcv prevalence ranged from . % to . %, in family replacement donors from . % to . %, autologous donors from % to . %. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is . % and women . % (p = . ). over the year periods the prevalence in men has a decline tendency ( . % to . %; p = . ) and increasing tendency in women ( . % to . %; p = . ). according to age group the anti-hcv is . % in - age group, . % in - age group, . % in - age group, . % in - age group (p = . ). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [ ] [ ] [ ] [ ] [ ] . a total ftd and ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the -year overall hbsag prevalence among first time blood donors was . %. and ad . %. among autologous blood donors was observed a decreasing hbv prevalence from . % to . % in . according to age the prevalence was higher in - year group . %, while according to sex was higher in man ( . %) than female . % (p < . ). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from - hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. ( : ) . the hbv dna-emia in hbsag negative samples was . ¥ - . ¥ copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high ( . %) therefore the decision to introduce routine hbv nat screening is justified. ( / ) with stored apheresis and whole blood derived platelet concentrates. of these failed results there were confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing / . the bacteria detected were staphylococcus or streptococcus sp. of the fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing . % or / , and were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing / . there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of / , and with a low false positive rate (< . %). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only . % of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot . (genelab diagnostics) results: results from a total of screened donors were analysed. hepatitis b surface antigen rates was . %; anti-hcv seropositivity was . %; anti-hiv seropositivity was . % and tpha seropositivity was . %. one study calculated this risk to be one in for hbv, one in for hiv and one in for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in - was . %. but it had decreased to approximately . % in . anti-hcv positivity has been reported to be . % between and . but it was approximately . % in . rpr positivity in blood donors in turkey was reported to be < . % in and . % in . in , the rpr rates was . %. in our study these rates are . %, . %, . % and . % respectively. anti-hiv seropositivity was found around . introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn , tpn and tpn ) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be %, after testing specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over titer by tppa). the specificity generated from testing hospitalised patients previously screened as tppa negative, was . %. testing a mixture of sera and plasma from random donor specimens, generated donor specificity figures of . %. the precision (cv%) with a positive control was . % ( % confidence interval: . - . %) by the standard -day nccls analysis (ep a ). in a study conducted at asahikawa medical college hospital, in which, positive and negative specimens were tested, concordance with fujirebio tppa was determined to be %. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of - days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns , ns ) and a sandwich test for core antigen detection. results are available within . hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of days earlier than the monolisa hcv plus v test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around . days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns , ns ) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products ( ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools ( bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was . ¥ e /pool with mean leucocyte count . ¥ e /pool. ebds installation and training occurred over a day period. platelet pools were tested for bacterial contamination over the subsequent weeks. ebds pouches were sterile connected onto platelet pools hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for hours on a shaking agitator at °c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, technician was able to make oxygen measurements in less than minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. ( ) was . the percentage collected from volunteer blood donors was % (n = ) and the rest % (n = ) was given from patient-related donors. the age of donors ranged from to years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of tested units, of them were found to be positive to at least one marker of hbv infection, that means the . % of the health adult population was infected in the past by the hbv. the . % (n = ) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and . % (n = ) were chronic carriers of the hbv with hbsag(+). the . % (n = ) of the positive donors were patient related donors and . % (n = ) were volunteer donors. in other words, of the not volunteers ( . %) and of the volunteers ( . %) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions ( . %) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, . % compared with the . % of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type , hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv- , hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv- (n = ), hcv (n = ), and hbv (n = ) seroconversion panels were tested neat and diluted ( : and : ) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv- , hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p- effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv- /hcv and hiv- /hcv/hbv (procleix ultrio) assays for the detection of hiv- rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, out of ( % of the positive, . % of total) were reactive for hcv rna only and out of ( % of the positive, . % of total) were reactive for hiv- rna only. none were positive for both hiv- and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv- /hcv assay. of the samples tested by the ultrio assay, ( . %) tested reactive for hiv- /hcv/hbv. in discriminatory assay testing, out of ( . % of the positive, . % of total) was reactive for hiv- rna, out of ( % of the positive, . % of total) were reactive for hcv rna, and out of ( . % of the positive, . % of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than virus. three out of positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in ), and hcv testing (introduced in ). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of patients tested, cases ( %) were hbsag positive, cases ( %) were anti-hcv positive, and cases ( %) were anti-hdv positive. co-infection of hbsag and hcv was found in cases ( %), whereas co-infection of hcv, hdv, and hbv was found in persons ( %). the highest rates of infections and coinfections were found in patients above years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: / ( . %) samples from rbd were anti hiv + nonreactive and rr for p ag both being nonreactive in the neutralization test, they were interpreted as false positives. / ( . %) sample from fbd was rr for p ag/anti hiv + nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion days later: p ag nonreactive, anti hiv + reactive and western blot positive. the only bd p ag positive/anti hiv + nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~ days), this suggests that some - infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in , the general incidence in rbd has dropped from . ‰ to . ‰, suggestive of a : escape rate. the prevalence in ftbd has stabilized at ± ‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that potentially infectious donation in rbd escapes the screening (= to a total of aprox. , annually). a limited investigation using hcv-antigen eia evidenced a ‰ escape rate in ftbds (= to a total of aprox. , annually table and are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than hours. background: the hbsag, anti-hcv, anti-hiv / , p antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: . hcv rna pcr testing in anti-hcv negative blood donations; . correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of samples. pools of ml were tested using the cobas ampliscreen hcv test v. . (roche). results: pools of samples from a-hcv serologically negative donations were tested from october to july . no one pool was initially reactive. invalid tests: ( . %) run failures were observed, due to: invalid internal controls ( . %) and invalid positive controls ( . %). invalid tests were repeated. in none of pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b , such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b by nat since april . methods: over the last years . million donations were screened for b by nat. samples with a virus load over iu/ml were defined as positive, whereas samples with a virus load between the detection limit ( iu/ml) and iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of b positive donors (case group) were determined over a time period of one year. virus load and b antibody status was compared with b negative donors (randomised control group). b antibodies (igg vp , igm vp , ns ) were analysed by two commercial antibody tests. results: overall b nat-positive donors were identified with a virus load over iu/ml out of . million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp igg was detected in . % and % of the case and control group, respectively (p = . ). these data demonstrated statistically significance (p = . ). all donor samples which were b nat positive for more than three months developed neutralizing vp antibodies. in contrast, ns antibodies were observed in % of the case group and in % of the control group (p < . ). ns antibodies were detected more frequently in samples, which were b nat positive for more than six months. conclusion: b nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below iu/ml. these data support our testing algorithm all components of high positive donations (virus load over iu/ml) were discarded. donors with ns antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: . ( . %)cases turned out negative with both technologies; . ( . %) cases were positive in both methods; . cases were positive only using tmpa recombinant [of which ( . %) were confirmed positive by tpha/fta abs. as seen we found samples ( . %) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the ¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between : to : per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least ¥ cfu. the positive control has to be stored at room temperature and is stable for years. after dilution in pbs, the preparation has to be used within hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was . in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per million transfusions, the risk for bacterial infections is estimated to be : to : . especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of cfu/ml to cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of cfu/ml and cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at °c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between cfu/ml for e. coli and cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation ( - h) at °c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within minutes when the sample was taken hours after spiking. background: since year our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from ml plasma by using either the qiagen biorobot and qiamp virus biorobot kit or the manual extraction with cobas ampliscreen hcv pcr kit v . . results: between march and december a total of seronegative donations ( pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs samples in hours where the manual method needs hours for samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than samples/day while preserving the availability of blood products. anti-hcv similarly was high till ( . - . %), but in trend to decrease afterwards ( . %). anti-hiv reflected the low endemicity of the disease in public setting and was % through the mentioned years. rpr test for syphilis was around . %. directed donors were % of all and volunteer donors consisted nearly %. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since . from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was of (% ) were female and (% ) male in age ranged from to years. results: sera tested, ( %) were found to be positive for toxoplasma igg antibodies and ( . %) were igm antibodies positive and of them ( . %) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group to years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are patients suffering from thalassemia major who are aged between and , and who have been receiving erythrocytic transfusion - times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia . ). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least transfusions a year. the tests in all patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years ( ) ( ) ( ) ( ) ( ) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv / was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv / positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: ( ) we observe that there is a decrease in all four infections. ( ) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches % in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least out of the estimate of first-time donors, involving blood transfusion centres from of the italian regions. each centre was required to enrol all first-time donors born before december st, , and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first months of the study the sites saw almost first-time donors, of whom . % belonged to the required age groups. among eligible donors, . % were both hbsag and anti-hbc positive, and . % were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, % were positive for anti-hbe, % had raised alt and % were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, % were negative and % were positive for anti-hbs. among anti-hbs positives, % showed values < miu/ml and % > miu/ml. the avidity index results suggested that approximately % of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately % of the italian first-time donors are older than years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that . % of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly % of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv / and tp ab. to c + ns + ns , ( . %) to c + ns , ( . %) to c + ns + ns , ( %) to ns + ns + ns , ( %) to c + ns + ns , ( %) to ns + ns . the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients ( . %) had anti-hcv ab for or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv & , anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october and january . the multi-marker qc sample 'pelispy s type ' (s ), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > ) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s lots were exported from edcnet and analysed. results: nearly results were submitted: all results were reactive (s/co > ). fifty of these results ( . %) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = ) or due to unacceptable results (n = )]. a further results were excluded because data provided by laboratories were inconsistent or incorrect. a total of results, reported using different prism reagent lots ( for anti-hiv, for anti-hcv, for anti-htlv and for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of < % for all s lots. results from prism anti-hcv and anti-htlv produced cvs between . % and . % for all s lots. data reported for s lot ps (n = , range for anti-htlv to for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with < % bias for the prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a + % bias. prism anti-hcv showed the greatest variability within reagent lot with results from of reagent lots showing a cv between % and %. conclusion: in results in a qc sample distributed to laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in , the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv- rna screening of all blood donors started late . aim of the study: to implement hiv- rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv- rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer ( ml/ml of each). from the final volume of the nucleic acid eluate ( ml) ml was used for the detection of hiv- rna (roche cobas ampliscreen) and ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv- rna ( iu/ml) and hcv rna ( iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv- rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = ) and minipool samples (n = ). results: detection limits of the hiv- and hcv assays ( % hit rate) were calculated to be . iu/ml and . iu/ml respectively. specificity for both assays was % and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of was below the recommendations by the council of europe for blood donor screening (for hiv- rna iu/ml and for hcv iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv- nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of donations, ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • out of donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in to the relevant institutions of the eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the - age limits for blood donation ( - for a first donation) were the rule in countries. in other countries the age limit could be brought forward to and extended to years old. the time interval between donations was identical for men and women in countries, and varied from to weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the / /ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be % correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from to months. previous transfusion history could not be a ci or could be one varying from months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the /ec/ directive on these practices in the eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average whole-blood donations are performed per inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection . % -bruise assessed using post-donation interview . %, sore arm - . %, nerve irritation . %, arterial puncture/pseudoaneurysm/arteriovenous fistula . - . % etc) are most commonly reported, while negative systemic reactions (vasovagal reactions . - . %, syncope . - . %) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol ; : - .) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare ( . %). fatigue ( . %- . %) and diminished physical working capacity ( . %) are reported to occur during days after the donation. a recent study of a consecutive sample of swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - .) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common ( % of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; % of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang ; : - ). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa- establishing a national adverse event reporting system for blood donors -a prospective study of . there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: . to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; . to define and categorise adverse events; . to record data systematically and prospectively using the existing computerised donor database. methods: from summer , a small project team of senior clinical and operational staff took months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over staff working on mobile collection teams, static sites and blood centres. daer was fully implemented by january . adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first months . million donors attended sessions throughout england and north wales. results were very consistent month on month. donors ( : ) had vasovagal symptoms but only % of these suffered syncope. % of all vasovagal reactions occurred in women. % occurred in donors aged - and a further % in donors aged - . (donors aged - represent only % of the total donor base.) donors ( : ) reported a delayed reaction, % of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in donors ( : ) and, more rarely, arterial puncture was diagnosed in donors ( : ). bruising was reported after the session by : donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about % of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only % of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around % of the global blood population has access to only % of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population ( participants). the survey was performed in november . results: five percent reported being active donors (had donated during the last months), % were passive donors (had not donated during the last months), % were non-donors with a positive attitude towards becoming donors, and % non-donors with no intentions ever to donate blood. in the youngest age group (age - ), % reported being active donors and % were passive donors. however, % of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and % reported they did not care about blood donation. in comparison only % of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and % reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion ( %) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: . a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. . specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. . consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to first-time and repeat blood donors, in order to be used as an indicant of donor's satisfaction. . first-time donors increased . % in the months of application. among them, . % were donors 'for relatives or friends' turned into volunteer donors and . % were first-time volunteer donors. the questionnaire analysis further revealed: • % were motivated by the use of magnetic card. • % appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • % were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • % considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. . the turnover of repeat donors also increased . % after replacing their plain old paper cards with new ones. further analysis revealed that: • % appreciated the quick cross-checking of donor's identity. • % were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in , greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa- emerging technologies in transfusion. dna based assays until the late s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short ( - nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing donors were genotyped for hpa- through - and ; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and , all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested ( epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons , , , , and . in all known weak d types, all six rhd specific exons are amplified (except for weak d type which lacks rhd exons and ), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between and , samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types ( t > g), ( g > c), ( c > g) and ( c > a). weak d type was identified by the combination of serology and absence of exons and by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all samples were subjected to the rhd mpx pcr and sample showed absence of rhd exons and , indicative of a weak d type when combined with serology. the remaining samples were analyzed by the weak d pcr-ssps, resulting in weak d type samples, weak d type samples, weak d type samples and weak d type sample. two samples remained undetermined and were sequenced for all rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau partial d phenotype ( g>a, g>a and c>t). the other sample had only one, not previously known mutation ( a>t), which is located intracellularly at the coohtail. extensive serology using the epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type . conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types , and should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, out of patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a . % incidence of anti-kel is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel acquired by transfusion/previous pregnancies: st case: in july , a -year-old woman (gravida , para ), rhdnegative, kel -negative was referred at weeks gestation. the father's phenotype was rhd-positive, kel -positive. a maternal antibody screen revealed d and kel alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron and exon of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel homozygosity; nd case: in august , a -year-old woman (gravida , para ) was referred at weeks gestation. she had a history of transfusion with rbcs units in -one of the donors was kel positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel -negative. the maternal antibody screen revealed anti-kel . doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron , exons , , and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel /kel by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel /kel result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of . g/dl was found to be favourable to one of g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger < . mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < . mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of months (hb transfusion trigger of hb < . mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then months. hrqol is measured after inclusion, after randomisation, weeks, , , , and months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july till june patients were included ( ra, rars, rcmd, raeb, cmmol) in general hospitals and university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and patients for the liberal transfusion policy. the mean follow up period in the liberal group was . months (inclusive run in period) and . months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group patients finished the study, received growth factors and patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about % reduction in amount of transfused red cells was found ( . units per pt per month in the liberal group vs . in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa- table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < . ). t-pa- thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches % at age . this rate is even better in well-treated patients, almost % of whom survive at age . regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the -year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from % to . % per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of adult patients with b-thalassaemia major transfused with a total of . rbc units from january to december . all units were fresh, stored less than days. . units were lr-rbc, . units were ld-rbc and . were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was . % and . %/per unit respectively, in those receiving ld-rbc was . % and . %/per unit respectively, while in those receiving washed lr-rbcs was . % and . %/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients . times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa- viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b ). pasteurisation (liquid heat treatment at °c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than nm pore size was introduced in the early s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately ml of plasma, whereas the improved set accommodates up to ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo ( ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below - °c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from % to %. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts- ), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained % to %. plasminogen and alpha -antiplasmin were retained % and %, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive / /ec require that fresh plasma prior to freezing contains < residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in fresh plasmas divided into groups: group : plasmas from leucoreduced whole blood, group : plasmas from packed cells after removal of buffy coat and specific filtration, group : : apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is . ¥ residual rbcs/l; linearity study with concentrations of . , . , . , . , . , . , and . ¥ rbcs/l showed excellent correlation between observed and expected values (r > . ); reproducibility study showed c.v of respectively . % and %. for values > ¥ rbcs/l, it appeared necessary to introduce a correction factor of . for the anti gpa pharmingen. quantification of rbcs in plasma: group (plasma from leucoreduced whole blood): . ± . rbcs/l; group (plasma from packed cells after removal of buffy coat and filtration): < . °¥ rbcs/l in all the cases; group (apheresis plasma): < . °¥ rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. . improving basic transfusion knowledge amongst health workers; . improving pre-operative preparation for surgery . strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao ) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o (hyperoxic ventilation) can be employed to rapidly raise cao by increasing the amount of physically dissolved o in plasma (hyperoxia). however, molecular o causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received homologous units in addition. the patients undergoing tkr and the patients undergoing removal of implants predeposited and units respectively, but finally and of them have been used. according to our patients data, the . % of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate autologous units. all of them underwent thr except one woman who also had a tkr months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of . homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran % is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f + . in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes / . subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo units three times per week (group b). rhuepo was given for a minimum of weeks or until icu discharge or death. the maximum duration of therapy was weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is > % chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa bb. ten percent of these women make anti-hpa a antibodies in their first hpa a incompatible pregnancy. in - new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of pregnancies show that only babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count < ¥ e /l. in previous studies, it is reported the ich appears in - % of the pregnancies where antibodies are present and that % of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets ( ¥ e mean) from donors with same blood group were pooled and divided equally into two bags, po- and control (pl , baxter), which have and ml/m *day*atm of oxygen permeability, respectively. on days , , , , , and of storage, swirling, mean platelet volume, po , pco , ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to days in po- ( / ) than in control ( / ) bags. dropped ph less than . on day was observed / in po- whereas / in the control. aggressive drop of glucose ( mmol/l) with prominent lactate accumulation ( mg/l) was also observed on day in of control bags. the po level in the control dropped more significantly by % ( . mmhg) on day than in po- ( . mmhg) compared with the initial level ( . mmhg) (p < . ). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po- bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day , and became a detrimental defect in of control bags on day . p-selectin expression was higher in control bags than in po- on days and with no statistical difference. in two control bags p-selectin expression reached > % and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for - days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa- background: maintenance of a neutral ph in the range of . - . is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of . - . cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph . ) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of bcs obtained from standard day -old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from - %. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields ( . ± . vs. . ± . °¥ e ) were similar (pags vs t-sol). statistically significant (p < . with paired ttest) improved platelet quality at days , , and of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at and days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = . for esc, and r = . for hsr at days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from to days provided the quality has been validated and bacterial screening is performed. irradiation up to gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to days is not known. method: two wbc reduced pcs, each made from buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at - °c. swirl, ph and cd p expression were determined on day , and . twelve experiments were performed and compared with a paired t-test, p < . was considered significant. results: see table (day values; mean ± sd; n = ). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day , the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day was < . in / pcs, versus / in group 'b' (not significant). the cd p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to days after blood collection. - b, - a, - b, pra %, donors. in patient -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient and were refractory to two and one hpa compatible platelet units respectively. in patient and compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: . the frequency of occurrence of anti-hpa antibodies in transfused patients was: - b, - b, - b, - a, - a; in three patients they were monospecific, in four polyspecific. . in patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. .the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa- the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b ). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen- or s- . the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about / of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of reactions in years with reactions more common in platelet pools ( : transfusions) than with single donor platelets ( : transfusions). these reactions caused fatalities in of cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the / of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march , the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of : true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: / repeats of x dilution and / repeats of x dilution were hcv rna negative, whereas / repeat of x dilution and / repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype a. the low hcv viremia (below iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a . % take-up rate ( . % where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd -enriched cord blood cells maintained at °c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at °c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd cells were cultured continuously at °c or °c for days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to °c, the cultures maintained at °c produced significantly more total cells ( . fold) and total mks ( fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets ( . fold). accordingly, the cells cultured at °c contained an increased frequency of cfc-mk ( fold) at day . cultures done at °c and °c were also more efficient than at °c but less than at °c. platelets produced in °c cultures could be normally activated by thrombin. as expected, the cells cultured at °c contained an increased amount of the heat shock protein hsp . control experiments showed that the culture of several cell lines was inhibited or unaffected by the °c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of > °c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at °c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than °c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only - % of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a . -year period. requests were received for deliveries from mothers and of these, collections were successful including pairs of twins. failed collection was most often due to a damaged cord at delivery. collections were made for siblings possibly requiring transplant (median age ) with for leukaemia, for erythroid disorders, for immune deficiency, for enzyme deficiency and others. the remaining collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was ml (range - , % exceeded mls) and mean tnc count was . ¥ (range . - . , % exceeded . ¥ ^ ). the mean cd +ve count was . ¥ (range . - . ). all collections were cryopreserved within hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was % (range - %) and the mean cd +ve viability post freezing was % (range - %). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v. . in an abi (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than %. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day . both test and control samples (n = ) were tested on day for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd +, cd +cd +, cd +cd +, cd + and cd +cd + human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd , was observed in . % (sd = . %) of control t cells upon activation with pma, while only a . % (sd = . %) of the test t cells increased cd expression. proliferation assays showed that h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il- , il- , il- b and il- cytokines after -h incubation in culture media increased significantly to pg/ml (sd = ), > pg/ml, pg/ml (sd = ) and > pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of . pg/ml (sd = . ) for il- , . pg/ml (sd = . ) for il- , pg/ml (sd = ) for il- b and pg/ml (sd = ) for il- . addition of lps further stimulated the release of tnf-a, il- , il- , il- b and il- in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k cells (k -rna). methods: dcs were derived from human pbmncs, which were incubated for days in the presence of gm-csf and il- , and then were transfected with k -rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k -rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared hours later, while the cells were expressing p bcr-abl protein and expressing increased cd , cd , cd , hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k cells. conclusion: human dendritic cells transfected with total rna of k cells in vitro could induce effective p bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl - donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in - % of relapsed patients. the success rate is % or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (< %) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately % of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately % to % of patients, is generally mild, and transient, but in < % of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to . j/ml uv light in the presence of um riboflavin, followed by storage under blood bank conditions with various concentrations of -deoxyglucose from to mm for days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without -dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days , , and . results: lactate production and glucose consumption increased from . mmol/ cells/h (sd = . ) and . mmol/ cells/h (sd = . ) for control samples to . (sd = . ) and . (sd = . ) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from . (sd = . ) for controls to . (sd = . ) for treated platelets at day , hsr from % (sd = . ) to % (sd = . ), esc from . % (sd = . ) to . % (sd = . ), swirl from . (sd = . ) to . (sd = . ), and increased p-selectin expression from . % (sd = . ) to . % (sd = . ). addition of -dog up to mm significantly reduced lactate production rate to . mmol/ cells/h (sd = . ) and glucose consumption rate to . mmol/ cells/h (sd = . ), and maintained ph above . (sd = . ) for days of storage. the effect of -dog exhibited a dose-dependent response. however, the addition of -dog had no effects on hsr ( . + . % at day ), esc ( . + . % at day ), swirl ( . + . at day ) and p-selectin expression ( . + . % at day ) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above % of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of -dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv- ( / ), international standards for hcv ( / ) and hbv ( / ) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv- subtypes other than hiv- b, for hiv- and for hepatitis b and c genotypes as well as specificity was evaluated with > specimens. results: results from this study indicate that high analytical sensitivities ( iu/ml hiv- m, cp/ml hiv- o and . cp/ml hiv- , iu/ml hcv and iu/ml hbv) and a specificity of > . % are accomplishable for the mpx test. the % detection rate for hiv- m subtype isolates (a through h) was between to iu/ml, for hcv genotype isolates ( a through ) between to iu/ml and for hbv genotype isolates (a through g and precore mutant) between to iu/ml. investigating seroconversion panels, hiv- rna was detected an average of and days earlier than hiv- antigen with abbott hivag- monoclonal and coulter p antigen tests, respectively, hcv rna an average of or days earlier than hcv antibody with the abbott hcv eia . or ortho eia . tests, hbv dna an average of days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october and september , apheresis ip units have been transfused in sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated ( . %). the population of patients receiving at least one transfusion (n = ) included . % of males, . % of females, the median age was (range - ). the most frequent broad diagnostic categories were hematology-oncology ( . %) and cardiovascular surgery ( . %). the patients received their transfusions either in regular hospital wards ( . %), intensive care units ( . %) or as outpatients ( . %). the number of transfusions by patient ranged from to (mean . ± . , median ). half of the patients ( . %) had previous transfusion experience and . % had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in . % (n = ) of the transfusions ( % ci . - . ), and . % of patients. only ( . %) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, . % ( % ci . - . ) of the transfusions were confirmed as having caused reactions in . % of patients, none of them serious. the most often reported symptoms were chills ( . %) and fever ( . %). itching, skin rash or urticaria was observed in . % of transfusions. of the serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio . , p = . ). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n mÜller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using single donor plasma units. for the evaluation of the plasma factors ml were drawn at different stages (before treatment, after plasma filtration with plas , after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha -antitrypsin complement: ch activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen ( . %), factor viii ( . %), and factor x ( . %). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days , and . the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was . ¥ ( . - . ¥ ; sd- . ). platelet pool volume was . ml (sd- . ). plasma % was within . % and . %. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had . ¥ (± . ). the average platelet loss was . ¥ . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for days. patients were randomized to receive one -day ip transfusion and one -day cp transfusion in random order. after each study transfusion, the hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, -hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included patients, randomized to the ip-cp sequence and to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence ( % vs %; p = . ). mean platelet dose (¥ e ) was . for ip and . for cp (p = . ). there was a significant period by treatment interaction (p = . ) at the . significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) -hour cci (¥ e ) was . ± . for ip vs . ± . for cp. the mean paired difference for both sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). for the first period only, mean -hour cci (¥ e ) was . ± . for ip vs . ± . for cp) the mean paired difference for the first period sequences was . ¥ e (p = . by non-inferiority test; upper bound of the % confidence interval = . ). the non-inferiority margin for the study was . ¥ e for mean treatment difference in cci (cp-ip). median time to next transfusion was h for ip vs h for cp following the first transfusion (p = . log-rank test; data censored at days after transfusion) and h ip vs h cp after the second transfusion (p = . ). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding -hour cci was not met. however, transfusion with -day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower -hour cci compared to -day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood ( ml/kg), across a major antigen (hgd) mismatch. high anti-s- ab titers (≥ : ) were induced after repeated immunization (days , , , , ) with klh-(s- ) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc ( ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken , , , , and days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = , g ), or o-srbc (n = , g ). no ab against o-srbc developed after biweekly transfusions over weeks in g rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = ; g ). transfused rabbits (g & g ) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s- abs were then induced by klh-hapten immunization in rabbits from g (n = ) and g (n = ), and in a group (n = , g ) of naïve rabbits. non-immunized rabbits (g , n = ), and (g , n = ) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g rabbits, but not any of the g rabbits. g rabbits exposed to o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g and g were transfused with m-srbc. viability of m-srbc in all g rabbits (hyper-immune or not) and the hyperimmune g rabbits was equivalent to crbc circulation in g rabbits. in pre-immunized rabbits with high titer anti-s- ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s- ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s- rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed + reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; + reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year are kept in computerized system that has patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr , rzr , r¢r¢, r¢r≤, r≤r≤, ge- , , and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge , anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally . examinees were typified. with o blood group were . ( . %); with blood group a were . ( . %); b blood group . ( . %) and ab blood group were . ( . %) examinees. totally . ( . %) were d positive and . ( . %) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system . ( . %) and d blood group in rh system with . ( . ) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence . % of anti-d in females versus . % in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than . %. however, the rare phenotype jk(a-b-) is more common in polynesians ( . %). individuals with jk(a-b-) phenotypes typically form anti-jk with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was . g/dl. she was given units of crossmatchcompatible rbc. on day her haemoglobin was . g/dl, with a total bilirubin of . mg/dl and lactate dehydrogenase of u/l. on th day an unexpected fall in hb ( . g/dl) occurred with an increase of bilirubin to . mg/dl and of lactate dehydrogenase to u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka: / and jkb: / ). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was g/dl. two months later her haemoglobin was . g/dl and anti-jk was present in the same titer. a year later her blood cell count was normal and the anti-jk was detected in a lessened titer ( / ). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at °c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer ( / ) were revealed. the rare anti-jk alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case . a years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer / ) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at °c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated . % red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated . % red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared . % enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc month to months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d( ), c( ), e( ), k( ), cw( )] and were tested with enzyme treated red cells in use and rbc to months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells to months post-expiration, except anti-k antibodies (negative with k positive cells - months post expiration). conclusion: most rbc antigens studied were detectable months after rbcs expiration date. tests with . % cells were valid in gel test (nacl/enzyme) for at least months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated . % red cells. background: wr(a) is a low-incidence blood group antigen ( : ) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: -year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in blood centres using different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard ng/ml anti rh (provided by cnrgs), a positive control anti kel and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions ( tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a + intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving blood donors from the university hospital of coimbra during the year . the incidence of weak d and rh (d) phenotype was determined in individuals who were rh (d) negative. the ab and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented . % of the total sample. we also describe ab , rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in s and s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from to times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about / of tested samples the number of igg molecules per rbc was small (< ) and the laboratory signs of haemolysis were present in . % of them as well as in . % samples with moderately coated red cells ( - igg/rbc). the large number of igg molecules per rbc (> ) was significantly associated with high frequency ( . %) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c d. in % of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in % of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting + with o rbcs at all phases and + with a rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c b,-c d (table ) . prewarming of test system did not change the reactivity ( + at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from + to + when tested with o red cells, as well as a significant reduction in antibody titer from : to : tested at immediate spin (table ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied . women, - years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in cases; moreover a rbc autoimmunization was found in women. anti-d ( cases), e ( ), c ( ), k ( ), c ( ), s ( ), d + jka ( ), d + s + e ( ), d + c + k ( ), m ( ), c + e ( ), d + c + g ( ) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in . % of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department ( . %) and ( %) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types , and were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum- , th- , ms- (bioscot/serologicals) and d e / - (immucor). results: out of samples molecular typing results were as follows: in samples dw were not determined, in samples, weak d type ; weak d type ; weak d type , weak d type ; weak d type and weak d type were determined. by all monoclonal reagents % weak d type , % weak d type , % weak d type and % weak d type negative results were given. by all monoclonal reagents weak d type and weak d type positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type , type and type . such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in > % of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in . according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting + - + s at c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs ( . % ficin) and no reactivity using . m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity > grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment ( . m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in samples from patients; ( . %) patients had > episode. these findings comprised . % of unexpected results found at pt and bg. incidences were . % at routine and . % at urgent bg ( and bgs, respectively), and . % both at routine and urgent pt ( and pts, respectively). . % patients were female, . % over , but . % < years, coming mostly from surgery ( . %), internal medicine ( . %), hematology ( . %), ginecology ( . %), cardiac diseases ( . %) and cardiac surgery ( . % patients). frequent diagnosis were solid tumors ( . %), cardiac diseases ( . %), hematologic malignancies ( . %), uraemia ( . %), orthopedic surgery ( . %) and hepatic diseases ( . % patients). . % patients were previously transfused, with only . % patients proved as not transfused or pregnant. subsequently positive antibody test during the study had . % tested patients. at pt positive crossmatch was found in . %, antibody screening in . % and both tests in . % cases. majority of reactions were ' +' ( % at pt and . % at bg); reactions ' +' or ' +' were found in only . % cases at pt, compared to % at bg. one crossmatch only was positive in . % positive crossmatches, with / patients having > crossmatched unit. ahg identification was positive in . % tested patients; in % of them papainized panel was also positive. lymphocytotoxic antibodies were found in . % tested patients; . % ( %- %) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in . %, hla lymphocytotoxic antibodies in . %, 'igg antibodies of unknown specificity' in . % (hla noncytotoxic antibodies in / elisa tested samples!), contaminated sample in . %, anti-bga in . %, non-specific cold antibodies in . %, subsequently recognized specific antibodies in . % ( lua, m, kpa, yka), non-specific autoantibodies in . %, carry-over of dat-positive cells and antibody to reagent in . % cases each, while in . % cases antibody screening and in . % cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < . ) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt ( . % vs . %), positive lct ( . % vs . %), lymphocytotoxic hla antibodies (lytxab) ( . % vs . %) and reactivity 'positive screening, negative panels' ( . % vs . %); in males non-specific cold antibodies ( . % vs . %) and antibodies to reagents ( . % vs ) were noted. age. in patients > vs < reactivity was often found at pt ( . % vs . %), caused by lytxab ( . % vs . %), anti-bga ( . % vs . %) or 'positive crossmatch, negative panels' ( . % vs ), but rarely by laboratory mistake ( . % vs . %) or later recognized antibody ( of patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt ( . % vs . % at bg), as positive antibody screening ( . % vs . % if positive crossmatch), with positive panels ( . % vs . % if negative). subsequent tests were positive in only . % patients with lytxab, % with anti-bga, of with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in % and . % tested samples found at urgent and routine pt (diamed), vs at bg. all cases due to lytxab, of anti-bga and of 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) . % cold antibodies and . % laboratory mistakes were found. positive antibody test: identification was negative in . % screening-only cases; % of them were caused by laboratory mistake. lytxab were found in . % crossmatch-only cases; . % reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in % cases with lytxab (in with papainized panel) and often due to 'igg antibodies of unknown specificity' ( . %), anti-bga ( . %), contaminated sample ( . %), but also to later recognized specific antibody ( . % cases). strength of reaction: laboratory mistake was noted in . % of 'w', . % of ' +', . % of ' +' and . % of ' + and +' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt ( % and . % of patients, respectively), due to positive crossmatch ( % and %; and . % patients with liver and cardiac diseases) and caused by lytxab ( . % and . %) or 'crossmatch/screening positive, panels negative' ( . % patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg ( . % and . % of patients), due to laboratory mistake ( % and %) or cold antibodies ( . % and %), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of patients ( male and female) were evaluated. the mean age of the patients was years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal ( / ) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in patients with the incidence of . %. antibodies were registered more frequently in females ( / , . %) than in males ( / , . %). patients ( . %) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table ). in patients ( . %) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. patients ( . %) were dat positive. autoantibodies were found in patients ( . %), all of which had specificity to rhesus system. cold agglutinins were positive in patients ( . %). no specificity could be assigned in patients ( . %), while in patients ( . %) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was . %. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about % of women form red cell allo-antibodies in pregnancy and . % of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that % of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and % of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case : a -year-old asian woman had three previous uneventful pregnancies. in her th pregnancy she presented with miscarriage at weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique ( . iu/ml by quantification using r r cells). in a th pregnancy, no antibodies were detected until weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case : a -year-old asian woman had a positive antibody screen post caesarean section in jan . no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at and weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was . iu/ml using rr cells and the anti-d level was < . iu/ml using r r cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of / . in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and ( ) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > % being s+. conclusion: there is a unique distribution of red cell antigen groups in the races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table . conjointly, in order to obtain the frequency of mi.v phenotype, we screened samples among with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype ( . %), there were also mi.i/ii phenotype ( . %), mi.v phenotype ( . %), mi.vi phenotype ( . %), mi.x phenotype ( . %), and most interestingly two miltenberger related not yet classified variants ( %). related variant a ( . %) was phenotypes as mia+, anek+ and hil+. related variant b ( . %) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore . % (table ). the discovery of unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period patient samples were screened, of the positive results were evaluated. blood samples with positive screen tests by the ortho auto vue tm method (av) performed with % cell suspension, and patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into categories according to antibody specificity; . rh system antibodies (n = ), . clinically significant non rh system antibodies (n = ), . clinically non significant antibodies (n = ), . auto antibodies (n = ), . not identified (ni) antibodies (n = ), . negative screening results by the diamed technique (n = ). the categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of samples were compared, . % of them gave stronger reactions by av, . % gave equal reaction strength and . % of them gave weaker reactions by av. positive screen tests by av only were detected in samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in samples, were rh system related, kell system related and not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to . % suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh and rh rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh or -rh antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague , † blood bank, faculty hospital m, prague , czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab grouping was performed with registered immucor reagents (immuclone: anti-a birma , anti-b lb ) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre . titration of reagents /+ aliquot of serum added/: anti-a: titre (both patient's serum and control); anti-b + patient's serum: titre , anti-b + control: titre . conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed % agreement between all three methods. samples were tested per min with the autovue innova, compared to min required for the same number of tests performed with manual testing and min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcÍa borrÁs, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples ( - years old, n = and > years old, n = ) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for h at °c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . , nrbc: . + . ; srbc: . + . . the values of am obtained with young individuals were: serbc: . + . , yrbc: . + . , rbcs: . + . , rbcws: . + . , nerbc: . + . , trbc: . + . . nrbc: . + . ; srbc: . + . . conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < . ) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < . ) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < . ) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < . ). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification ¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by d-fluorescence microscopy after lipid extraction with triton x- in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa- tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at nm the flim-fret from alexa tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from to %, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in transfused sickle cell patients. methods: all the patients included in the study had received at least unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was - units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c c, c d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the patients. the dat was positive in ( %) of the patients. in cases ( %), the dat was igg type, in case ( %) igg + c d and in case ( %) igg + c c + c d. the acid elution was performed in cases. the eluate was positive in cases ( %). in ( %), of the cases, autoantibodies were pointed out whose specificity were specificity public, anti-e (rh ), anti-c (rh ), anti-ce (rh ). in cases ( %) we found alloantibodies whose specificity were anti-e(rh ), anti-k(k ), anti-c (rh ) and anti-jka (jk ). in the cases ( %) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in ( . %) of the patients. of these, ( %) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of patients was %. seventeen patients ( %) of the patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang , (suppl. ), ). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having distinct incubation wells, application zones and detection areas. microliters of % suspensions of reagent red cells for reverse grouping (reverse-cyte a , a , b, , medion diagnostics, switzerland) are mixed in each incubation well with microliters of plasma. the resulting suspensions are incubated for min, followed by the transfer of microliters each to the application zones, where migration starts immediately. results can be read after min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho screening rbc since the year . aim: (i) to summarize the performance of the autovuetm system for rbc abs, during years ( ) ( ) ( ) ( ) ( ) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in . % of , blood donations that were screened, during - , using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in . % ( . % of the donations). an increased percentage of confirmed tests is noted, since , probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, samples with low positive results (agglutination degree of . ) were retested twice on the autovuetm. / ( %) gave negative results in both repeats. only / was confirmed positive and anti-m was identified by diamed gel test. the remaining samples had at least one positive repeat. / ( . %) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and blood donors of azeri nationality were examined. patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee ( . %) •• ccdee ( . %) phenotypes were twice as much higher than the occurrence of those in the donors ( % and . % respectively). phenotype ccdee occurs more frequently in the donors ( %), whereas it is significantly rare in the patients ( . %). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors ( . % and . %) than in thalassemic patients ( . % and . %). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c c and c d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c c or anti-c d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of heterozygous sickle patients and homozygous patients were analysed. of the homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. ( ) and are present in more than % of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a years old swedish woman, a rh negative, at th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre ( : ) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from . /ul to . /ul) was observed. this pattern showed no variation until the end of the pregnancy. at th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to . /ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p- rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of alloantibodies on blood samples over . these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia ( . %), e ( %), le a ( . %), le b ( . %), p ( . %), m ( . %), d ( . %), c ( . %), jka ( . %) and c ( . %). within the chinese community, the most frequent alloantibodies were similar: mia ( . %), e ( . %), le a ( %), le b ( . %) and p ( . %). in the malays, the most frequent alloantibodies were le a ( %), le b ( . %), mia ( . %), e( . %) and p ( . %); while in the indians, these were mia ( . %), le b ( . %), le a ( . %), d ( . %), and e ( . %), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians ( %) compared to chinese ( . %). anti jka tended to occur mainly in the malay race and anti-c was rare in all (< %) reflecting the high prevalence of c in the singapore population (r r phenotype). the ratio of alloimmunised male to female (m : f) is : . most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at : . . alloimmunisation increased with age for mia, e, k, p , jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies ( or more) within the alloimmunised subjects is . %. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a years period, from / / - / / , we studied newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in ( %) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in ( . %) (newborns of group a with mothers of group o), anti-b antibody in ( %) (newborns of group b with mothers of group o), anti-d antibody in ( . %) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in newborns and in the rest , no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d ( ), anti-e ( ), anti-k ( ), anti-fya ( ), anti-s ( ), anti-jkb ( ), anti-n ( ), anti-kpb ( ). in two patients antibodies were identified, while in / ( . %) no antibody was identified (unspecific). it is remarkable that only in out of patients with both dat and iat positive, an irregular antibody was identified, while the rest patients had unspecific antibodies. in patients with only iat positive, had an irregular antibody and had unspecific antibodies. in out of patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is . % and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. . % of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than % in china) were reported from % to %. moreover all del individuals in this population were found mainly carrying a same allele, rhd a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in rh-positive donors with cde/cde and cde/cde genotypes, respectively, and del phenotype individuals carrying rhd a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and isoforms were detected in rh-positive and del, respectively. among them, isoforms have identical sequences, which are transcripts with exon , exons and , exons and , and exons to spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon or exons - were spliced out, but both possess a bp segment of sequence from intron of rhd. through additional reversedtrancriptase pcrs, which amplified exon to ¢-region and exon to ¢-region, the results showed that exon did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd a allele since the exon was always spliced out in all isoforms. all transcripts in del maintain a normal open reading frame and encode proteins with different numbers of amino acid residues and different c-terminals (genbank ay , ay , ay , ay , ay , ay ). among them, the sequence of del (isoform with exon spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in . it encodes amino acid residues and has amino acids more than normal rhd. it is different from rhd after codon . in normal rhd protein, the amino acids after ( residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del that was found in del. (iii) there is only one polymorphism in the region of bp segment between rhd intron and of the del transcripts, which indicated that other polymorphisms may exist in intron of rhd a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh ( pooled we reported earlier that metabolic arrest followed by incubation at °c reduces the platelet storage lesion (badlou et al. transfusion ) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium ( min, °c) followed by storage ( h, °c) and recovery with glucose ( h, °c). controls were (i) platelets in glucose-rich medium stored for h at °c and recovery with glucose (c ) and (ii) platelets stored for h at °c (c ) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp- cells ( °c). binding was measured by facs analysis of cd b/cd positive particles, and phagocytosis by counting mepacrine/cd positive particles. binding of msp, c , c was ± , ± , ± % of total platelets. phagocytosis of msp, c and c was ± , ± , ± % of total macrophages (means ± sem, n = ). before recovery of msp, binding/phagocytosis was % higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in patients with leukemia and lymphoma, males and females with mean age ± years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of compatibility tests were performed, of which were compatible. twenty five compatible platelet concentrates out of were clinically evaluated. twenty from compatible crossmatches ( %) were resulted in successful transfusion while only from ( %) in unsuccessful. the incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~ . ). additionally patients out of ( . %) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june and is still in course: in january , blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the blood samples analysed, were reactive and the specificity of the antibodies were: anti hpa a: , anti hpa b: , anti hpa a + hla: , anti hpa b. , anti hla: , auto hpa- b: . specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa- , , , , , ) using pcr-ssp or pcr-rflp. the infants with hpa immunization suffered from severe (plt count - /ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa- and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa- a is the most frequently implicated antigen. other antigens such as hpa- a, or hpa- a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa- bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa- bw (maxa+) mismatch as the sole antigenic incompatibility in out of families. in the last one, incompatibility was found for hpa- without anti hpa- b maternal alloantibody. as the father was found to be hpa- bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa- bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa- bw (maxa+) fmait is not uncommon and was found to be around % of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa- a antibodies after non-myeloablative allogeneic hsct. a year-old male patient was diagnosed with multiple myeloma in / . treatment consisted of cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received cycles of bortezomib, and after complete remission a stem cell allograft ( . ¥ /kgbw cd + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine ( ¥ mg/m ) and alkeran ( mg/m ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d but was accompanied with a continuous decrease of plt counts. between d and d the patient received seven plt transfusions, containing a median of . ¥ plt/unit (range , - , ¥ plt/unit) from random donors. the corrected plt count increments at to h after these transfusions were < /ml. therefore, and because of even a further decline of platelet counts to /ml on d we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak ®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa- a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa- , - , - and - of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d after hsct reacted strongly with the donor's plt due to anti-hpa- a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa- bb, - aa, - ab, and - aa; the donor was hpa- ab, - aa, - aa, and - aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d , d and d after hsct contained antibodies against hla class i antigens but hpa- a antibodies were not anymore detectable. no hla antibodies were detectable on d after hsct. the severe thrombocytopenia was caused by hostderived hpa- a antibodies. fortunately, plt counts started to increase on d spontaneously and the patient could be discharged at d (plt . /ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa- b antibodies: case report p moncharmont, m vignal, y mÉrieux and d rigal efs rhone alpes site de lyon, lyon cedex , france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa- b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a -year old mother, gestation /partum , gave birth to a male neonate by caesarean section at weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted . g, was . cm tall and had a head circumference of . cm. the apgar score was , , , at , , and min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during h. the plt count which was . , . and . giga/l at day (d) , d and d respectively dropped dramatically at . giga/l at d . simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa- a/a and anti-hpa- b ab were detected in her serum. the baby was heterozygous, hpa- a/b. plt were transfused to the baby and the plt count rose to . , . and . giga/l at d , and respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa- b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor antibodies. hit type ii occurs in up to % of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf antibody test (diamed id-card). results: from the studied patients, ( . %) developed thrombocytopenia (determined by a decrease in the platelet count below %, after the introduction of heparin therapy); ( . %) did not show decrease in the platelet count. six ( . %) out of thrombocytopenic patients were positive for anti-heparin/pf antibody. three ( . %) out of non thrombocytopenic patients were positive for anti-heparin/pf antibody. the results demonstrate that ( . %) patients were positive for anti-heparin/pf antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects . - in live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to % of reported cases). the commonest antibodies are anti-hpa- a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week of gestation to a healthy mother after first uneventful pregnancy; birth weight g, apgar score . immediately after birth, severe thrombocytopenia ( ¥ /l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa- a/a) were given. twenty-four hours later platelet count rose to ¥ /l and no new petechiae were observed. on third day of life the blood platelet count was ¥ /l and the newborn received ivig g/kg and corticosteroides. twenty-four hours later the platelet count rose to ¥ /l and further clinical course was uneventful. natp due to hpa- a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa- a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of . % to % in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: samples were referred after being characterized as weak d. in cases we obtained a positive result, with a preponderance of weak d type ( . %) over type ( . %), ( . %) and ( . %). two samples were not categorized. the high incidence of weak d type in our population is in marked contrast to studies performed in other european populations where weak d type was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a -bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to ¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the ¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a -bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = ). results: ( . %) rhd-positive and ( . %) rhd-negative samples were phenotyped. of the rhd-positive donors, ( . %) were rhd homozygous and ( . %) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee ( . % vs . %) and dccee ( . % vs . %) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in . % of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with fluorescent primer pairs. positive results were obtained for rhd-exons - , , and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons to and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v. . in an abi (applied biosystems). results: see table . the substitutions of rhce-specific nucleotides in exons , and with their rhd-specific counterparts lead to different new rhc-antigens with weakened expression. since one amino acid change in allele lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a -year old mother (w.o.g. ) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron , exons and were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron , exon and exon while sample of fetus ii lacked rhd-specific intron . therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons - , & ; polymorphisms for dweak type - , d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi . rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons , , , and were possible and failed only in exon - . determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron pcr. results: weak d type was not identified in our research population. weak d type was identified in cn, weak d type was identified in cn and weak d type was found in cn, bsa, be, bc and saa. conclusions: although weak d types to represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type in all populations. however, regarding rhd phylogeny, the weak d type mutations ( c > g and t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from a, b, ab subgroups, and provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon and , and allele separation were performed on these samples. results: abo*a allele was observed in an aint subgroup. two new a alleles that showed g > a base change and c > t of intron and a polymorphism of c > t in a(pro) intron were discovered. o and o alleles were also observed. in b subgroups, a silent substitution c > t (leu leu) was observed as a new b allele. another new b allele which showed g > a was also found in b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > . ) verifying that the monoclonal anti-ab associa- study aim: to prevent the sensitization to rh (d), to a d-patient who was transfused with d+ blood. material and method: on september , ( / / ), we admitted to our hospital through air-carriage, a female of years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: %) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally units ( ml) of d+ condensed red cells, out of that were initially asked. before the h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh (d). the indicted dose was - iu/ ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = ) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table ) . for the long-term follow-up samples, additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with copies of negative dna in a single amplification tube for all primer pairs. we were also able to calculated the clinical sensitivity that using donor-recipient pairs. . % of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with . % of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to -chloro- - dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag- /common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with million viral particles of murine cmv (mcmv). at different post-transfusion time-points ( h, weeks, weeks, weeks, weeks), different female recipients plus non-transfused female knock-out controls were challenged with million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp ( mg sc once or q weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day and day after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ % during blood donations and blood donation was not performed if ht < %. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was . € in the aranesp group and . € in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from operations in patients with anti-erythroid antibodies. materials: three patients ( male and females), aged between - years old, had to undergo selective operations, total hip replacement surgery and aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of consecutive patients (group ) who donated two red cell units (rbc) of autologous blood and weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ ) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ . ; postop-hb = hb ( - h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as g/l) < (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next patients who were also assigned for pabd (group ). pabd was refused if the probability of rbc requirement (prr) was < %. between - % one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded % two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group ( vs and vs g/l). % of autologous blood of group were discarded, / patients needed additional allogeneic rbc. hb-loss caused by rp was ± g. mean prr in group was . %. / patients donated one rbc, which was later discarded, and no patients donated two rbc. / of group needed allogeneic rbc. mean prr of these patients was % (range . - . ). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other ( . %) patients who required autologous and homologous blood, had average predonation hb level of (sd ± . ) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = . ), predonation htc values (p = . ), weight (p = . ) and gender (p = . ): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = . ). ( . %) patients with ptka were transfused with autologous blood only, and had average predonation hb level of (sd ± . ) g/l. other ( . %) patients transfused with autologous and homologous blood had average predonation hb level of (sd ± . ) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = . ): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = . ) predonation htc values (p = . ), gender (p = . ) and age (p = . ) of patients and the need for postoperative transfusion. conclusions: our results show that over % of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight - g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, cm proximal and cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, patients, including males and females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about - percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at - day intervals. the blood volume taken from patients in every collection differed from - ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, cc to cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately % of the total blood volume withdrawn] had platelet counts of - /mm . the procedures of agp labelling were performed in laminar flow chamber. to seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed h after application of labelled agp (early scan) and at , , , h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at , h and at lapse time = h for days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every - weeks. by the th week of gestation the patient had donated four units of blood, her hematocrit was %, anti-ku titre was / and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were and at and min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day . the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above °c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+ °c, - °c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature + °c, + °c and + °c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+ °c) or freezer (- °c). the temperature was observed in min intervals for three hours, first measurement was min after putting into freezer or termostat. results: (see table ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since , we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between - %, the mean volume about ml and the mean platelet-content is . ¥ per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from to days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: twins made out of pools from buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at °c, and sampled at day , day and day . ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day with cb was . , with nfb . ; glucose with cb . mmol/l, with nfb . mmol/l; lactate with cb . mmol/l, and with nfb . mmol/l, hsr with cb %, with nfb %; p-selectin with cb % and with nfb %. the new platelet storage bag showed better results of in vitro quality markers, especially after day of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at °c or rt simplifies the preparation process. filtration at + °c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, % of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by . mm. a decrease of the process time is expected. methods: units of whole blood were collected with the new t 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within min in all cases. after donation whole blood units were stored at room temperature for h. subsequently, whole blood filtration was performed by gravity at a standard height of cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at g ¥ min by an heraeus cryofuge i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx -abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table . results are given as mean and standard deviation. whole blood filtration was completed within min in all cases. red cell units were transfused after a mean of days to patients affected from transfusion dependent ( %), post-surgery ( %), and post chemotherapy anemias ( %). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day , , and , and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day , and , and cd expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years ( , .) aberrations from these principles have taken place (self-sufficiency). nbti collected x = ( ) blood units into blood bags. in serbia x = ( ) blood units. retrospective analysis: ldpc-bc was administered x = % with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted ( %). increase of the use of leukocyte poor red blood cells was also registered ( % introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at °c with platelets stored at °c, and to determine the in vitro-effects of pre-incubation at °c for h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at - degree on a flat bed agitator; (ii) at - degree on a flat bed agitator and pre-incubated for h prior to analysis; (iii) at °c; and (iv) at °c and pre-incubated for h prior to analysis. this paired in vitro study (n = ) over days include volume, platelet counts, mpv, volume, ph, po , pco , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf ). results: platelet count (day and ; p < . ), mpv (day ; p < . ), ph (day and ; p < . ), pco (day and ; p < . ), bicarbonate (day ; p < . ), glucose (day , , , and ; p < . ), atp (day and ; p < . ) was significantly higher in platelets stored at °c and platelets stored at °c with preincubation. ldh (day ; p < . ), bicarbonate (day and ; p < . ), lactate (day , , , and ; p < . ), ph (day and ; p < . ), esc (day , , , and ; p < . ), hsr (day , and ; p < . ) was significantly lower in platelets stored at °c and platelets stored at °c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf was significantly higher in platelets stored at °c than in platelets stored at °c (day , , , and ; p < . ). hsr (day and ; p < . ) and esc (day , , and ; p < . ) was significantly higher in preincubated platelets stored at °c compared with platelets stored at °c. conclusion: platelets stored at °c maintain metabolic and cellular characteristics to a great extent during days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at °c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: whole blood donations were collected in a whole blood filtration set with cpda- and stored at room temperature for h before filtration at room temperature. the leucodepleted whole blood was stored for days. following parameters were analysed on day , , : free haemoglobin in%, k. in addition leucocyte count was performed on day and a blood culture on day (see table) . blood cultures on day remained negative and all counts of residual leucocytes were below ¥ (exponential) /unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day . however the results were within the required specifications from the european and swiss guidelines up to day . we conclude that autologous leucodepleted whole blood can be stored in cpda- -for days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a -month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: ( ) blood of female donors was not used for ffp production, and plasma was only used for fractionation ( ) plasma of female donors was not used for kt-bc pools ( ) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: %*; %** ffp produced by plasmapheresis: %*; . %** female donor units on cell separator: . %*; . %** ffp from total plasma units: %*; %** plasma units used for bc-pc pools: %*; %** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~ %), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of days. extended storage to days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd p and cd b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = ) using apheresis technique. the pc from each donor was divided in units, one served as control and the other was gamma irradiated with gy. the pcs were stored on a flatbed agitator at °c for days. samples were taken on day (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day , and were also analysed for hrs, cd p (p-selectin) and cd b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day , and . the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd b expressing cells were well maintained for days of storage. glucose decreased and lactate increased significantly during the storage period, from . mmol/l to . mmol/l for lactate and from . mmol/l to . mmol/l for glucose. the percent cd p expressing cells increased significantly during storage from % on day to % on day . po was well maintained but ph increased and pco decreased significantly between day and whereafter ph decreased and pco continued to decrease. the for parameters g'max and time to g'max increased significantly between day and and the time to g'max continued to increase significantly between day and . the results indicate a well preserved platelet quality after storage for days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b (transforming growth factor b ) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from buffy coats. following overnight storage at - °c, buffy coats were pooled with ml t-sol (baxter). forty-two pcs were produced either manually (n = ) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = ) using the orbisac validation bc set (gambro) equipped with the lrp leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl series analyser (radiometer). samples for testing of cytokines were centrifuged for min at g, °c; supernatants were harvested and frozen at - °c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days , and . results: platelet concentrate volume (mean): m-pcs: ml, a-pcs: ml. platelet yield was found to be . ¥ for m-pcs and . ¥ for a-pcs (p < . ). in all pcs ph levels were between . - . . glucose consumption and lactate production from days - and days - did not differ significantly. rantes levels (pg pr plts) were significantly higher in a-pcs than in m-pcs (p = . , repeated measures analysis of variance), but no significant difference was found in tgf-b levels (pg pr plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/ - . pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd + cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in °c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd + cell count were evaluated by flow cytometry with a no-wash, single-platform technique and aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h , stemcell technologies) and colonies were counted after days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables , and . post-thawing tnc and mnc, as well as cd + cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd + cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd + cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: cord blood units ( from males and from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb . blood units were also randomly typed for hla-dqa and dqb at high resolution. results: mean birth weight was g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was . g. babies were < th centile ( g) and were > th centile ( g). comparing the hla allele distribution in these extreme bands we found that hla drb * was significantly associated with high relative birth weight: . % in th centile vs . % in th centile, p = . . on the contrary, hla-drb * and dqb * were associated with low relative birth weight: . % and . % respectively in th centile vs . % and . % in th centile p = . and p = . . all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb * and higher relative birth weight (mean . vs . ; p = . ) as well as the association of hla-drb * with lower relative birth weight (mean . vs . , p = . ). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb * and hla-drb * genes encode for different amino acid sequences in the pocket of the molecule (aa , , ). this implies distinct functional restriction patterns. the sequence motif of hla-dr is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retarda- which provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a %- % dextran solution in . % saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation °c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was % to % when stained with gimsa or wright staining method. the viability of isolated pmnls was also % too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during to . at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged g to g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: exchange transfusions were carried out. four out of et due to abo incompatibility mother-newborn. five out of due to rhesus incompatibility mother-newborn and eight out of due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at °c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: samples were taken from newborns at first h of life for abo blood group typing during - . simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in out of newborns were found detected antibodies anti-a, anti-b. in out of newborns no antibodies were found. in out of were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from + . to + . conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab incompatibilities, we have discovered alloimmunized women with the following specificities: anti-d, anti-c, anti-c, anti-e, anti-jka, anti-d + anti-jka, anti-d + anti-s, anti-d + anti-c, anti-d + anti-c + anti-k, anti-s, anti-k, anti-m, anti-c + anti-e and anti-d + anti-c + anti-g. the most severe hdn were the d + c + g, the c + e and out of c newborns, with mean hemoglobin between and g/l, bilirubin = . g/l, reticulocyte count = %. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, years of age, in th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the th gestation week covered with mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < % or hemoglobin (hb) < g/dl and reticulocytes < %; b) hct < % or hb < g/dl in these conditions: o required < %, recurrent apnoea and bradicardia, cardiac rate > bpm and respiratory rate > bpm for more h; c) hct < % or hb < g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of % after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume ( ml per kg if premature or ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of - weeks) show a range of weight from to . g, while at term newborns from . to . g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always - ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of neonates admitted during one year, ( male, female) received blood components. fifty four percent received one, % two and % received three types of blood components. the frequency of transfusions were times. the most common used blood products were fresh frozen plasma ( %), red blood cell ( %), whole blood ( %) and platelets ( %). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were %, %, % and % respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january and december we studied d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of d-negative studied samples, ( . %) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that patients ( . %) had in their serum other anti-d associated alloantibodies: anti-k ( . %), anti-c ( . %), anti-e ( . %) and anti-c + e ( . %). during the immunohematologic follow up, it was determined that in / patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (- °c and - °c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at ¥ g for min within h of collection. plasma was removed. four units of plasma were pooled into a ml bag, mixed, divided into parts (aprox. . ml) and immediately frozen. two of these units were stored at - °c and units of ffp at - °c. after one month the plasma was thawed at °c during - h. the fibrinogen concentrates ( - ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at - °c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored - °c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center ( donations per year and utilization of approximately of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in the national center for the production of blood products was established, under the direction of elias politis and years later in begun the production of dried plasma from greek donors. by the year the center started the production of fibrinogen and by the year the production of antihaemophilc factor. in all the activities of the center settled down due to administrative aspects. at the beginning of s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early s, when techniques for the isolation and concentration of clotting factors were improved. in , matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl , is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation ( ¥ g for min) of whole blood. four units of plasma were pooled into a ml bag, mixed, divided into parts and immediately frozen. one of them was stored at - °c and after one month the plasma was thawed at °c during - h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well- (light cycler, roche diagnostic systems, nj) was used for the identification of the c y, h d and the s c point mutations of the hemochromatosis gene, and were based on protocols developed, for c y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c y allele was obtained ( . %) in individuals with genotype , however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than . patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c y, h d and s c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from % to - %, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of samples ( mothers and newborns), from / / and / / , were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the cases studied it was found cases without incompatibility ( %). from the incompatibilities, . % were abo, . % were rhd, . % were other rh incompatibilities, and . % were due to auto-antibodies. % of the mothers were rhd+ and % rhd-. conclusion: of the pregnant women studied, only were rhd-. from this group ( %) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the pregnant women rhd-, % had incompatibility abo, which decreases to near % the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a haplotype expression has been found about - % in caucasian and in greek population particularly, . %. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a expression. in of these donors the expression of cd , cd , cd , cd , cd , cd , hla dr, cd +cd +, cd +cd +, cd -cd +, cd +cd -cd +, cd -cd -cd +, cd -cd -cd + was examined. meanwhile, patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a expression, while in of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a expression in healthy donors was . % and in cancer patients % (p > . ). in table the mean, standard error, t-test and p of the two groups are included. (see table ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd expression, which was higher in cancer patients. summary and conclusion: the expression of hla a in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in ), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in , dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march . after keeping it for days at storage, blood was forwarded on a two-day journey to a small town, kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are - nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deter- objective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd ) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = ) and umbilical cordon (n = ) were used. several dilutions oh hailuronic acid sodium salt solution % (sigma l- h ) were confronted with % erythrocyte suspension in phosphate saline buffer (pbs) ph . . the rbcs were previously treated with an enzymatic solution of % bromeline in pbs ph . (sigma l h ). agglutination readings' were been by slow sharking after of h incubation at °c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à si. di- . - (i = , , . . .) where si represent the score and di- is dilution inverse. the adult' rbcs showed a = ± , while en the newborn the parameter was a = ± . our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd ) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during min at °c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p -dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig , is induced by p through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele ( tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (< %) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele ( repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a +/ + reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a + reaction in a test with monoclonal anti-d and a +/ + reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against anti-d reagents in the id-partial d typing system. one of these (number ) gave negative reactions and the remaining five gave positive reactions (ranging from +/ + to +), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a +/ + reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk applied conventional and no conventional (hemolytic medium time) techniques in sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in ae from b patients and in ae from ab patients; a epithopes in ae from ab patient and in ae from a patients; p and p epithopes in ae and only p epithopes in ae. these patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in sera and anti a immune antibodies in sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c a and c a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during - period all anemic patients on hemodialysis were divided in groups. the first group pts with febrile non-hemolitic transfusion reaction. the second group- pts immunized toward leukocyte and hla antigen. the third group young candidates for kidney transplantation for prevention of hla immunization. the fourth group pts with sle (for immune-complexes and autoantibodies). total patients ( males and females) received units of rbc with residual number of leukocytes. commercial filters of baxterÔ (lekostop lds) and terumoÔ (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with - microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until days of collection. result: aabb permits maximum < ¥ wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of ¥ the le reduction of - . %. the number of rbc after filtration is minimum % - g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g a, mthfr c t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients ( . % heterozygous, . % homozygous) compared to controls ( . % heterozygous), p < . . the or for heterozygous carriers was . ( % ci . - . ), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients ( . %) and controls ( . %), or . ( % ci . - . ), p = . . although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c t mthfr than the control group ( . % vs . %), or was not significant ( . , % ci . - . ), p = . . analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c t mthfr gene (tt and ct) (or . , % ci . - . ), p < . . conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to ¥ /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease ( % atipical plasma cells). to treat heavy anaemia (hgb g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol mg per day). coagulation parameters were tested: pt . inr, aptt s, fibrinogen . g/l, trb ¥ /l, d-dimer mg/l, atiii %. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with dose of cryoprecipitate, dose of fresh frozen plasma, iu atiii, doses of red blood cells and vitamin k mg. two days after the substitution therapy we saw pt . inr, aptt s, fibrinogen < . g/l, trb ¥ /l, atiii %. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till th day of therapy, fibrinogen was below . g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with mg/kg/dose of rfviia (*) for days, after which there was clinical and endoscopic recovery, and an inh decrease to . ub/ml (fviii dosage %) . he began to take meprednisone ( mg/kg/day) for days, after which the inh titre was . ub/ml. (table a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of . ub/ml and was treated with mg/kg/dose of rfviia (*) for days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to . ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was . ub/ml. (table a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a -year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio ( : ). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, to november, , we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than °b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo < ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of patients (ra, rars and raeb < % blasts) receiving epo ± gcsf between and and having serum epo < ui/l, the response rate at week (iwg criteria) was %. six variables were associated with response to epo ± g-csf for mds: age > years (p = . ), number of prior red blood cell transfusions < packs/months (p = . ), serum epo level < ui/l (p = . ), percentage of blasts < % (p = . ), percentage of erythroblasts > % (p = . ) and low ipss score (p = . ). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p- pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on february , during the course of the first general meeting of the pbsa, which comprised of founding patient organizations. the objectives of the pbsa are: . to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. . to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl - gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl - gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to - u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than patients with haemophilia have now been treated in phase gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, < u/dl rise, in / patients enrolled were observed, although less factor ix concentrate was needed in / subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (> % of normal) for > year in mice following a single treatment. in / patients treated repeated factor viii rises ( . - . u/dl) were seen, but no improvements lasted beyond months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. / patients enrolled sustained levels of factor viii > u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~ u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. con- reference: petz ld, garratty g. immune hemolytic anemias. nd ed. philadelphia: churchill livingstone, , pp - . w-pa- autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [ ] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [ ] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [ ]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (< h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [ ] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about % of cases. gift detects igg antibodies and is positive in about % of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa- practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab ( + = adsorption; + = adsorptions; + = adsorptions; + = or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time ( - h) does not allow for adsorptions, one can dilute the patient's serum (e.g. in , or in ) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that - % of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [ ] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [ ] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [ ] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [ ] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb (cd ) and gp - . the respective antigens, frequencies and alleles are illustrated in table . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [ ] . references are available from the author upon request. w-pa- a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a -yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c -independent binding of c b complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia ( and neutrophils/ml). the diagnosis of annanti hna- a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day and , respectively. omphalitis was successfully treated with -day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for days without success. this was followed by an attempt at neutrophil count increase with -week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at -week intervals. in spite of prolonged neutropenia ( and months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: ( ) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; ( ) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna- a immunization, and ( ) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors ( . %), aged between and years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during , % of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the % of them were successfully processed to ffp. during , % of the donations were fulfilling the specifications and . % of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc / ). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about % during - , suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc / hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa- transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september , , with almost deaths and more than injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the -year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than units of blood used in the first to h. in in new york, the five hospitals closest to the disaster site admitted only disaster victims. the new york blood center, which supplies percent of blood for the city's hospitals, added units to routine inventory at hospitals. the center received telephone calls and collected more than units of blood in the first h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september , america's blood centers collected more units of blood, and the american national red cross collected more units than in the same period the previous year. more than units were collected for the disaster victims, but only units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach %s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june , , a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august , , as shown in the following sections: . the development of a voluntary blood donor system. . removal of the upper age limit for blood donors. . funding for blood donations. from january , , each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa- can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from to %. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around . % of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv , hhv ), parvovirus b , hepatitis g ( . %) and tt (transfusion transmitted) virus ( . %). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around : to : depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values < . g/dl in the first and third trimesters, and < . g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa- impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris , bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach / above years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than % bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts > %) evolving in a short timespan with severe cytopenias, and to aml in approximately % of cases. at diagnosis, % of mds patients are anemic, with an hemoglobin level less than g/l, and % of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of - units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) ( - u/kg tiw, or a - u as single weekly dose) are typically used in low-risk mds. the response rate to epo is - %, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than g/l). absent or low rbc transfusion needs and a serum epo level less than u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable ( - months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to - %, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in , compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin or ug/week) with response rates varying from % to % in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over years and backed by postmarketing experience in countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa- iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss > l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (< weeks) of the anemia is desirable in cases with transferrin saturation < % and ferritin levels < mg/l. with this regime it is possible to collect up to autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed red cell apheresis in patients with diagnosis of hereditary hemochromatosis ( ¥ c y homozygotes, ¥ c y + h d heterozygote) using haemonetics mcs p cell separator (protocol tae) in which red cells are removed from patients in - cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december , and february , have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter and days after drbc procedure. donors suitable to drbc apheresis had to have: age between and years, weight > kg, hb > . g/dl and serum ferritin between and ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of donors who donated drbc during the study period, only males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb . ± . g/dl, ferritin ± ng/ml, serum iron ± microg/dl. on day mean hb was . ± . g/dl (p < . ). on day mean hb was . ± . g/dl (p < . ), ferritin ± ng/ml (p < . ), serum iron ± (p ns). only out of donors ( %) had a ferritin value > ng/ml. in the studied donors the collection of units of rbcs induced an expected reduction of about grams of hb, however only % of this reduction was recovered after days (p < . ). similarly, also iron stores have not been restored after months from donation, as shown by a % reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of units of rbcs (approximately - mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively % of subjects at first blood donation and, in these subjects, hfe mutations prevalence was . for c y and . for h d (velati et al., ) . aims: the role of the c y mutation is well known in the patho-genesis of iron overload, whereas the role of the h d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h d mutation in comparison to a population of blood donors wt/wt for the h d mutation. methods: a total of repeat blood donors were examined in italian transfusion centers ( in northern italy and in southern) for c y and h d mutations. out of those, blood donors heterozygous for the h d mutation and wt/wt for the same hfe mutation, both groups wt/wt for the c y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table summarizes the allelic frequencies in the blood donors. table reports the haematological evaluation in the subjects heterozigous for h d mutation and the wt/wt for the same mutation. conclusions: these data suggest that subjects with h d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h d heteroxygotes or even homozygotes when available, in order to get further insights on the h d role in iron metabolism. background: cd is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and females aged to (median ). twenty of them had never been transfused (group a) and were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd b, anti-cd , anti-cd , and anti-cd . leukocytes were gated according to cd . we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < . ) and controls (p = . ). this downregulation concerned the proportion of cd + cells, that was lower in the transfused patients than the non-transfused (p < . ) and controls (p = . ), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < . ) and group c (p = . ). negative correlation was observed between the cd expression and cd b (p = . ) and cd (p = . ). cd b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = . and . respectively) while the percentage of cd b cells did not differ significantly between the various groups. increased expression of cd was also found in the group a compared to group b (p < . ) and c (p = . ). the proportion of cd + cells did not differ between the various groups. the levels of immuno-reactive svcam- as determined by elisa were found . + . in group a, . + in group b and . + . in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in centers and the gfm has the intention to extend the study to a larger population in at least centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after blood donations or plasma donations. before , each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g % for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g % for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g % for intravenous use (ivig) has been used in the study. the preparation is liquid, % stabilised with glucose of a ph value of . ± . . it is used in those cases where sepsis developed after surgery. both an ivig group (n = ) and a control group (n = ) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = ), and the control group (n = ); pancreatitis (n = ) control group (n = ); intestinal obstruction (n = ) control group (n = ); abdominal organ perforation (n = ) control group (n = ); abdominal perforate injuries (n = ) control group (n = ); serious abdominal interventions (n = ) control group (n = ). the period of hospitalisation of the patients in the ivig group was ± days while the period of hospitalisation in the control group was ± days. the mortality rate in the ivig group was % counter . % in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g % for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: pc were produced by this device and some parameters were studied. for the preparation of pc, bc were pooled using the orbisac set, with an integrated filter (pall lrp ). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol / . the pc was stored in a gambro elp bag. results: the average platelet count per unit was . ¥ e . the platelet recovery from pooled bc was . % (range . %- . %). all products of the tested pc containing < ¥ e wbc (by flow cytometry). the values of ph on day and of storage were . and . . the swirling phenomenon was good until day °. the average loss of haemoglobin per bc was . g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood ( ml) was collected in top-and-bottom bags, and randomly selected to be processed by either ( ) current routine or ( ) atreus c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc d (atreus). lr-rccs were stored at °c and sampled until day . various in vitro measurements were performed (n = per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was % lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained ± ¥ platelets in ± ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. . ± . . ± . < < . aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day . on day , either , , , or pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days , and for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein b (gp b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of pools were studied, each of , , and pc's. the mean platelet yield was . ¥ e with a range of . - . ¥ e . the five pc's had a mean yield of . ¥ e and all maintained a ph > . on day . all products had less than ¥ e residual wbc. platelet quality data is presented in the table. data are the mean ± sd, n = . conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may . the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were blood units (ek) issued for the mentioned period to pediatric clinic for pediatric patients (~ , % units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to - bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was - ml er (depends on age and body weigh). ek was issued as washed concentrates, ek were filtered and ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than times than obstetric ones ( vs ) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk is an immunohaematological analyser for detection of red blood cells antigens of ab , rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: blood samples of donors were tested between july and january . all samples were analysed for ab blood group. samples were tested for rhd antigen and ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab , rh (d, c, c, e, e) and k were investigated truly by first testing at . % samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about . % samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify bp in sregion of hbv genome in the first pcr and bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to . iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of % (hc-ii; %) and a specificity of % (hc-ii; %) (p < . ). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p- blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in . since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from . january to . december . results: in whole blood participated in the consumption with . %, packed red blood cells with (rbc) only . %, washed rbc were used in . % of the cases. in whole blood participated in the consumption with . %, packed rbc with . %, washed rbc with . % and rbc in additive solution with . %. as far as plasma preparations are concerned, there has been, since , a great consumption of plasma -witch was separated from whole blood in period up to five day in . % cases, and small consumption of fresh frozen plasma (ffp) only . %. since , there has completely been cancelled the production of five day old plasma, only ffp is being used. from to for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from units to units per year. in , after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from units in to units in . conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we total febrile non haemolitic male transfusion reaction f e m a l e alloimunisation on le/hla male antigens f e m a l e kandidates of renal male transplantation female lupus nephritis male f e m a l e total female male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, cgy of gamma radiation reduce lymphocyte response to mitogens by %.the aim of the study: . to estimate potassium level increment in stored irradiation blood units. . to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. . to evaluate the expiratory date of blood units post irradiation. the study included units of blood collected in cpd-adsol (as- ). in twenty units the blood collection bag was with inline leucodepletion, while the other units were non leucodepleted. all the units were irradiated using caesium as a source of irradiation, with a dose of - cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from st day post irradiation and continues to day post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day , p value of more than . . . gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. . there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within h post irradiation. . there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. . an out date of days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within - h post irradiation. . the percentage of irradiated blood units requested by our physicians ( . %) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from % ( ) to . % ( ) and increasing trend of using packed red cells component therapy from . % ( ) to . % ( ) . there is also increasing trend of using fresh frozen plasma (ffp) from . % ( ) to . % ( ) . comparing and year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during year period, a total of units of blood and units of f.f.p were used. more specifically, the results can be shown in the following table . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a -year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt s, pt %, fibrinogen . g/l, fv % (other factors were in normal range), platelet count ¥ /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about . bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of mg/kg (~ ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding ( %), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted ( %) along with the presence of factor v inhibitor at a level of . bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of : respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr t/t cpd/sagm (macopharma). whole blood ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. ± °c for to h. after component separation (beckman coulter j mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. ± °c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit . wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr and abc leucoflex lcr ( . and . ) is highly efficient. the use of abc leucoflex lcr showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = . < . ). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = . < . ). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable : , rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst system and leucoflex lst system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed donations with the lst system and donations with the abc lst system. all the donors were random male volunteers and they were meant to donate mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows: . no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst system compared to the abc lst system. . the duration of filtration has been found without statistically significant differences between the two systems. . the loss of volume in the filter of lst is higher than in the abc lst (p = . < . , which is statistically significant). . there was very good leucodepletion with the lst systems (median reduction of the wb cells . log) but there was a superiority of the abc lst over the lst concerning the leucodepletion per litter and per unit (p: . and p: . respectively). . the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst . evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged - days, filtered and consecutively washed with ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were: . the duration of priming and filtering the rccs. . the loss of volume in the filter. . the efficiency of the filtration. . the acceptance of the personnel of a new (to them) filtration system.our results have as follows: . we counted the duration of priming and filtration. median time of priming was s (range - ) and of filtration was min (range - ). . the median volume lost in the filter (as calculated) was . ml (ranging from . to . ). this narrow range is apparently due to the non-flexible cell of the filter. . the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was . ¥ (range . - . ¥ ) and per unit was . ¥ (range . - . ¥ ). the median reduction of the wbc count was . log (range . - . ). . the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly blood banks and blood stations in whole country. the overall blood collected at those centers is about . . units. nearly one in third is being collected at government hospitals ( . %), nearly the same is collected at university hospital blood banks ( . %). the third major group is the red crescent society blood centers-rcsbc ( . %), followed by the social security hospitals ( . %). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) > . . . - . . . - . . . - . . < . . conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among topics in five broad categories. the top two topics were selected for the forum from the completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field.